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Sample records for armed replicating adenoviruses

  1. shRNA-armed conditionally replicative adenoviruses: a promising approach for cancer therapy

    Science.gov (United States)

    Zhang, Jie; Ding, Meng; Xu, Kai; Mao, Lijun; Zheng, Junian

    2016-01-01

    The small-interfering RNAs (siRNAs) have been employed to knockdown the expression of cancer-associated genes and shown some promise in cancer therapy. However, synthetic siRNA duplexes or plasmid mediated delivery of siRNAs have several problems, such as short half-life, low transfection efficiency and cytotoxicity associated with transfection. Conditionally replicating adenovirus (CRAds) as the delivery vector for short hairpin RNAs (shRNAs) could overcome these limitations and have shown augmented anti-tumor effects in experimental studies and preclinical trials. In this review, we summarize recent progress in the development of CRAds-shRNA for cancer treatment. Combination of CRAds-shRNA with chemotherapeutics, radiation, dendritic cells, monoclonal antibodies and small-molecule inhibitors will be necessary to eradicate cancer cells and cancer stem cells and achieve superior outcomes. The use of CRAd platform for efficient delivery of shRNAs and foreign genes will open a new avenue for cancer therapy. PMID:26980708

  2. Vaccine Design: Replication-Defective Adenovirus Vectors.

    Science.gov (United States)

    Zhou, Xiangyang; Xiang, Zhiquan; Ertl, Hildegund C J

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies. PMID:27076309

  3. 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.

  4. Localization of adenovirus DNA replication in KB cells

    NARCIS (Netherlands)

    Vlak, J.M.; Rozijn, Th.H.; Spies, F.

    1975-01-01

    The localization of adenovirus type 5 DNA replication has been investigated by both fractionation of isolated nuclei and electron-microscope autoradiography. Nuclear fractionation by means of the M-band-technique of Tremblay et al. (Tremblay, G. Y., Daniels, M. J., and Schaechter, M. (1969). J. Mol.

  5. 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.

  6. CCL21/IL21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells.

    Science.gov (United States)

    Li, Yang; Li, Yi-Fei; Si, Chong-Zhan; Zhu, Yu-Hui; Jin, Yan; Zhu, Tong-Tong; Liu, Ming-Yuan; Liu, Guang-Yao

    2016-07-15

    Multigene-armed oncolytic adenoviruses are capable of efficiently generating a productive antitumor immune response. The chemokine (C-C motif) ligand 21 (CCL21) binds to CCR7 on naïve T cells and dendritic cells (DCs) to promote their chemoattraction to the tumor and resultant antitumor activity. Interleukin 21 (IL21) promotes survival of naïve T cells while maintaining their CCR7 surface expression, which increases their capacity to transmigrate in response to CCL21 chemoattraction. IL21 is also involved in NK cell differentiation and B cell activation and proliferation. The generation of effective antitumor immune responses is a complex process dependent upon coordinated interactions of various subsets of effector cells. Using the AdEasy system, we aimed to construct an oncolytic adenovirus co-expressing CCL21 and IL21 that could selectively replicate in TERTp-positive tumor cells (Ad-CCL21-IL21 virus). The E1A promoter of these oncolytic adenoviruses was replaced by telomerase reverse transcriptase promoter (TERTp). Ad-CCL21-IL21 was constructed from three plasmids, pGTE-IL21, pShuttle-CMV-CCL21 and AdEasy-1 and was homologously recombined and propagated in the Escherichia coli strain BJ5183 and the packaging cell line HEK-293, respectively. Our results showed that our targeted and armed oncolytic adenoviruses Ad-CCL21-IL21 can induce apoptosis in TERTp-positive tumor cells to give rise to viral propagation, in a dose-dependent manner. Importantly, we confirm that these modified oncolytic adenoviruses do not replicate efficiently in normal cells even under high viral loads. Additionally, we investigate the role of Ad-CCL21-IL21 in inducing antitumor activity and tumor specific cytotoxicity of CTLs in vitro. This study suggests that Ad-CCL21-IL21 is a promising targeted tumor-specific oncolytic adenovirus. PMID:27157859

  7. 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.

  8. HSF1 overexpression enhances oncolytic effect of replicative adenovirus

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    Deng Youwen

    2010-05-01

    Full Text Available Abstract Background E1B55kD deleted oncolytic adenovirus was designed to achieve cancer-specific cytotoxicity, but showed limitations in clinical study. To find a method to increase its efficacy, we investigated the correlation between oncolytic effect of such oncolytic adenovirus Adel55 and intracellular heat shock transcription factor 1 (HSF1 activity. Methods In the present study, human breast cancer cell line Bcap37 was stably transfected with constitutively active HSF1 (cHSF1 or HSF1 specific siRNA (HSF1i to establish increased or decreased HSF1 expression levels. Cytotoxicity of Adel55 was analyzed in these cell lines in vitro and in vivo. Furthermore, Adel55 incorporated with cHSF1 (Adel55-cHSF1 was used to treat various tumor xenografts. Results Adel55 could achieve more efficient oncolysis in cHSF1 transfected Bcap37 cells, both in vitro and in vivo. However, inhibition of HSF1 expression by HSF1i could rescue Bcap37 cell line from oncolysis by Adel55. A time course study of viral replication established a correlation between higher replication of Adel55 and cytolysis or tumor growth inhibition. Then, we constructed Adel55-cHSF1 for tumor gene therapy and demonstrated that it is more potent than Adel55 itself in oncolysis and replication in both Bcap37 and SW620 xenografts. Conclusions cHSF1 enhances the Adel55 cell-killing potential through increasing the viral replication and is a potential therapeutic implication to augment the potential of E1B55kD deleted oncolytic adenovirus by increasing its burst.

  9. Non-Replicating Adenovirus-Vectored Anthrax Vaccine

    International Nuclear Information System (INIS)

    As bioterrorism is emerging as a national threat, it is urgent to develop a new generation of anthrax vaccines that can be rapidly produced and mass administered in an emergency setting. We have demonstrated that protective immunity against anthrax spores could be elicited in mice by intranasal administration of a non-replicating human adenovirus serotype 5 (Ad5)-derived vector encoding Bacillus anthracis protective antigen (PA) in a single-dose regimen. The potency of an Ad5 vector encoding PA was remarkably enhanced by codon optimization of the PA gene to match the tRNA pool found in human cells. This nasal vaccine can be mass-administered by non-medical personnel during a bioterrorist attack. In addition, replication-competent adenovirus (RCA)-free Ad5-vectored anthrax vaccines can be mass produced in PER.C6 cells in serum-free wave bioreactors and purified by column chromatography to meet a surge in demand. The non-replicating nature of this new generation of anthrax vaccine ensures an excellent safety profile for vaccines and the environment.(author)

  10. ADENO-ASSOCIATED SATELLITE VIRUS INTERFERENCE WITH THE REPLICATION OF ITS HELPER ADENOVIRUS

    Science.gov (United States)

    Parks, Wade P.; Casazza, Anna M.; Alcott, Judith; Melnick, Joseph L.

    1968-01-01

    Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80°C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This

  11. Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

    International Nuclear Information System (INIS)

    The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

  12. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    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.

  13. 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

  14. Targeting Mesothelioma Using an Infectivity Enhanced Survivin-Conditionally Replicative Adenoviruses

    OpenAIRE

    ZHU, Zeng B.; Makhija, Sharmila K.; Lu, Baogen; Wang, Minghui; Wang, Shuyi; Takayama, Koichi; Siegal, Gene P.; Reynolds, Paul N.; Curiel, David T.

    2006-01-01

    Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells ...

  15. 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......-less viruses replicated with higher efficiency in Ras-mutant cells, as compared to cell lines without Ras mutation. However, several exceptions to this rule were observed, arguing against a direct inhibition of PKR by mutant Ras. Phosphorylation of the PKR-substrate eIF2alpha was observed regardless of the Ras...

  16. Studies on the mechanism of replication of adenovirus DNA. IV. Discontinuous DNA chain propagation

    NARCIS (Netherlands)

    Vlak, J.M.; Rozijn, Th.H.; Sussenbach, J.S.

    1975-01-01

    The replication of adenovirus type 5 DNA occurs by discontinuous chain propagation via short pieces of DNA. These pieces accumulate if the infected cells are treated with hydroxyurea. They have a sedimentation coefficient of 11 S corresponding to a molecular weight of about 700,000, and they contain

  17. Inhibitory effects of conditionally replicative adenovirus armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control on human renal carcinoma OSRC cells%荷载管家基因shRNA的双调控溶瘤腺病毒抑制肾癌细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    李连涛; 张宝福; 李慧忠; 郑骏年

    2012-01-01

    Objective To study the effects of suppressing cell proliferation and GAPDH gene expression on human renal carcinoma OSRC cells with TD - GAPDH, a conditionally replicative adenovirus ( CRAds ) armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control . Methods Renal carcinoma OSRC cells and human proximal tubular epithelial cells ( HK - 2 ) were infected with TD - GAPDH, TD55, ZD55 - EGFP. The protein expressions of E1A were detected by Western blot. GAPDH expressing levels of OSRC cells were detected by RT -PCR and Western blot. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTT method. OSRC cells were stained with crystal violet to detect cytotoxici effect . Results Western blot assay of E1A expression indicated OSRC cells infected with TD - GAPDH, TD55, ZD55 - EGFP expressed E1A, not detected E1A protein expression in HK - 2 cells. Significant reduction of GAPDH mRNA and protein content was observed in the lysates from OSRC cells in -fected with TD - GAPDH. TD - GAPDH treatment of OSRC cells resulted in increased apoptotie cell death than TD 55 and ZD55 - EGFP treatment. Results of crystal violet stain and MTT assay demonstrated that the antitumor effect of TD-GAPDH was more potent than both TD55 and ZD55 - EGFP. Conclusion CRAds armed with shRNA against GAPDH gene could duplicate in tumor cells , which has both the lytic ability of oncolytic adenovirus and the capacity to deliver shR -NA. TD - GAPDH armed with shRNA gene can significantly inhibit tumor cell growth , which may provide a new platform for cancer gene therapy with shRNA and mayprove to be a useful novel tool for renal cancer therapy .%目的 研究荷载管家基因GAPDH shRNA的溶瘤腺病毒(TD-GAPDH)对人肾癌细胞OSRC增殖抑制的作用.方法 病毒TD-GAPDH、TD55、ZD55-EGFP感染OSRC细胞、人肾小管上皮细胞HK-2.Western blot法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法分别检测GAPDH

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. The p53 Protein Does Not Facilitate Adenovirus Type 5 Replication in Normal Human Cells

    OpenAIRE

    Chahal, Jasdave S.; Flint, S J

    2013-01-01

    Although several adenovirus type 5 (Ad5) proteins prevent deleterious consequences of activation of p53, it has been reported that viral replication proceeds more efficiently when human tumor cells produce wild-type compared to mutant p53. We have now exploited RNA interference and lentiviral vectors to achieve essentially complete knockdown of p53 in normal human cells: no effects on the kinetics or efficiency of viral gene expression or production of infectious particles were observed.

  3. 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.

  4. 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.

  5. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ran-yi, E-mail: liuranyi@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhou, Ling [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhang, Yan-ling [School of Biotechnology, Southern Medical University, Guangzhou 510515 (China); Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Li, Li-xia [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010 (China); Fu, Xiang; Wu, Jiang-xue [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Huang, Wenlin, E-mail: hwenl@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Guangdong Provincial Key Laboratory of Tumor-Targeted Drug, Doublle Bioproducts Inc., Guangzhou 510663 (China)

    2013-12-13

    Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

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

    Directory of Open Access Journals (Sweden)

    Saki Kondo

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

  7. Tumor promoters alter the temporal program of adenovirus replication in human cells.

    OpenAIRE

    Fisher, P B; Young, C S; Weinstein, I B; Carter, T. H.

    1981-01-01

    In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) ...

  8. 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.

  9. Unique conditionally replication competent bipartite adenoviruses-cancer terminator viruses (CTV): efficacious reagents for cancer gene therapy.

    Science.gov (United States)

    Sarkar, Devanand; Su, Zao-Zhong; Fisher, Paul B

    2006-07-01

    The frequent resistance of aggressive cancers to currently available therapies, such as radiotherapy and chemotherapy, mandates development of targeted, nontoxic and more efficacious treatment protocols. Conditionally replication competent adenoviruses (CRCAs) that induce oncolysis by cancer-specific replication are currently being evaluated in clinical trials. However, a single modality approach may not be sufficient to completely eradicate cancer in a patient, because most cancers arise from abnormalities in multiple genetic and signal transduction pathways. The promoter region of rodent progression elevated gene-3 (PEG-3), cloned and characterized in our laboratory, embodies the unique property of increased activity in a broad range of tumor cells, both rodent and human, when compared to normal counterparts. Bipartite adenoviruses were engineered to express the E1A gene, necessary for viral replication, under control of the PEG-3 promoter (PEG-Prom) and simultaneously express a second transgene in the E3 region that encodes an apoptosis-inducing and immunomodulatory cytokine, either immune interferon (IFN-gamma) or melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). These conditionally replication competent bipartite adenoviruses, referred to as cancer terminator viruses (CTVs), facilitated cancer-selective adenovirus replication, robust transgene expression and apoptosis induction with complete eradication of both primary and distant (metastatic) human cancers xenotransplanted in athymic nude mice. These findings suggest that CTVs might prove efficacious for the therapy of primary and advanced neoplastic diseases. PMID:16861924

  10. Immunotherapeutic effects of cytokine-induced killer cells combined with CCL21/IL15 armed oncolytic adenovirus in TERT-positive tumor cells.

    Science.gov (United States)

    Ye, Jun-Feng; Lin, Yuan-Qiang; Yu, Xiu-Hua; Liu, Ming-Yuan; Li, Yang

    2016-09-01

    The effective antitumor immune responses are dependent on coordinate interaction of various effector cells. Thus, the combination of adoptive immunotherapy and target gene therapy is capable of efficiently generating a productive antitumor immune response. We investigated whether combination of cytokine-induced killer (CIK) cells adoptive immunotherapy and CCL21/IL15 armed oncolytic adenovirus could induce the enhanced antitumor activity. The CCL21/IL15 co-expression oncolytic adenoviruses were constructed by using the AdEasy system, which uses homologous recombination with shuttle plasmids and full length Ad backbones. This conditionally replicating adenoviruses CRAd-CCL21-IL15 could induce apoptosis in TERTp-positive tumor cells for viral propagation, but do not replicate efficiently in normal cells, because the E1A promoter was replaced by telomerase reverse transcriptase promoter (TERTp). Our results showed that the combination of CIK cells and CRAd-CCL21-IL15 could induce higher antitumor activity than either CIK cells or CRAd-CCL21-IL15 alone. This combined treatment could induce the tumor specific cytotoxicity of CTLs (cytotoxic T lymphocytes) in vitro. Moreover, the treatment of established tumors with the combined therapy of CIK cells and CRAd-CCL21-IL15 resulted in tumor regression. This study suggests that the combined treatment by adoptive immunotherapy and gene therapy is a promising strategy for the therapy of tumor. PMID:27380620

  11. Double-detargeted oncolytic adenovirus shows replication arrest in liver cells and retains neuroendocrine cell killing ability.

    Directory of Open Access Journals (Sweden)

    Justyna Leja

    Full Text Available BACKGROUND: We have previously developed an oncolytic serotype 5 adenovirus (Ad5 with chromogranin-A (CgA promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control. METHODOLOGY/PRINCIPAL FINDINGS: Ad[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter. CONCLUSIONS/SIGNIFICANCE: A combination of transcriptional (promoter and post-transcriptional (miRNA target regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.

  12. Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FM...

  13. 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.

  14. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  15. The study of RNAi-mediated by conditionally replicating adenovirus silencing on Survivin gene in colon cancer cell lastingly

    Institute of Scientific and Technical Information of China (English)

    Chunyi Wang; Zhongxue Fu

    2008-01-01

    Objective: To investigate the effect of RNAi-mediated Survivin gene with conditionally replicating adenovirus silencing on Survivin gene in colon carcinoma cell lines HT-29 lastingly.Methods: We transfected Ad-delElb55KD-shRNA/ Survivin-EGFP to HT-29 (control was replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA/Survivin-EGFP).The expressions of EGFP, Survivin mRNA and Survivin protein in HT29 were detected at the 1st, 7th, 14th and 28th days after transfection.Results: The expression of EGFP, the inhibition of Survivin mRNA and Survivin protein in HT-29 were high in each group at the 7th day after transfection, among the total, the effect of Ad-delE1b55KD-shRNA/Survivin-EGFP group was the highest; at the 14th day, the effects of replication defective adenovirus group and liposome vector group were decreased obviously, and it was still high in Ad-delE1b55KD-shRNA/Survivin-EGFP group; at the 28th day, the effects of control groups were disappeared, and it was still high in Ad-delE1b55KDshRNA / Survivin-EGFP group like before (P<0.05).Conclusion: RNAi-mediated Survivin gene with conditionally replicating adenovirus can silence Survivin gene in colon carcinoma call lines HT-29 lastingly.

  16. Production and purification of non replicative canine adenovirus type 2 derived vectors

    OpenAIRE

    Szelechowski, Marion; Bergeron, Corinne; Gonzalez Dunia, Daniel; Klonjkowski, Bernard

    2013-01-01

    Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central ne...

  17. Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

    OpenAIRE

    Kaner, Robert J.; Santiago, Francisco; Rahaghi, Franck; Michaels, Elizabeth; Moore, John P.; Crystal, Ronald G.

    2009-01-01

    Heterologous viruses may transactivate or suppress human immunodeficiency virus (HIV)–1 replication. An adenovirus type 5 gene transfer vector (Ad5) HIV-1 vaccine was recently evaluated in a clinical trial, without efficacy. In this context, it is relevant to ask what effect Ad vectors have on HIV-1 replication, particularly in cells that are part of the innate immune system. Infection of HIV-1–infected human alveolar macrophages (AMs) obtained from HIV-1+ individuals with an Ad vector contai...

  18. Intravenous administration of the conditionally replicative adenovirus Ad5-Δ24RGD induces regression of osteosarcoma lung metastases

    Directory of Open Access Journals (Sweden)

    Gerritsen Winald R

    2008-01-01

    Full Text Available Abstract Metastatic osteosarcoma (OS has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Δ24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Δ24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Δ24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1–3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Δ24RGD-treated mice (p = 0.15. Moreover, mice treated at weeks 5–7 showed a significantly reduced lung weight (decrease of tumor mass, p 149, p = 0.12 compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Δ24RGD might be a promising new treatment strategy for metastatic osteosarcoma.

  19. Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Huajun Jin

    Full Text Available Highly selective therapy for hepatocellular carcinoma (HCC remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4% and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011(let7T, by introducing eight copies of let-7 target sites (let7T into the 3' untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels of the SG7011(let7T was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011(let7T was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68 expressing high level of let-7 (>300 folds, whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5 with low level of let-7. Consequently, the cytotoxicity of SG7011(let7T to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011(let7Tin vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011(let7T may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.

  20. Molecular basis for viral selective replication in cancer cells: activation of CDK2 by adenovirus-induced cyclin E.

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    Full Text Available Adenoviruses (Ads with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL, promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

  1. Production and purification of non replicative canine adenovirus type 2 derived vectors.

    Science.gov (United States)

    Szelechowski, Marion; Bergeron, Corinne; Gonzalez-Dunia, Daniel; Klonjkowski, Bernard

    2013-01-01

    Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo. PMID:24326926

  2. Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative

    OpenAIRE

    Sanchez-Cespedes, Javier; Moyer, Crystal L.; Whitby, Landon R.; Boger, Dale L.; Nemerow, Glen R.

    2014-01-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to i...

  3. 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.

  4. Avian influenza in ovo vaccination with replication defective recombinant adenovirus in chickens: vaccine potency, antibody persistence, and maternal antibody transfer.

    Science.gov (United States)

    Mesonero, Alexander; Suarez, David L; van Santen, Edzard; Tang, De-Chu C; Toro, Haroldo

    2011-06-01

    Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibody persistence, transfer of maternal antibodies (MtAb), and interference between MtAb and active in ovo or mucosal immunization with RCA-free recombinant Ad expressing a codon-optimized AIV H5 HA gene from A/turkey/WI/68 (AdTW68.H5(ck)). Vaccine coverage and intrapotency test repeatability were based on anti-H5 hemagglutination inhibition (HI) antibody levels detected in in ovo vaccinated chickens. Even though egg inoculation of each replicate was performed by individuals with varying expertise and with different vaccine batches, the average vaccine coverage of three replicates was 85%. The intrapotency test repeatability, which considers both positive as well as negative values, varied between 0.69 and 0.71, indicating effective vaccination. Highly pathogenic (HP) AIV challenge of chicken groups vaccinated with increasing vaccine doses showed 90% protection in chickens receiving > or = 10(8) ifu (infectious units)/bird. The protective dose 50% (PD50) was determined to be 10(6.5) ifu. Even vaccinated chickens that did not develop detectable antibody levels were effectively protected against HP AIV challenge. This result is consistent with previous findings ofAd-vector eliciting T lymphocyte responses. Higher vaccine doses significantly reduced viral shedding as determined by AIV RNA concentration in oropharyngeal swabs. Assessment of antibody persistence showed that antibody levels of in ovo immunized chickens continued to increase until 12 wk and started to decline after 18 wk of age. Intramuscular (IM) booster vaccination with the same vaccine at 16 wk of age significantly increased the antibody responses in breeder hens, and these responses were maintained at high

  5. Immunological effects of a tumor necrosis factor alpha-armed oncolytic adenovirus.

    Science.gov (United States)

    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.

  6. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    Science.gov (United States)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  7. Interferon-β-armed oncolytic adenovirus induces both apoptosis and necroptosis in cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hongling Huang; Tian Xiao; Lingfeng He; Hongbin Ji; Xin-Yuan Liu

    2012-01-01

    Interferon-β (IFN-β) has been widely used in cancer therapy,but the clinical trial results are generally disappointing.Our previous studies have shown that an oncolytic adenovirus carrying IFN-β (ZD55-IFN-β) exhibits significant anti-tumor activities.However,the underlying mechanisms are not clear.Here we showed that ZD55-IFN-β infection-induced S-phase cell cycle arrest in a p53-dependent manner by activating the ataxia telangiectasia mutated-dependent DNA damage pathway.In addition, ZD55-IFN-β infection could initiate both caspase-dependent apoptosis and necroptosis in cancer cells.More importantly,ZD55-IFN-β showed a synergistic effect on cancer cells when combined with doxorubicin.These results suggest that the combination of ZD55-IFN-β with doxorubicin may represent a promising clinical strategy in cancer therapy.

  8. Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model

    International Nuclear Information System (INIS)

    Purpose: The purpose of this study was to evaluate the efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in an adjuvant setting with external beam radiation therapy (EBRT) in an experimental prostate cancer model in preparation for a Phase I clinical study in humans. Methods: For efficacy studies, i.m. DU145 and intraprostatic LNCaP C4-2 tumors were established in immune-deficient mice. Tumors were injected with the lytic, replication-competent Ad5-CD/TKrep adenovirus containing a cytosine deaminase (CD)/herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene. Two days later, mice were administered 1 week of 5-fluorocytosine + ganciclovir (GCV) prodrug therapy and fractionated doses of EBRT (trimodal therapy). Tumor control rate of trimodal therapy was compared to that of EBRT alone. For toxicology studies, immune-competent male mice received a single intraprostatic injection (1010 vp) of the replication-competent Ad5-CD/TKrep adenovirus. Two days later, mice were administered 4 weeks of 5-fluorocytosine + GCV prodrug therapy and 56 Gy EBRT to the pelvic region. The toxicity of trimodal therapy was assessed by histopathologic analysis of major organs and clinical chemistries. Results: In both the i.m. DU145 and intraprostatic LNCaP C4-2 tumor models, trimodal therapy significantly improved primary tumor control beyond that of EBRT alone. In the DU145 model, trimodal therapy resulted in a tumor growth delay (70 days) that was more than twice that (32 days) of EBRT alone. Whereas EBRT failed to eradicate DU145 tumors, trimodal therapy resulted in 25% tumor cure. In the LNCaP C4-2 tumor model, EBRT slowed the growth of intraprostatic tumors, but resulted in no tumor cures, and 57% of the mice developed retroperitoneal lymph node metastases at 3 months. By contrast, trimodal therapy resulted in 44% tumor cure and reduced significantly the percentage (13%) of lymph node metastases relative to EBRT alone. Overall

  9. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G., E-mail: chinnag@slu.edu

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.

  10. 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.

  11. Enhanced antitumoral efficacy and immune response following conditionally replicative adenovirus containing constitutive HSF1 delivery to rodent tumors

    Directory of Open Access Journals (Sweden)

    Fan Rong

    2012-05-01

    Full Text Available Abstract Background Oncolytic adenoviruses are promising as anticancer agents but have limited clinical responses. Our previous study showed that heat shock transcription factor 1 (HSF1 overexpression could increase the anti-tumor efficacy of E1B55kD deleted oncolytic adenovirus through increasing the viral burst. Due to the important roles of heat shock proteins (HSPs in eliciting innate and adaptive immunity, we reasoned that besides increasing the viral burst, HSF1 may also play a role in increasing tumor specific immune response. Methods In the present study, intra-dermal murine models of melanoma (B16 and colorectal carcinoma (CT26 were treated with E1B55kD deleted oncolytic adenovirus Adel55 or Adel55 incorporated with cHSF1, HSF1i, HSP70, or HSP90 by intra-tumoral injection. Tumors were surgically excised 72 h post injection and animals were analyzed for tumor resistance and survival rate. Results Approximately 95% of animals in the Adel55-cHSF1 treated group showed sustained resistance upon re-challenge with autologous tumor cells, but not in PBS, Adel55, or Adel55-HSF1i treated groups. Only 50–65% animals in the Adel55-HSP70 and Adel55-HSP90 treated group showed tumor resistance. Tumor resistance was associated with development of tumor type specific cellular immune responses. Adel55-cHSF1 treatment also showed higher efficacy in diminishing progression of the secondary tumor focus than Adel55-HSP70 or Adel55-HSP90 treatment. Conclusions Besides by increasing its burst in tumor cells, cHSF1 could also augment the potential of E1B55kD deleted oncolytic adenovirus by increasing the tumor-specific immune response, which is beneficial to prevent tumor recurrence. cHSF1 is a better gene for neoadjuvant immunotherapy than other heat shock protein genes.

  12. A pRb-responsive, RGD-modified, and Hyaluronidase-armed Canine Oncolytic Adenovirus for Application in Veterinary Oncology

    OpenAIRE

    Laborda, Eduardo; Puig-Saus, Cristina; Rodriguez-García, Alba; Moreno, Rafael; Cascalló, Manel; Pastor, Josep; Alemany, Ramon

    2014-01-01

    Human and canine cancer share similarities such as genetic and molecular aspects, biological complexity, tumor epidemiology, and targeted therapeutic treatment. Lack of good animal models for human adenovirotherapy has spurred the use of canine adenovirus 2-based oncolytic viruses. We have constructed a canine oncolytic virus that mimics the characteristics of our previously published human adenovirus ICOVIR17: expression of E1a controlled by E2F sites, deletion of the pRb-binding site of E1a...

  13. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    Science.gov (United States)

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Jasdave S Chahal

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

  16. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  17. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Science.gov (United States)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  18. 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.

  19. The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: Implications for the mechanism of DNA replication

    NARCIS (Netherlands)

    Steenbergh, P.H.; Sussenbach, J.S.

    1979-01-01

    The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the

  20. 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.

  1. 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.

  2. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

    Science.gov (United States)

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J Michael; Kanerva, Anna; Hemminki, Akseli

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors.

  3. CXCR4启动子的条件复制型腺病毒对肺癌细胞的靶向杀伤作用%Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

    Institute of Scientific and Technical Information of China (English)

    李龙光; 李书华; 王红艳; 龙捷; 谢晓斌; 张雅洁

    2015-01-01

    [ ABSTRACT] AIM:To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically.METHODS:Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP.The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP.PCR was used to detect the target gene fragments, and the viral titer was determined.A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR.CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL.CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay.RE-SULTS:The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully.Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus.PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome.The virus in the supernatant reached a titer of 1 ×1013 PFU/L.The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group.Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group

  4. Packaging and anti-tumor effect of double-targeted conditionally replicative adenovirus%双重靶向条件复制型腺病毒的包装及抗肿瘤作用

    Institute of Scientific and Technical Information of China (English)

    王宏芳; 刘扬; 李金华; 吴嘉慧; 李艳博; 王志成; 刘威武; 龚守良

    2013-01-01

    Objective To construct and package double-targeted conditionally replicative adenovirus (CRAd.p) regulated by hTERT promoter El A protein (CR2 missing) expression and to study its inhibitory effect on tumor cells. Methods The hTERT promoter was amplified by RT-PCR and cloned into the shuttle vector of pShuttle-hTERT-ElA-ElBp-ElB55K. Then it was transfected into HEK293 to package CRAd. p. Anti-tumor effect of CRAd. p was detected by CCK-8 assay. Results The sequence of amplified hTERT was consistent with that published on GenBank, indicating that the hTERT was cloned successfully. Furthermore, the recombinant plasmid pShuttle-hTERT-ElA-ElBp-ElB55K was identified by endonuclease digestion and PCR, which could be digested into two fagments of Kpn I . One was 1 542 bp and the other was 6 775 bp. The length of fragments amplified by PCR was 250 bp, 1 148 bp and 298 bp, which were consistent with those amplified fragments of recombinant adenovirus. CRAd.p was identified by PCR. These results were in agreement with the plan. The results of CCK-8 assay showed that the inhibitory effects of CRAd. p on the MCF-7, MDA-MB-231 and HepG2 cells were significantly different from their control groups (P<0.01), especially on MDA-MB-231 cells. The inhibition of the recombinant adenovirus on HCT-8 cells was irregular. The MDA-MB-231 cells were infected with different titers of recombinant adenovirus at 24, 48 and 72 hours after infection. Their results showed that with the increase of adenovirus doses, the inhibition of cellular growth increased gradually. Conclusion A double-targeted oncolytic virus is packed successfully, which may make gene therapy targeting and effective.%目的 构建并包装以人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子调控E1A(CR2区缺失)蛋白表达的肿瘤细胞双重靶向条件复制型腺病毒(conditionally replicating adenovirus,CRAd),并探讨其对肿瘤细胞的抑制作用.方法 采用PCR技术扩增hTERT启

  5. 表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用%The antitumor effects of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy in nude mouse model

    Institute of Scientific and Technical Information of China (English)

    薛松; 刘斌; 高超; 顾玉明; 郑骏年; 李望; 李海龙; 朱海涛; 辛勇; 刘俊杰; 徐为; 宋文哲

    2009-01-01

    目的 观察表达针对端粒酶逆转录酶(hTERT)基因的小干扰RNA(hTERT siRNA)的溶瘤腺病毒(ZD55-hTERT)抑制肾癌移植瘤生长作用.方法 荷肾癌裸鼠随机分4组,每组8只.瘤体内分别注射ZD55-hTERT、增殖缺陷型腺病毒(Ad-hTERT)、溶瘤腺病毒ZD55-EGFP及磷酸盐缓冲液(PBS),每次注射病毒7×108pfu/只,连续注射3 d.注射后第7天,每组处死3只取肿瘤组织,免疫组织化学检测肿瘤hTERT、E1A表达及凋亡.第50天时处死动物测量肿瘤体积.结果 ZD55-hTERT、Ad-hTERT、ZD55-EGFP及PBS处理组肿瘤体积(mm3)分别为:124.1±27.5、609.0±102.5、499.8±77.1、1552.1±206.4,ZD55-hTERT处理组与各组之间差异有统计学意义(P<0.01).Ad-hTERT处理组肿瘤无E1A表达,ZD55-hTERT处理组E1A大量表达,表明病毒复制.ZD55-hTERT处理组肿瘤hTERT表达显著低于Ad-hTERT处理组,凋亡细胞阳性率均显著高于Ad-hTERT处理组.结论 表达hTERT siRNA的溶瘤腺病毒ZD55-hTERT具有更强的抑制肾癌生长作用.%Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups

  6. Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

    OpenAIRE

    Wold, William S.M.; Toth, Karoly

    2013-01-01

    Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vector...

  7. 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...

  8. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

    Science.gov (United States)

    Okamba, Faust René; Arella, Maximilien; Music, Nedzad; Jia, Jian Jun; Gottschalk, Marcelo; Gagnon, Carl A

    2010-07-01

    Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines. PMID:20472025

  9. Construction of conditionally replicative adenovirus vector mediated by dual specific promoters%双特异性启动子调控条件复制腺病毒载体的构建

    Institute of Scientific and Technical Information of China (English)

    李玮; 谭建

    2013-01-01

    Objective To construct and identify the conditionally replicative adenovirus vectors which may induce the human sodium iodide symporter (hNIS) expression in the early region 1A (E1A) gene of the human telomerase reverse transcriptase (hTERT) and the glial fibrillary acidic protein (GFAP)promoter regions.Methods Immunoblotting assay was employed to detect the variation in GFAP and telomerase protein expression prior to and following viral infection of the cerebral stellar glioblastoma cells (U87),neuroglioma cells (U251) and human embryonic lung fibroblasts (MRC-5) cells.The hTERT and GFAP promoters and the hNIS genes were amplified by polymerase chain reaction for synthesis of adenoviral E1A genes.The recombinant plasmids containing hTERT and GFAP gene promoters were adopted for transfection into MRC-5,U251 and U87,which entailed assessment of the hTERT and GFAP promoter activity via fluorescent analysis after 24 h.This was followed by ligation with the E1A and hNIS genes and subsequent cloning into the plasmid pDC311 for construction of the recombinant plasmid pDC311-Tp-E1A-Gp-NIS that was identified by double enzyme digestion (EcoR Ⅰ and Sal Ⅰ) and gene sequencing.This recombinant plasmid was co-transfected with adenoviral genomic plasmid pBHGlox△E1-3Cre into the human embryonic kidney 293 cells forming the conditionally replicative recombinant adenovirus Ad-Tp-E1A-Gp-NIS.This Ad-Tp-E1A-Gp-NIS was employed to transfect the 293,MRC-5,U87 and U251 cells,whose conditional replicability was measured by plaque forming assay.The recombinant virus Ad-Tp-E1A-Gp-NIS and Ad-CMV-EGFP controls were used to transfect the U251,U87 and MRC-5 cells for detection of the capacity of 125I uptake by using a γ-ray counter.Results The expression of the 120 000 telomerase and 49 000 GFAP protein could be found in U87 and U251 cells,but not in MRC-5 cells.Glioma target gene expression could be induced by the GFAP and hTERT promoters,the efficiency of which was (62.10±6.26)

  10. Synergistic suppression effect on tumor growth of ovarian cancer by combining cisplatin with a manganese superoxide dismutase-armed oncolytic adenovirus

    Science.gov (United States)

    Wang, Shibing; Shu, Jing; Chen, Li; Chen, Xiaopan; Zhao, Jianhong; Li, Shuangshuang; Mou, Xiaozhou; Tong, Xiangmin

    2016-01-01

    Gene therapy on the basis of oncolytic adenovirus is a novel approach for human cancer therapeutics. We aim to investigate whether it will synergistically reinforce their antiovarian cancer activities when the combined use of ZD55-manganese superoxide dismutase (MnSOD) and cisplatin was performed. The experiments in vitro showed that ZD55-MnSOD enhances cisplatin-induced apoptosis and causes remarkable ovarian cancer cell death. Apoptosis induction by treatment with ZD55-MnSOD and/or cisplatin was detected in SKOV-3 by apoptotic cell staining, flow cytometry, and western blot analysis. In addition, the cytotoxicity caused by ZD55-MnSOD to normal cells was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and western blot analysis. Animal experiment further confirmed that combination of ZD55-MnSOD and cisplatin achieved significant inhibition of SKOV-3 ovarian tumor xenografted growth. In summary, we have demonstrated that ZD55-MnSOD can sensitize human ovarian cancer cells to cisplatin-induced cell death and apoptosis in vitro and in vivo. These findings indicate that the combined treatment with ZD55-MnSOD and cisplatin could represent a rational approach for antiovarian cancer therapy.

  11. 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.

  12. A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

    OpenAIRE

    Gallaher, Sean D.; Berk, Arnold J.

    2013-01-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activi...

  13. 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

  14. Rapid generation of fowl adenovirus 9 vectors.

    Science.gov (United States)

    Pei, Yanlong; Griffin, Bryan; de Jong, Jondavid; Krell, Peter J; Nagy, Éva

    2015-10-01

    Fowl adenoviruses (FAdV) have the largest genomes of any fully sequenced adenovirus genome, and are widely considered as excellent platforms for vaccine development and gene therapy. As such, there is a strong need for stream-lined protocols/strategies for the generation of recombinant adenovirus genomes. Current genome engineering strategies rely upon plasmid based homologous recombination in Escherichia coli BJ5183. This process is time-consuming, involves multiple cloning steps, and low efficiency recombination. This report describes a novel system for the more rapid generation of recombinant fowl adenovirus genomes using the lambda Red recombinase system in E. coli DH10B. In this strategy, PCR based amplicons with around 50 nt long homologous arms, a unique SwaI site and a chloramphenicol resistance gene fragment (CAT cassette), are introduced into the FAdV-9 genome in a highly efficient and site-specific manner. To demonstrate the efficacy of this system we generated FAdV-9 ORF2, and FAdV-9 ORF11 deleted, CAT marked and unmarked FAdV-9 infectious clones (FAdmids), and replaced either ORF2 or ORF11, with an EGFP expression cassette or replaced ORF2 with an EGFP coding sequence via the unique SwaI sites, in approximately one month. All recombinant FAdmids expressed EGFP and were fully infectious in CH-SAH cells. PMID:26238923

  15. 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.

  16. 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...

  17. Improved Production of Gutted Adenovirus in Cells Expressing Adenovirus Preterminal Protein and DNA Polymerase

    OpenAIRE

    Hartigan-O’Connor, Dennis; Amalfitano, Andrea; Chamberlain, Jeffrey S.

    1999-01-01

    Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis...

  18. Structure of human adenovirus

    Energy Technology Data Exchange (ETDEWEB)

    Nemerow, Glen R.; Stewart, Phoebe L.; Reddy, Vijay S. (Scripps); (Vanderbilt)

    2012-07-11

    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 {angstrom} resolution. The virus structure revealed the location, folds, and interactions of major and minor (cement proteins) on the inner and outer capsid surface. This new structural information sheds further light on the process of adenovirus capsid assembly and virus-host cell interactions.

  19. Adenovirus (For Parents)

    Science.gov (United States)

    ... respiratory tract as well, causing bronchiolitis , croup , or viral pneumonia, which is less common but can cause serious illness in infants. Adenovirus can also produce a dry, harsh cough that can resemble whooping cough (pertussis) . Gastroenteritis is an inflammation of the stomach and the ...

  20. Armoring CRAds with p21/Waf-1 shRNAs: the next generation of oncolytic adenoviruses

    OpenAIRE

    Höti, N; Chowdhury, WH; Mustafa, S.; Ribas, J; Castanares, M; Johnson, T.; Liu, M.; Lupold, SE; Rodriguez, R.

    2010-01-01

    Conditionally replicating adenoviruses (CRAds) represent a promising modality for the treatment of neoplastic diseases, including Prostate Cancer. Selectively replicating viruses can be generated by placing a tissue or cancer-specific promoter upstream of one or more of the viral genes required for replication (for example, E1A, E1B). We have previously reported multiple cellular processes that can attenuate viral replication, which in turn compromises viral oncolysis and tumor kill. In this ...

  1. Human adenovirus type identification

    OpenAIRE

    Banik U; Adhikary AK

    2015-01-01

    Urmila Banik,1 Arun Kumar Adhikary21Unit of Pathology, 2Unit of Microbiology, Faculty of Medicine, AIMST University, Bedong, Kedah, MalaysiaThe published paper in your journal entitling “Human adenovirus type 8 epidemic keratoconjunctivitis with large corneal epithelial full-layer detachment: an endemic outbreak with uncommon manifestations” has come into our attention.1 The article provides interesting clinical presentation of corneal epithelial layer detachment among 25%...

  2. Synergistic Antitumor Efficacy of Oncolytic Adenovirus Combined with Chemotherapy

    Institute of Scientific and Technical Information of China (English)

    LI Yue-min; QIAN Qi-jun; SONG San-tai; JIANG Ze-fei; ZHANG Qi; QU Yi-mei; SU Chang-qing; ZHAO Chuan-hua; LI Zhi-qiang; GE Fei-jiao

    2007-01-01

    Objective: Chemotherapy is an effective means of treating breast cancer, and cancer-specific replicative adenovirus is also a promising antitumor agent in recent years. Our investigation aims to demonstrate that CNHK300 can mediate selective antitumor efficacy and produce synergistic cytotoxicity with chemotherapy on HER-2 over-expressing breast cancer. Methods: We engineered the telomerase-dependent replicative adenovirus CNHK300 by placing the E1A gene under the control of the human hTERT promoter. By analysis of E1A expression, we proved the fidelity of hTERT promoter in adenovirus genome and the selective expression of E1A in telomerase-positive breast cancer cells but not in normal fibroblast cells. By proliferation test, we further showed efficient replication of CNHK300 in breast cancer cells with apparently attenuated proliferation in normal fibroblast cells. Finally, we demonstrated by MTT methods that CNHK300 virus caused potent cytolysis and produced synergistic cytotoxicity with chemotherapy in breast cancer cells with attenuated cytotoxicity on normal cells. Results: In this virus, the E1A gene is successfully placed under the control of the human hTERT promoter. CNHK300 virus replicated as efficiently as the wild-type adenovirus and caused intensive cell killing in HER-2 over-expressing breast cancer cells in vitro. In contrast, telomerase-negative normal fibroblast cells, which expressed no hTERT activity, were not able to support CNHK300 replication. Combined treatment of CNHK300 with paclitaxel improved cytotoxicity on cancer cells. Conclusion: We conclude that CNHK300 can produce selective antitumor efficacy and enhance the in vitro response of chemotherapy on HER-2 overexpressing breast cancer.

  3. 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.

  4. New Adenovirus in Bats, Germany

    OpenAIRE

    Sonntag, Michael; Mühldorfer, Kristin; Speck, Stephanie; Wibbelt, Gudrun; Kurth, Andreas

    2009-01-01

    We tested 55 deceased vespertilionid bats of 12 species from southern Germany for virus infections. A new adenovirus was isolated from tissue samples of 2 Pipistrellus pipistrellus bats, which represents the only chiropteran virus isolate found in Europe besides lyssavirus (rabies virus). Evidence was found for adenovirus transmission between bats.

  5. Bicalutamide Activated Oncolytic Adenovirus for the Adjuvant Therapy of High Risk Prostate Cancer

    OpenAIRE

    Johnson, Tamara Jane; Hoti, Naser Uddin; Liu, Chunyan; Chowdhury, Wasim H.; Ying LI; Zhang, Yonggang; Lupold, Shawn E.; DeWeese, Theodore; Rodriguez, Ronald

    2013-01-01

    Conditionally replicating adenoviruses (CRAds) utilize tissue specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer gene therapy require androgens to activate prostate specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen suppressive therapy. We describe a novel prostate specific CRAd generated by fusin...

  6. Combined use of chemotherapeutics and oncolytic adenovirus in treatment of AFP-expressing hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chen-Yu Mao; Han-Ju Hua; Ping Chen; De-Chao Yu; Jiang Cao; Li-Song Teng

    2009-01-01

    BACKGROUND: Hepatocellular carcinoma (HCC) which is always refractory to most chemotherapeutic agents may result in poor survival of patients with advanced HCC. Oncolytic adenovirus is a new form for cancer gene therapy via its ability to replicate and kill tumor cells in a tumor-speciifc manner. In order to eradicate tumors effectively, the combination of chemotherapeutic agents and oncolytic adenovirus has been considered. This study aimed to systematically analyze the possibility of synergistic cytotoxicity of oncolytic adenoviruses in combination with chemotherapeutic agents. METHODS: Several types of human HCC cell lines were used to determine the speciifcity and cytotoxicity of oncolytic adenovirus Ad5-HC and Ad5-AFP (IRES) by measuring cell viabilityin vitro and antitumor efifciency in vivo. The cytotoxicity of Ad5-HC and Ad5-AFP (IRES) combined with chemotherapeutic agents were also assessed by the methyl thiazolyl tetrazolium assay. RESULTS: Both Ad5-HC and Ad5-AFP (IRES) were signiifcantly cytotoxic to HCC cells with great speciifcity in vitro andin vivo. The combination of oncolytic adenovirus with 5-FU, doxorubicin, and paclitaxel was synergistically effective for the killing of HCC cells. CONCLUSIONS: These data suggest that oncolytic adenovirus sensitize tumors to chemotherapy and the combination therapy of chemotherapeutic agents and oncolytic adenovirus has an enhanced antitumor effect on HCC cells.

  7. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲

    2016-01-01

    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  8. [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.

  9. 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.

  10. Tracking adenovirus infections in reptiles

    OpenAIRE

    Ball, Inna

    2015-01-01

    The purpose of this project was to screen reptiles for the presence of adenovirus (AdV) infection, develop serological tests for the detection of antibodies against AdVs in squamate reptiles and to examine the serological relationships between lizard and snake AdVs, helping to ensure the establishment and maintenance of healthy populations. An additional aim of the project was the establishment of an agamid cell line and isolation of adenoviruses from bearded dragons (Pogona vitticeps). A...

  11. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    OpenAIRE

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the vi...

  12. 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.

  13. Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli

    OpenAIRE

    Wildner Oliver; Hoffmann Dennis

    2006-01-01

    Abstract Background Oncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 reg...

  14. 表达猪圆环病毒2型Cap蛋白重组复制缺陷型人5型腺病毒的构建及小鼠免疫试验%Construction and Immunogenicity Analysis of Recombinant Replication-defective Human Adenovirus Type 5 Bearing the Porcine Circovirus Type 2 Cap Protein Gene

    Institute of Scientific and Technical Information of China (English)

    宫婷; 张守峰; 刘晔; 孙程龙; 杨洋; 陈奇; 钱方; 刘博浩; 扈荣良

    2013-01-01

    To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up. To 108'5 TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 107 TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.%构建表达猪圆环病毒2型Cap蛋白重组复制缺陷型人5型腺病毒,评价其对小鼠免疫效果.本研究将编码PCV2-Cap蛋白的ORF2克隆到腺病毒表达系统穿梭质粒,构建了重组穿梭质粒pacAd5CMV-Cap.将被限制性内切酶PacⅠ线性化后的骨架质粒和重组穿梭质粒共转染HEK293AD细胞,两者在真核细胞内同源重组并包装出携带PCV2-Cap基因的复制缺陷型重组人5型腺病毒,命名为rAd5-Cap;以同样方法重组获得了不含任何靶基因的

  15. Construction of an Survivin-Conditionally Replicative Oncolytic Adenovirus Targeting Prostate Cancer and its Anti-Tumor Effect in Vitro%含Survivin启动子条件复制型腺病毒的构建及其对前列腺癌抑瘤效应的体外研究

    Institute of Scientific and Technical Information of China (English)

    文博; 李晓铭; 黄丽兴; 黄小佳; 李春; 冯海航; 余贵亮

    2011-01-01

    Objective; To construct a conditional rephcative oncolytic adenovirus regulated by sumvin promotor. And to test its oncolytic effects on prostate cancer celL Methods-Survivin promotor was amplified by PCR from the genome of-survivin positive prostate cancer cell line LNCaP. pGL3Bsurvivin plasmid was constructed and transfected into LNCaP cell, the survivin transcriptional responses was tested by Luciferase Reporter Gene Assay. pGL3Bsurvivin was subclone into pShuttle vector, and reADGL3Bsurvivin was constructed by homologous recombination with El null pAdEasy-1 in BJ5183. Adenoviral plasmid was propagated in293cells and purified. Oncolytic effect of reADGL3Bsurvivin on LNCaP was tested by CCK-8 test and flow cytometry. Results :Survivin-conditional replicative oncolytic adenovirus reADGL3Bsurvivin was successfully constructed, which confirmed by PCR and luciferase reporter gene assay. CCK-8 test showed that re-ADGL3Bsurvivin could specifically inhibit proliferation and killed survivin positive prostate cancer cell line LNCaP cell, but showed no significant oncolytic effect on survivin negative normal prostate epithelia cell. Conclusions:Constructed re-ADGL3Bsurvivin could specifically killed survivin positive prostate cancer cell , which may be used in gene therapy against prostate cancer in the future.%目的:构建携带有Survivin启动子和报告基因的条件复制型腺病毒并观察该病毒对前列腺癌细胞的特异性溶瘤作用.方法:以前列腺癌细胞系LNCaP细胞全基因组DAN为模板,PCR扩增Survivin启动子,构建pGL3BSurvivin质粒表达载体,荧光素酶检测系统观察前列腺癌细胞中Survivin启动子活性.将pGL3BSurvivin亚克隆至穿梭质粒pShuttle中,构建含有Survivin启动子的重组腺病毒reADGL3BSurvivin.转染HEK293细胞进行重组病毒的扩增、纯化和滴度检测.利用CCK-8法检测重组腺病毒对前列腺癌细胞的生长抑制作用,以正常前列腺细胞为对照.结果:经多种限制

  16. Effects of hCD80-Adenovirus on B16-F10 Cells

    Institute of Scientific and Technical Information of China (English)

    田长富; 李殿俊; 刘旭

    2002-01-01

    @@ To study the biological characteristics of B16-F10 cells transduced with hCD80 gene in vitro, a kind of replication-deficient recombinant adenovirus bearing hCD80 gene (hCD80-rAd) was constructed and several means were utilized to observe the changes of biological characteristics after gene transduced.

  17. Adenovirus serotype 5 vectored foot-and-mouth disease subunit vaccines: the first decade

    Science.gov (United States)

    Here we present the results of the first decade of development of a replication-defective human adenovirus (Ad5) containing the capsid and 3C protease coding regions of foot-and-mouth disease virus (FMDV) as a vaccine candidate. In proof-of concept studies we demonstrated that a single inoculation w...

  18. 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...

  19. 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.

  20. 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.

  1. 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

    replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation......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...... for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection....

  2. A CD46-binding chimpanzee adenovirus vector as a vaccine carrier.

    Science.gov (United States)

    Tatsis, Nia; Blejer, Ariella; Lasaro, Marcio O; Hensley, Scott E; Cun, Ann; Tesema, Lello; Li, Yan; Gao, Guang-Ping; Xiang, Zhi Q; Zhou, Dongming; Wilson, James M; Ertl, Hildegund C J

    2007-03-01

    A replication-defective chimeric vector based on the chimpanzee adenovirus serotype C1 was developed and tested as a vaccine carrier in mice. The AdC1 virus is closely related to human adenoviruses of subgroup B2 and uses CD46 for cell attachment. To overcome poor growth of E1-deleted AdC1 vectors on cell lines that provide the E1 of adenovirus of the human serotype 5 (AdHu5) virus in trans, the inverted terminal repeats and some of the early genes of AdC1 were replaced with those from AdC5, a chimpanzee origin adenovirus of subfamily E. The chimeric AdC1/C5 vector efficiently transduces CD46-expressing mouse dendritic cells (DCs) in vitro and initiates their maturation. Transduction of DCs in vivo is inefficient in CD46 transgenic mice. The AdC1/C5 vector induces transgene product-specific B- and CD8(+) T-cell responses in mice. Responses are slightly higher in wild-type mice than in CD46 transgenic mice. Transgene product-specific T-cell responses elicited by the AdC1/C5 vector can be increased by priming or boosting with a heterologous adenovirus vector. Pre-existing immunity to adenovirus of the common human serotype 5 does not affect induction of cell-mediated immune responses by the AdC1/C5 vector. This vector provides an additional tool in a repertoire of adenovirus-based vaccine vectors. PMID:17228314

  3. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    OpenAIRE

    Gahéry-Ségard, H; Molinier-Frenkel, V.; Le Boulaire, C; Saulnier, P.; Opolon, P; Lengagne, R.; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A.; Schatz, C; Courtney, M; Le Chevalier, T.; Tursz, T; Guillet, J G

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galact...

  4. Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever.

    Science.gov (United States)

    Warimwe, George M; Gesharisha, Joseph; Carr, B Veronica; Otieno, Simeon; Otingah, Kennedy; Wright, Danny; Charleston, Bryan; Okoth, Edward; Elena, Lopez-Gil; Lorenzo, Gema; Ayman, El-Behiry; Alharbi, Naif K; Al-dubaib, Musaad A; Brun, Alejandro; Gilbert, Sarah C; Nene, Vishvanath; Hill, Adrian V S

    2016-01-01

    Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs. PMID:26847478

  5. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  6. Evaluation of fiber-modified adenovirus vector-vaccine against foot-and-mouth diseaes in cattle

    Science.gov (United States)

    Novel vaccination approaches against foot-and-mouth-disease (FMD) include the use of a replication-defective human adenovirus type 5 vector (Ad5) that contains the capsid encoding regions of FMD virus (FMDV). An Ad5.A24 has proven effective as a vaccine against FMD in swine and cattle. However, ther...

  7. Adeno-associated virus Rep78 restricts adenovirus E1B55K-mediated p53 nuclear exportation

    Institute of Scientific and Technical Information of China (English)

    Jingjing Wang; Wenjuan Li; Ran Wang; Jinglun Xue; Jinzhong Chen

    2013-01-01

    Inactivation of p53 is needed during adenovirus type 5 DNA replication.E1B55K,an adenovirus early protein,has been reported to interact with p53 and inhibit p53 transactivation.Previous studies have shown that adenoassociated virus (AAV) type 2 could reduce the transforming potential of adenovirus by rescuing p53 from adenovirus-mediated degradation,but the details are not clear yet.We detected the Rep78-p53 interaction by co-immunoprecipitation assay.The co-localization assay revealed that Rep78 inhibits E1B55K-mediated p53 nuclear exportation.However,Rep78 did not detectably influence p53 stability and could not relieve the transcriptional inactivation of p53,as E1B55K could not be replaced from the p53-E1B55K complex by Rep78.Our results reveal a new possible mechanism that AAV-2 Rep78 inhibits adenovirus 5 by relocalizing p53 in the nucleus,which may shed some light on the regulatory mechanism of AAV-2 on its helper virus,adenovirus.

  8. Polymerase chain reaction detection of adenovirus DNA sequences in human lymphocytes

    Directory of Open Access Journals (Sweden)

    Araújo Aufra A.

    2001-01-01

    Full Text Available Human lymphoid cells frequently carry adenoviruses and DNA sequences have been identified in peripheral blood lymphocytes by Southern blot and PCR, although these cells are not permissive for virus replication, suggesting persistence of the viral genome. In order to investigate this phenomenon we screened non-symptomatic volunteers for adenovirus DNA presence and E1A gene expression. DNA samples extracted from peripheral blood mononuclear cells of 51 volunteers were submitted to PCR using primers for a conserved hexon sequence, followed by nested PCR. Adenovirus sequences were detected in 27 samples (52.9%. After more than one year, new samples of these positive volunteers were analyzed and in 70.8% of the cases the result was maintained. Since this could be due to a possible persistence we checked if the early gene E1A was involved analyzing its expression by RT-PCR. For that purpose we developed a pair of primers to target a conserved region in the E1A gene. The RT-PCR results for E1A were negative for all samples. Using these primers it was possible to detect adenovirus sequences directly by PCR in DNA samples and we found 84% agreement in comparison to the hexon analysis. Our data suggest a high occurrence and persistence of adenovirus genome sequences in human lymphoid cells, and an indication that a region other than E1A is involved in persistence. We also can say that E1A gene is a good choice for amplification as a tool in adenovirus detection, avoiding the high risk of contamination in the nested PCR procedure necessary for hexon detection.

  9. Replicating vaccines

    Science.gov (United States)

    Early work on fish immunology and disease resistance demonstrated fish (like animals and humans) that survived infection were typically resistant to re-infection with the same pathogen. The concepts of resistance upon reinfection lead to the research and development of replicating (live) vaccines in...

  10. 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.

  11. 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.

  12. 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

  13. An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy: An in vitro Evaluation

    Institute of Scientific and Technical Information of China (English)

    Fei-qun Zheng; Yin Xu; Yi-de Qin; Ren-jie Yang; Jun Han

    2009-01-01

    Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage.Methods: A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase (hTERT), the coding sequence of E1A gene, an internal ribosome entry site sequence (IRES) and the coding sequence of herpes simplex virus-thymidine kinase (HSV-TK) was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry.Results: Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir (GCV) induced an obvious cell death.Conclusion: An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.

  14. Fatal adenovirus 32 infection in a bone marrow transplant recipient.

    OpenAIRE

    Charles, A K; Caul, E. O.; Porter, H J; Oakhill, A

    1995-01-01

    A case of disseminated adenovirus type 32 infection causing severe hepatitis, gastrointestinal ulceration and also with respiratory involvement is reported in a bone marrow transplant recipient. Typical viral inclusions were seen in the postmortem histological sections and adenovirus infection was confirmed using in situ hybridisation and isolation of adenovirus type 32 from separate organs at necropsy. This is the first case in which adenovirus 32 was the cause of fatal disseminated disease ...

  15. A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

    Directory of Open Access Journals (Sweden)

    Kelvin K W To

    2014-12-01

    Full Text Available Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1 was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention

  16. A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

    Science.gov (United States)

    To, Kelvin K W; Tse, Herman; Chan, Wan-Mui; Choi, Garnet K Y; Zhang, Anna J X; Sridhar, Siddharth; Wong, Sally C Y; Chan, Jasper F W; Chan, Andy S F; Woo, Patrick C Y; Lau, Susanna K P; Lo, Janice Y C; Chan, Kwok-Hung; Cheng, Vincent C C; Yuen, Kwok-Yung

    2014-12-01

    Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be

  17. Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissues

    Science.gov (United States)

    Recombinant replication-defective human adenovirus type 5 (Ad5) vaccines containing capsid-coding regions from foot-and-mouth disease virus (FMDV) have been demonstrated to induce effective immune responses and provide homologous protective immunity against FMDV in cattle. However, basic mechanisms ...

  18. 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...

  19. Vectors based on Semliki Forest virus for rapid and efficient gene transfer into non-endothelial cardiovascular cells : comparison to adenovirus

    NARCIS (Netherlands)

    Roks, AJM; Pinto, YM; Paul, M; Pries, F; Stula, M; Eschenhagen, T; Orzechowski, HD; Gschwendt, S; Wilschut, J; vanGilst, WH

    1997-01-01

    Objective: Replication-deficient, recombinant adenovirus is used as a carrier for gene transfer, but it is unspecific and the onset of transgene expression is relatively late. Here, we evaluated the efficiency and selectivity of gene transfer mediated by recombinant Semliki Forest virus (SFV). Metho

  20. Impact of Adenovirus infection in host cell metabolism evaluated by (1)H-NMR spectroscopy.

    Science.gov (United States)

    Silva, Ana Carina; P Teixeira, Ana; M Alves, Paula

    2016-08-10

    Adenovirus-based vectors are powerful vehicles for gene transfer applications in vaccination and gene therapy. Although highly exploited in the clinical setting, key aspects of the adenovirus biology are still not well understood, in particular the subversion of host cell metabolism during viral infection and replication. The aim of this work was to gain insights on the metabolism of two human cell lines (HEK293 and an amniocyte-derived cell line, 1G3) after infection with an adenovirus serotype 5 vector (AdV5). In order to profile metabolic alterations, we used (1)H-NMR spectroscopy, which allowed the quantification of 35 metabolites in cell culture supernatants with low sample preparation and in a relatively short time. Significant differences between both cell lines in non-infected cultures were identified, namely in glutamine and acetate metabolism, as well as by-product secretion. The main response to AdV5 infection was an increase in glucose consumption and lactate production rates. Moreover, cultures performed with or without glutamine supplementation confirmed the exhaustion of this amino acid as one of the main causes of lower AdV5 production at high cell densities (10- and 1.5-fold less specific yields in HEK293 and 1G3 cells, respectively), and highlighted different degrees of glutamine dependency of adenovirus replication in each cell line. The observed metabolic alterations associated with AdV5 infection and specificity of the host cell line can be useful for targeted bioprocess optimization. PMID:27215342

  1. 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

  2. The therapy and mechanisms of replication-deficient recombinant adenovirus Ad-p14ARF in hepatocellular carcinoma%复制缺陷型腺病毒Ad-p14ARF治疗肝细胞癌的实验研究及作用机制的探讨

    Institute of Scientific and Technical Information of China (English)

    Haili Qian; Haifeng Song; Xueyan Zhang; Xiao Liang; Ming Fu; Chen Lin

    2007-01-01

    Objective: To study the therapeutic effect and mechanisms of recombinant adenovirus Ad-p14ARF in hepatocellular carcinoma cell lines. Methods: Morphology and trypan blue assay were adopted to evaluate the proliferation of different liver cancer cells after Ad-p14ARF infection. Cell apoptosis was confirmed by detecting phosphatidylserine (PS) externalization with Annexin V/PI double staining. Western blotting assay analyzed the expression of related proteins. Subcutaneous tumor model of BEL7402 was established to evaluate the therapeutic ability of Ad-p14ARF. Results: Ad-p14ARF suppressed cell growth, proliferation and promoted cell apoptosis of cancer cell lines with different genetic background. Ad-p14ARF inhibited growth of liver cancer cells (HepG2, BEL7402) in a dose-dependent manner. Ad-p14ARF leaded to overexpression of Bax and p21, which were the downstream regulating genes of p53. Ad-p14ARF suppressed tumor growth significantly in the experimental therapy in nude mice bearing subcutaneous tumor of BEL7402. Conclusion: P14ARF gene is a powerful tumor suppressor gene to be used in cancer gene therapy. It may play an important role in gene therapy against the malignancies in the future.

  3. Robotic arm

    Science.gov (United States)

    Kwech, Horst

    1989-04-18

    A robotic arm positionable within a nuclear vessel by access through a small diameter opening and having a mounting tube supported within the vessel and mounting a plurality of arm sections for movement lengthwise of the mounting tube as well as for movement out of a window provided in the wall of the mounting tube. An end effector, such as a grinding head or welding element, at an operating end of the robotic arm, can be located and operated within the nuclear vessel through movement derived from six different axes of motion provided by mounting and drive connections between arm sections of the robotic arm. The movements are achieved by operation of remotely-controllable servo motors, all of which are mounted at a control end of the robotic arm to be outside the nuclear vessel.

  4. Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wildner Oliver

    2006-08-01

    Full Text Available Abstract Background Oncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 regions are modified in a time consuming sequential fashion. Results Based on the widely used adenoviral cloning system AdEasy we generated a novel transfer vector that allows efficient and rapid generation of conditionally replication-competent adenovirus type 5 based vectors with the viral E1 and E4 genes under the transcriptional control of heterologous promoters. For insertion of the promoters of interest our transfer vector has two unique multiple cloning sites. Additionally, our shuttle plasmid allows encoding of a transgene within the E1A transcription unit. The modifications, including E1 mutations, are introduced into the adenoviral genome by a single homologous recombination step in Escherichia coli. Subsequently infectious viruses are rescued from plasmids. As a proof-of-concept we generated two conditionally replication-competent adenoviruses Ad.Ki•COX and Ad.COX•Ki with the promoters of the Ki-67 protein and the cyclooxygenase-2 (COX-2 driving E1 and E4 and vice versa. Conclusion We demonstrated with our cloning system efficient generation of double heterologous promoter controlled oncolytic adenoviral vectors by a single homologous recombination step in bacteria. The generated viruses showed preferential replication in tumor cells and in a subcutaneous HT-29 colon cancer xenograft model the viruses demonstrated significant oncolytic activity comparable with dl327.

  5. Identification of a novel downstream binding protein implicated in late-phase-specific activation of the adenovirus major late promotor.

    OpenAIRE

    Mondesert, G; Tribouley, C; Kedinger, C

    1992-01-01

    The adenovirus major late promotor (MLP) is induced to very high levels after the onset of the viral DNA replication. Previous studies have identified sequence elements located downstream of the MLP startsite (DE1, between +85 and +98; DE2, between +100 and +120) implicated, together with the upstream promoter element, in this late-phase-specific transcriptional activation. One protein (DEF, now renamed DEF-A), induced during the late phase of viral infection, has been identified and shown to...

  6. 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.

  7. Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

    Science.gov (United States)

    Padi, Megha; Korkhin, Anna; James, Robert L.; Adelmant, Guillaume; Yoon, Rosa; Guo, Luxuan; Berrios, Christian; Zhang, Ying; Calderwood, Michael A.; Velmurgan, Soundarapandian; Cheng, Jingwei; Marto, Jarrod A.; Hill, David E.; Cusick, Michael E.; Vidal, Marc; Florens, Laurence; Washburn, Michael P.; Litovchick, Larisa; DeCaprio, James A.

    2012-01-01

    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. PMID:23093934

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

    Science.gov (United States)

    Fine, Debrah A; Rozenblatt-Rosen, Orit; Padi, Megha; Korkhin, Anna; James, Robert L; Adelmant, Guillaume; Yoon, Rosa; Guo, Luxuan; Berrios, Christian; Zhang, Ying; Calderwood, Michael A; Velmurgan, Soundarapandian; Cheng, Jingwei; Marto, Jarrod A; Hill, David E; Cusick, Michael E; Vidal, Marc; Florens, Laurence; Washburn, Michael P; Litovchick, Larisa; DeCaprio, James A

    2012-01-01

    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. PMID:23093934

  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. Effect of Preexisting Immunity to Adenovirus Human Serotype 5 Antigens on the Immune Responses of Nonhuman Primates to Vaccine Regimens Based on Human- or Chimpanzee-Derived Adenovirus Vectors▿

    OpenAIRE

    McCoy, Kimberly; Tatsis, Nia; Korioth-Schmitz, Birgit; Lasaro, Marcio O; Hensley, Scott E.; Lin, Shih-Wen; Li, Yan; Giles-Davis, Wynetta; Cun, Ann; Zhou, Dongming; Xiang, Zhiquan; Letvin, Norman L.; Ertl, Hildegund C J

    2007-01-01

    In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T...

  11. Mouse adenovirus type 1 infection of macrophages

    NARCIS (Netherlands)

    Ashley, S.L.; Welton, A.R.; Harwood, K.M.; Rooijen, van N.; Spindler, K.R.

    2009-01-01

    Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus.

  12. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    Science.gov (United States)

    Gao, Peng

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  13. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice.

    Science.gov (United States)

    Xie, Yinli; Gao, Peng; Li, Zhiyong

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  14. 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.

  15. 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.

  16. Novel Viral Disease Control Strategy: Adenovirus Expressing Alpha Interferon Rapidly Protects Swine from Foot-and-Mouth Disease

    OpenAIRE

    Chinsangaram, Jarasvech; Mauro P. Moraes; Koster, Marla; Grubman, Marvin J.

    2003-01-01

    We have previously shown that replication of foot-and-mouth disease virus (FMDV) is highly sensitive to alpha/beta interferon (IFN-α/β). In the present study, we constructed recombinant, replication-defective human adenovirus type 5 vectors containing either porcine IFN-α or IFN-β (Ad5-pIFNα or Ad5-pIFNβ). We demonstrated that cells infected with these viruses express high levels of biologically active IFN. Swine inoculated with 109 PFU of a control Ad5 virus lacking the IFN gene and challeng...

  17. Replication in Economics

    OpenAIRE

    Hamermesh, Daniel S.

    2007-01-01

    This examination of the role and potential for replication in economics points out the paucity of both pure replication -- checking on others' published papers using their data -- and scientific replication -- using data representing different populations in one's own work or in a Comment. Several controversies in empirical economics illustrate how and how not to behave when replicating others' work. The incentives for replication facing editors, authors and potential replicators are examined...

  18. 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.

  19. 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.

  20. 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

  1. Adenovirus serotype 5 hexon mediates liver gene transfer.

    Science.gov (United States)

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  2. 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

  3. Adenovirus: an emerging factor in red squirrel Sciurus vulgaris conservation

    OpenAIRE

    Everest, David J.; Shuttleworth, Craig M.; Stidworthy, Mark F.; Sylvia S Grierson; Duff, J. Paul; Kenward, Robert E.

    2014-01-01

    1. Adenovirus is an emerging threat to red squirrel Sciurus vulgaris conservation, but confirming clinically significant adenovirus infections in red squirrels is challenging. Rapid intestinal autolysis after death in wild animals frequently obscures pathology characteristic of the disease in animals found dead. 2. We review the available literature to determine current understanding of both subclinical and clinically significant adenovirus infections in free-living wild and captive red sq...

  4. Mouse Adenovirus Type 1 Infection of Macrophages

    OpenAIRE

    Ashley, Shanna L.; Welton, Amanda R.; Harwood, Kirsten M.; van Rooijen, Nico; Spindler, Katherine R.

    2009-01-01

    Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus. Here we determined the extent and functional importance of macrophage infection by MAV-1. Bone marrow-derived macrophages expressed MAV-1 mRNAs and proteins upon ex vivo infection. Adherent perito...

  5. Isolation and Epidemiology of Falcon Adenovirus

    OpenAIRE

    Oaks, J. Lindsay; Schrenzel, Mark; Rideout, Bruce; Sandfort, Cal

    2005-01-01

    An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2...

  6. Oncolytic Adenovirus Loaded with L-carnosine as Novel Strategy to Enhance the Antitumor Activity.

    Science.gov (United States)

    Garofalo, Mariangela; Iovine, Barbara; Kuryk, Lukasz; Capasso, Cristian; Hirvinen, Mari; Vitale, Andrea; Yliperttula, Marjo; Bevilacqua, Maria Assunta; Cerullo, Vincenzo

    2016-04-01

    Oncolytic viruses are able to specifically replicate, infect, and kill only cancer cells. Their combination with chemotherapeutic drugs has shown promising results due to the synergistic action of virus and drugs; the combinatorial therapy is considered a potential clinically relevant approach for cancer. In this study, we optimized a strategy to absorb peptides on the viral capsid, based on electrostatic interaction, and used this strategy to deliver an active antitumor drug. We used L-carnosine, a naturally occurring histidine dipeptide with a significant antiproliferative activity. An ad hoc modified, positively charged L-carnosine was combined with the capsid of an oncolytic adenovirus to generate an electrostatic virus-carnosine complex. This complex showed enhanced antitumor efficacy in vitro and in vivo in different tumor models. In HCT-116 colorectal and A549 lung cancer cell lines, the complex showed higher transduction ratio and infectious titer compared with an uncoated oncolytic adenovirus. The in vivo efficacy of the complex was tested in lung and colon cancer xenograft models, showing a significant reduction in tumor growth. Importantly, we investigated the molecular mechanisms underlying the effects of complex on tumor growth reduction. We found that complex induces apoptosis in both cell lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in future studies also for delivery of other bioactive drugs. Mol Cancer Ther; 15(4); 651-60. ©2016 AACR. PMID:26861248

  7. 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.

  8. 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).

  9. Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication

    Science.gov (United States)

    Several studies have demonstrated that administration of type I, II, or III interferons (IFN) delivered using a replication defective human adenovirus 5 (Ad5) vector is effective to control Foot-and-Mouth Disease (FMD) in cattle and swine during experimental infections. However, high doses are requi...

  10. Anti-HBV activity of TRL mediated by recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    Wei-Dong Gong; Ya Zhao; Jun Yi; Jin Ding; Jun Liu; Cai-Fang Xue

    2005-01-01

    AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGlox(delta)E1,3Cre to acquire RAd/TRL, TR, HBVcand hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs 1.60±0.47, P = 0.0266, <0.05) andother control groups (0.63±0.14 vs 8.50±2.78, 8.25±2.26,8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assaysuggested that there were no significant differences in cell metabolic activity between groups (P>0.05).CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

  11. 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.

  12. Replicability of Experiment

    Directory of Open Access Journals (Sweden)

    John D. Norton

    2015-06-01

    Full Text Available The replicability of experiment is routinely offered as the gold standard of evidence. I argue that it is not supported by a universal principle of replicability in inductive logic. A failure of replication may not impugn a credible experimental result; and a successful replication can fail to vindicate an incredible experimental result. Rather, employing a material approach to inductive inference, the evidential import of successful replication of an experiment is determined by the prevailing background facts. Commonly, these background facts do support successful replication as a good evidential guide and this has fostered the illusion of a deeper, exceptionless principle.

  13. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    Science.gov (United States)

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

  14. Archaeal DNA replication.

    Science.gov (United States)

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  15. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  16. Development of a model based on oncolytic adenovirus loaded with L-carnosine as a drug delivery system for cancer therapy

    OpenAIRE

    Garofalo, Mariangela

    2015-01-01

    Oncolytic viruses are viruses that are able to replicate specifically and infect and destroy only tumor cells. Many clinical studies have shown that the oncolytic approach alone could not efficiently destroy the large tumor mass, thus by limiting an efficacy virotherapy. Combination of oncolytic adenoviruses (Ads) and chemotherapeutic drugs has shown promising therapeutic results due to the synergistic action of virus and drug and is considered as a potential approach for cancer therapy. In t...

  17. PEGylated Adenoviruses: From Mice to Monkeys

    Directory of Open Access Journals (Sweden)

    Piyanuch Wonganan

    2010-02-01

    Full Text Available Covalent modification with polyethylene glycol (PEG, a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

  18. Bicalutamide Activated Oncolytic Adenovirus for the Adjuvant Therapy of High Risk Prostate Cancer

    Science.gov (United States)

    Johnson, Tamara Jane; Hoti, Naser Uddin; Liu, Chunyan; Chowdhury, Wasim H.; Li, Ying; Zhang, Yonggang; Lupold, Shawn E.; DeWeese, Theodore; Rodriguez, Ronald

    2013-01-01

    Conditionally replicating adenoviruses (CRAds) utilize tissue specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer gene therapy require androgens to activate prostate specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen suppressive therapy. We describe a novel prostate specific CRAd generated by fusing the E1A gene to the androgen receptor (AR) cDNA with a point mutation in codon 685 (C685Y). The E1A-AR fusion neutralizes the previously described mutual inhibition of E1A & AR, and the C685Y point mutation alters specificity of steroid ligand binding to the AR, such that both androgens and non-steroidal anti-androgens can activate viral replication. We demonstrate that the mutated E1A-AR retained the ability to function in regulating AR responsive genes and E1A responsive viral genes. In combination therapy of virus, bicalutamide (anti-androgen) and radiation, a profound impact on cell death by viral oncolysis was seen both in vitro and tumor xenografts. To our knowledge, this is the first gene therapy engineered to be enhanced by anti-androgens, and a particularly attractive adjuvant strategy for intensity modulated radiation therapy (IMRT) of high-risk prostate cancers. PMID:23764901

  19. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    Science.gov (United States)

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

  20. 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.

  1. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    OpenAIRE

    Yinli Xie; Peng Gao; Zhiyong Li

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expr...

  2. 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.

  3. Adenovirus Ad-p53AIP1-mediated gene therapy and its regulation of p53-MDM2 interactions

    OpenAIRE

    Jiang, Yunbo; Chen, Huihua; JIA, HAIQUAN; XU, YUANJI; Liu, Gang; Wang, Yan; Yang, Xiaohe; Yinglin LU

    2010-01-01

    We generated replication-defective adenovirus Ad-p53AIP1 and studied its anti-tumor efficacy both in vitro and in vivo. We demonstrated that Ad-p53AIP1 infection elicited high levels of p53AIP1 expression in cancer cells. We also found that Ad-p53AIP1 expression induced marked apoptosis and cell cycle arrest in HepG2 cells. Moreover, Ad-p53AIP1 infection significantly inhibited the tumorigenesis of 4T1 mouse mammary cancer cells in vivo. In particular, we discovered that p53AIP1 overexpressio...

  4. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    Institute of Scientific and Technical Information of China (English)

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍

    2003-01-01

    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  5. Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01.

    Directory of Open Access Journals (Sweden)

    Beatriz Vera

    Full Text Available Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease.

  6. Construction and Identification of Recombinant Adenovirus Vector Containing the hVEGF165 Gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF165), the cDNA for hVEGF165 was subcloned into pACCMV·pLpA. Subsequently, this recombinant pACCMV·hVEGF was co-transfected into 293 cells together with pJM17 to obtain the replication-deficient recombinant adenovirus containing hVEGF gene-AdCMV·hVEGF. The VEGF gene expression was detected by using RT-PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV·hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV·hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV·hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro. Successfully prepared AdCMV·hVEGF165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.

  7. MOLECULAR REPLICATOR DYNAMICS

    OpenAIRE

    BÄRBEL M. R. STADLER; Stadler, Peter F

    2003-01-01

    Template-dependent replication at the molecular level is the basis of reproduction in nature. A detailed understanding of the peculiarities of the chemical reaction kinetics associated with replication processes is therefore an indispensible prerequisite for any understanding of evolution at the molecular level. Networks of interacting self-replicating species can give rise to a wealth of different dynamical phenomena, from competitive exclusion to permanent coexistence, from global stability...

  8. Infection with E1B-mutant adenovirus stabilizes p53 but blocks p53 acetylation and activity through E1A

    DEFF Research Database (Denmark)

    Savelyeva, I.; Dobbelstein, M.

    2011-01-01

    Wild-type adenovirus type 5 eliminates p53 through the E1B-55 kDa and E4-34 kDa gene products. Deletion or mutation of E1B-55 kDa has long been thought to confer p53-selective replication of oncolytic viruses. We show here that infection with E1B-defective adenovirus mutants induces massive...... to the suppression of p21 transcription. Depending on the E1A conserved region 3, E1B-defective adenovirus impaired the ability of the transcription factor Sp1 to bind the p21 promoter. Moreover, the amino terminal region of E1A, binding the acetyl transferases p300 and CREB-binding protein, blocked p53 K382...... acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p21 mRNA levels. Our findings argue that adenovirus attenuates p53-mediated p21 induction, through at least two E1B-independent mechanisms. Other virus species and cancer cells may employ analogous...

  9. 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.

  10. Self-replicating systems.

    Science.gov (United States)

    Clixby, Gregory; Twyman, Lance

    2016-05-01

    Over the past 25 years, there has been a surge of development in research towards self-replication and self-replicating systems. The interest in these systems relates to one of the most fundamental questions posed in all fields of science - How did life on earth begin? Investigating how the self-replication process evolved may hold the key to understanding the emergence and evolution of living systems and, ultimately, gain a clear insight into the origin of life on earth. This introductory review aims to highlight the fundamental prerequisites of self-replication along with the important research that has been conducted over the past few decades. PMID:27086507

  11. Clinical characteristics analysis of adult human adenovirus type 7 infection

    Institute of Scientific and Technical Information of China (English)

    张乃春

    2014-01-01

    Objective To investigate the clinical characteristics of patients infected with human adenovirus type 7 and to provide guidance for early diagnosis and timely control of the outbreak.Methods A total of 301 patients infected with the human adenoviruses who were quarantined in hospital from December 2012 to February 2013 were observed.Epidemiological questionnaires were used to collect data of clinical features of the disease including

  12. New Adenovirus Species Found in a Patient Presenting with Gastroenteritis▿

    OpenAIRE

    Jones, Morris Saffold; Harrach, Balázs; Ganac, Robert D.; Gozum, Mary M. A.; Dela Cruz, Wilfred P.; Riedel, Brian; Pan, Chao; Delwart, Eric L.; David P Schnurr

    2007-01-01

    An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible. Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with ...

  13. 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.

  14. Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus.

    Science.gov (United States)

    Matoq, Amr; Salahuddin, Asma

    2016-01-01

    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. PMID:27340581

  15. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang;

    2013-01-01

    It has been reported that adenovirus (Ad) primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells directly comparing these cells to CD8 T cells induced through...... adenoviral boosting and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad-vaccination will lead to even higher numbers of memory cells. In this case, the vaccination...... an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells....

  16. 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.

  17. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    International Nuclear Information System (INIS)

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer

  18. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Freytag, Svend O., E-mail: sfreyta1@hfhs.org [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Stricker, Hans [Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan (United States); Lu, Mei [Public Health Sciences, Henry Ford Health System, Detroit, Michigan (United States); Elshaikh, Mohamed; Aref, Ibrahim; Pradhan, Deepak; Levin, Kenneth; Kim, Jae Ho [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Peabody, James [Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan (United States); Siddiqui, Farzan; Barton, Kenneth; Pegg, Jan; Zhang, Yingshu; Cheng, Jingfang [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Oja-Tebbe, Nancy; Bourgeois, Renee [Public Health Sciences, Henry Ford Health System, Detroit, Michigan (United States); Gupta, Nilesh; Lane, Zhaoli [Pathology, Henry Ford Health System, Detroit, Michigan (United States); Rodriguez, Ron [Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); DeWeese, Theodore [Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); and others

    2014-06-01

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

  19. 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.

  20. Radiomimetic Effect of Oncogenic and 'Non-Oncogenic' Human and Simian Adenoviruses

    International Nuclear Information System (INIS)

    The capacity of human (AD2, 4, 7, 12, 16, 18, 31) and simian (SA7, SV20) adenoviruses to induce chromosome aberrations and to affect the proliferative capacity and cloning efficiency was examined using permissive (AV-3, HEp-2, CV-1 cell strains) and non-permissive (BHK-21, primary Syrian hamster cultures) cell systems. The comparative study revealed (1). that genetically abnormal cells are only induced when the viral replication cycle is incomplete or when cells are infected with defective or experimentally (UV-irradiation, hydroxylamine treatment) impaired viruses; (2) that the chromosome damaging capacity of one virus is comparable to that of about 2 rads of X-ray; and (3) that the viral genome, which can survive in the host cells, represents a self- replicating mutagenic agent capable of continuous induction of new cell mutants. Frequency of cells with chromosome aberrations, incidence of chromatid breaks per cell, loss of cloning efficiency and the formation of virus-induced T(tumour)-antigen, which is coded for by the parental viral DNA molecule but is apparently not a part of the viral coat, depend on the viral dose. The chromosome damaging factors) seem not to reside in the virus particle but appears to be a viral product(s) from which transcription occurs in the early stages of the viral replicative cycle. It is suggested that viruses might represent an important etiologic factor in the mutagenesis of higher organisms. (author)

  1. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  2. Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75

    Directory of Open Access Journals (Sweden)

    Ramesh Kumar

    2015-02-01

    Full Text Available Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm. Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5. Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01. Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was

  3. 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.

  4. Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

    Science.gov (United States)

    Zhang, Nian-Hua; Peng, Rui-Qing; Ding, Ya; Zhang, Xiao-Shi

    2016-08-01

    The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity. PMID:27373420

  5. An Artificially Designed Interfering lncRNA Expressed by Oncolytic Adenovirus Competitively Consumes OncomiRs to Exert Antitumor Efficacy in Hepatocellular Carcinoma.

    Science.gov (United States)

    Li, Xiaoya; Su, Yinghan; Sun, Bin; Ji, Weidan; Peng, Zhangxiao; Xu, Yang; Wu, Mengchao; Su, Changqing

    2016-07-01

    Endogenous miRNAs, especially oncogenic miRNAs (OncomiR), have been molecular targets for cancer therapy. We generated an artificially designed interfering long noncoding RNA (lncRNAi), which contains the sequences that can complementarily bind to multiple OncomiRs and is expressed by cancer-selectively replicating adenovirus. The adenovirus-expressed lncRNAi with high levels in hepatocellular carcinoma (HCC) cells competes with OncomiR target genes to bind to and consume OncomiRs, thereby achieving the targeted anti-HCC efficacy. With the targeting replication of adenovirus in HCC cells, lncRNAi was highly expressed and resulted in decreased abilities of proliferation, migration, and invasion, induced cell-cycle changes and apoptosis, and markedly changed the cellular mRNA and miRNA expression profiles in HCC cells. The optimal antitumor effect was also demonstrated on HCC cell line xenograft models and HCC patient-derived xenograft (PDX) tumor models in nude mice. This strategy has established a technology platform with a reliable therapeutic effect for HCC therapy. Mol Cancer Ther; 15(7); 1436-51. ©2016 AACR.

  6. Patterns of replication in the neo-sex chromosomes of Drosophila nasuta albomicans

    Indian Academy of Sciences (India)

    G Mahesh; N B Ramachandra; H A Ranganath

    2000-09-01

    Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species of Drosophila.

  7. Late-replicating X-chromosome: replication patterns in mammalian females

    Directory of Open Access Journals (Sweden)

    Tunin Karen

    2002-01-01

    Full Text Available The GTG-banding and 5-BrdU incorporation patterns of the late-replicating X-chromosome were studied in female dogs and cattle, and compared to human female patterns. The replication patterns of the short arm of the X-chromosomes did not show any difference between human, dog and cattle females. As to the long arm, some bands showed differences among the three studied species regarding the replication kinetics pattern. These differences were observed in a restricted region of the X-chromosome, delimited by Xq11 -> q25 in humans, by Xq1 -> q8 in dogs, and by Xq12 -> q32 in cattle. In an attempt to find out if these differences in the replication kinetics could be a reflection of differences in the localization of genes in that region of the X-chromosome, we used the probe for the human androgen receptor gene (AR localized at Xq12, which is in the region where we observed differences among the three studied species. We did not, however, observe hybridization signals. Our study goes on, using other human probes for genes located in the region Xq11 -> Xq25.

  8. Vaccination to conserved influenza antigens in mice using a novel Simian adenovirus vector, PanAd3, derived from the bonobo Pan paniscus.

    Directory of Open Access Journals (Sweden)

    Alessandra Vitelli

    Full Text Available Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP and matrix 1 (M1. We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.

  9. Isolation and characterization of adenoviruses persistently shed from the gastrointestinal tract of non-human primates.

    Directory of Open Access Journals (Sweden)

    Soumitra Roy

    2009-07-01

    Full Text Available Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported together with 22 complete adenovirus genomes available from GenBank revealed that (a the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b there was evidence for intraspecies recombination between adenoviruses, and (c the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.

  10. Host Immune Responses to Chronic Adenovirus Infections in Human and Nonhuman Primates ▿ †

    OpenAIRE

    Calcedo, Roberto; Vandenberghe, Luk H.; Roy, Soumitra; Somanathan, Suryanarayan; Wang, Lili; Wilson, James M.

    2008-01-01

    Recent studies indicate that great apes and macaques chronically shed adenoviruses in the stool. Shedding of adenovirus in the stool of humans is less prevalent, although virus genomes persist in gut-associated lymphoid tissue in the majority of individual samples. Chimpanzees have high levels of broadly reactive neutralizing antibodies to adenoviruses in serum, with very low frequencies of adenovirus-specific T cells in peripheral blood. A similar situation exists in macaques; sampling of gu...

  11. Chromatin Immunoprecipitation of Replication Factors Moving with the Replication Fork

    OpenAIRE

    Rapp, Jordan B.; Ansbach, Alison B.; Noguchi, Chiaki; Noguchi, Eishi

    2009-01-01

    Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization ...

  12. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  13. Abiotic self-replication.

    Science.gov (United States)

    Meyer, Adam J; Ellefson, Jared W; Ellington, Andrew D

    2012-12-18

    The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic

  14. Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

    NARCIS (Netherlands)

    Haisma, Hidde J.; Kamps, Jan A. A. M.; Plantinga, Josee A.; Rots, Marianne G.; Bellu, Anna Rita; Kamps, Grietina

    2008-01-01

    Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the

  15. 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.

  16. Viability of human adenovirus from hospital fomites.

    Science.gov (United States)

    Ganime, Ana Carolina; Carvalho-Costa, Filipe A; Santos, Marisa; Costa Filho, Rubens; Leite, José Paulo G; Miagostovich, Marize P

    2014-12-01

    The monitoring of environmental microbial contamination in healthcare facilities may be a valuable tool to determine pathogens transmission in those settings; however, such procedure is limited to bacterial indicators. Viruses are found commonly in those environments and are rarely used for these procedures. The aim of this study was to assess distribution and viability of a human DNA virus on fomites in an Adult Intensive Care Unit of a private hospital in Rio de Janeiro, Brazil. Human adenoviruses (HAdV) were investigated in 141 fomites by scraping the surface area and screening by quantitative PCR (qPCR) using TaqMan® System (Carlsbad, CA). Ten positive samples were selected for virus isolation in A549 and/or HEp2c cell lines. A total of 63 samples (44.7%) were positive and presented viral load ranging from 2.48 × 10(1) to 2.1 × 10(3) genomic copies per millilitre (gc/ml). The viability was demonstrated by integrated cell culture/nested-PCR in 5 out of 10 samples. Nucleotide sequencing confirmed all samples as HAdV and characterized one of them as specie B, serotype 3 (HAdV-3). The results indicate the risk of nosocomial transmission via contaminated fomites and point out the use of HAdV as biomarkers of environmental contamination.

  17. 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.

  18. Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors.

    Science.gov (United States)

    Solanki, Manish; Zhang, Wenli; Jing, Liu; Ehrhardt, Anja

    2016-01-01

    Commonly used human adenovirus (HAdV)-5-based vectors are restricted by their tropism and pre-existing immunity. Here, we characterized novel HAdV-5 vectors pseudotyped with hypervariable regions (HVRs) and surface domains (SDs) of other HAdV types. Hexon-modified HAdV-5 vectors (HV-HVR5, HV-HVR12, HV-SD12 and HV-SD4) could be reconstituted and amplified in human embryonic kidney cells. After infection of various cell lines, we measured transgene expression levels by performing luciferase reporter assays or coagulation factor IX (FIX) ELISA. Dose-dependent studies revealed that luciferase expression levels were comparable for HV-HVR5, HV-SD12 and HV-SD4, whereas HV-HVR12 expression levels were significantly lower. Vector genome copy numbers (VCNs) from genomic DNA and nuclear extracts were then determined by quantitative real-time PCR. Surprisingly, determination of cell- and nuclear fraction-associated VCNs revealed increased VCNs for HV-HVR12 compared with HV-SD12 and HV-HVR5. Increased nuclear fraction-associated HV-HVR12 DNA molecules and decreased transgene expression levels were independent of the cell line used, and we observed the same effect for a hexon-modified high-capacity adenoviral vector encoding canine FIX. In conclusion, studying hexon-modified adenoviruses in vitro demonstrated that HVRs but also flanking hexon regions influence uptake and transgene expression of adenoviral vectors. PMID:26519158

  19. 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.

  20. Investigating variation in replicability: A "Many Labs" replication project

    NARCIS (Netherlands)

    Klein, R.A.; Ratliff, K.A.; Vianello, M.; Adams, R.B.; Bahnik, S.; Bernstein, M.J.; Bocian, K.; Brandt, M.J.; Brooks, B.; Brumbaugh, C.C.; Cemalcilar, Z.; Chandler, J.; Cheong, W.; Davis, W.E.; Devos, T.; Eisner, M.; Frankowska, N.; Furrow, D.; Galliani, E.M.; Hasselman, F.W.; Hicks, J.A.; Hovermale, J.F.; Hunt, S.J.; Huntsinger, J.R.; IJzerman, H.; John, M.S.; Joy-Gaba, J.A.; Kappes, H.B.; Krueger, L.E.; Kurtz, J.; Levitan, C.A.; Mallett, R.K.; Morris, W.L.; Nelson, A.J.; Nier, J.A.; Packard, G.; Pilati, R.; Rutchick, A.M.; Schmidt, K.; Skorinko, J.L.M.; Smith, R.; Steiner, T.G.; Storbeck, J.; Van Swol, L.M.; Thompson, D.; Veer, A.E. van 't; Vaughn, L.A.; Vranka, M.; Wichman, A.L.; Woodzicka, J.A.; Nosek, B.A.

    2014-01-01

    Although replication is a central tenet of science, direct replications are rare in psychology. This research tested variation in the replicability of 13 classic and contemporary effects across 36 independent samples totaling 6,344 participants. In the aggregate, 10 effects replicated consistently.

  1. Hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA,ε, as template, and depends on cellular chaperones;moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids.This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV),now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately,not be complemented by three-dimensional structural information on the involved components. However, at least for the s RNA element such information is emerging,raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal,will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

  2. Adenovirus-Vectored Vaccine as a Rapid-Response Tool Against Avian Influenza Pandemic

    International Nuclear Information System (INIS)

    Influenza viruses in nature undergo genetic mutation and reassortment. Three pandemics of avian influenza in man were recorded in the twentieth century. Highly pathogenic avian influenza (HPAI) viruses currently in circulation pose a threat for another world-wide pandemic, if they become transmissible from man to man. Manufacturing protective vaccines using current egg-based technology is often difficult due to the virulence of the virus and its adverse effects on the embryonating egg substrate. New technologies allow the creation of safe and protective pandemic influenza vaccines without the need for egg based substrates. These technologies allow new vaccines to be created in less than one month. Manufacturing is in tissue culture, not eggs. Vaccine can be administered to man non-invasively, without adjuvants, eliciting a rapid and protective immune response. Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad5)-derived vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 and H5N2 HPAI virus challenges. Mass-administration of this bird flu vaccine can be streamlined with available robotic in ovo injectors. Vaccination using this vaccine could protect the the largest host reservoir (chickens) and greatly reduce the exposure of man to avian influenza. In addition, Ad5-vectored vaccines can be produced rapidly and the safety margin of a non-replicating vector is superior to that of a replicating counterpart. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural AI virus infections. In addition to mass immunization of poultry, both animals and humans have been effectively immunized by intranasal administration of Ad5-vectored influenza vaccines without any appreciable side effects, even in mice and human volunteers with

  3. A Novel Molecular Therapy Using Bioengineered Adenovirus for Human Gastrointestinal Cancer

    Directory of Open Access Journals (Sweden)

    Fujiwara,Toshiyoshi

    2011-06-01

    Full Text Available Replication-selective tumor-specific viruses constitute a novel approach for treatment of neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Human telomerase is highly active in more than 85オ of primary cancers, regardless of their tissue origins, and its activity correlates closely with human telomerase reverse transcriptase (hTERT expression. We constructed an attenuated adenovirus 5 vector (Telomelysin, OBP-301, in which the hTERT promoter element drives expression of E1 genes. Since only tumor cells that express telomerase activity would activate this promoter, the hTERT proximal promoter would allow for preferential expression of viral genes in tumor cells, leading to selective viral replication and oncolytic cell death. Lymphatic invasion is a major route for cancer cell dissemination, and adequate treatment of locoregional lymph nodes is required for curative treatment in patients with gastrointestinal tumors. We demonstrated that intratumoral injection of Telomelysin mediates effective in vivo purging of metastatic tumor cells from regional lymph nodes. Moreover, using noninvasive whole-body imaging, we found that intratumoral injection of Telomelysin followed by regional irradiation induces a substantial antitumor effect, resulting from tumor cell-specific radiosensitization, in an orthotopic human esophageal cancer xenograft model. These results illustrate the potential of oncolytic virotherapy as a promising strategy in the management of human gastrointestinal cancer.

  4. Crystallization and preliminary X-ray diffraction analysis of human adenovirus

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, V.S.; Natchiar, S.K.; Gritton, L.; Mullen, T.-M.; Stewart, P.L.; Nemerow, G.R. (Scripps); (Vanderbilt)

    2010-07-22

    Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in {approx}25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100 mM HEPES pH 7.0, 200 mM Ca(OAc){sub 2}, 14% PEG 550 MME, 15% glycerol in 100 mM Tris-HCl 8.5. Freshly grown crystals diffracted well to 4.5 {angstrom} resolution and weakly to 3.5 {angstrom} at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a = 854.03 {angstrom}, b = 855.17 {angstrom}, c = 865.24 {angstrom}, {alpha} = 119.57{sup o}, {beta} = 91.71{sup o}, {gamma} = 118.08{sup o} with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

  5. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection.

    Science.gov (United States)

    Wechman, Stephen L; Rao, Xiao-Mei; Cheng, Pei-Hsin; Gomez-Gutierrez, Jorge G; McMasters, Kelly M; Zhou, H Sam

    2016-01-01

    Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties. PMID:27314377

  6. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection

    Directory of Open Access Journals (Sweden)

    Stephen L. Wechman

    2016-06-01

    Full Text Available Oncolytic adenoviruses (Ads have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties.

  7. Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

    1982-12-01

    Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

  8. Infecciones por adenovirus en Cuba (2000-2008)

    OpenAIRE

    González Muñoz, Grehete

    2013-01-01

    Las infecciones por adenovirus causan un variado espectro clínico en el hombre, con un rango que incluye desde la infección asintomática hasta la enfermedad diseminada con peligro para la vida. En el presente estudio, se analizó el comportamiento de las infecciones por adenovirus en síndromes de etiología viral como la infección respiratoria aguda, la miocarditis viral, la conjuntivitis hemorrágica y el síndrome neurológico infeccioso. Además, se estudió la participación de estos agentes en l...

  9. Comparative study of adenoviruses with monoclonal antibodies Estudo comparativo de diferentes tipos de adenovirus através de anticorpos monoclonais

    OpenAIRE

    Terezinha Maria de Paiva; Sueko Takimoto; María Akíko Ishida; María Candida Oliveira de Souza; Tuneo Ishimaru; Jorge Neumann; Jorge Kalil

    1992-01-01

    The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and on...

  10. Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

    Energy Technology Data Exchange (ETDEWEB)

    Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E. [Department of Viroscience, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015 GE Rotterdam (Netherlands); Leeuwen, M. van [Viroclinics Biosciences, Marconistraat 16, 3029 AK Rotterdam (Netherlands); Boheemen, S. van [Department of Viroscience, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015 GE Rotterdam (Netherlands); Jong, A.A.W. de [Department of Pathology, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015 GE Rotterdam (Netherlands); Osterhaus, A.D.M.E.; Smits, S.L. [Department of Viroscience, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015 GE Rotterdam (Netherlands); Viroclinics Biosciences, Marconistraat 16, 3029 AK Rotterdam (Netherlands); Kuiken, T., E-mail: t.Kuiken@erasmusmc.nl [Department of Viroscience, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015 GE Rotterdam (Netherlands)

    2013-05-25

    Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. - Highlights: ► Lesions typical for adenovirus infection detected in cloacal bursa of dead gulls. ► Confirmation of adenovirus infection by electron microscopy and deep sequencing. ► Sequence analysis indicates that it is a novel adenovirus in the genus Aviadenovirus. ► The novel (Gull) adenovirus was detected in multiple organs of two species of gulls.

  11. 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

  12. Identification and characterization of a novel adenovirus in the cloacal bursa of gulls.

    Science.gov (United States)

    Bodewes, R; van de Bildt, M W G; Schapendonk, C M E; van Leeuwen, M; van Boheemen, S; de Jong, A A W; Osterhaus, A D M E; Smits, S L; Kuiken, T

    2013-05-25

    Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

  13. A targeting ligand enhances infectivity and cytotoxicity of an oncolytic adenovirus in human pancreatic cancer tissues.

    Science.gov (United States)

    Yamamoto, Yuki; Hiraoka, Nobuyoshi; Goto, Naoko; Rin, Yosei; Miura, Kazuki; Narumi, Kenta; Uchida, Hiroaki; Tagawa, Masatoshi; Aoki, Kazunori

    2014-10-28

    The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

  14. Growing Through Innovation Replicability

    DEFF Research Database (Denmark)

    Hsuan, Juliana; Lévesque, Moren

    2012-01-01

    We propose a formal model of firm growth through replication that considers the extent of the investment to adapt routines as replication unfolds and the portion of this investment that goes toward innovation in the routines. The use of these two investment constructs brings about four types...... of growth policies. We use a utility function that considers proxies for both growth and failure potential to uncover the role played in selecting these policies by the economic environment of the targeted market for expansion. Our analysis further reveals the importance of the innovation...

  15. 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.)

  16. Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

    Institute of Scientific and Technical Information of China (English)

    Fei-qun ZHENG; Yin XU; Ren-jie YANG; Bin WU; Xiao-hua TAN; Yi-de QIN; Qun-wei ZHANG

    2009-01-01

    Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors.We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting.Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo.Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma.

  17. 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.

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

    Science.gov (United States)

    Feduchi, E; Carrasco, L

    1987-04-01

    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.

  19. 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

  20. 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

  1. Co-expression of sCD40LIg and CTLA4Ig mediated by adenovirus prolonged mouse skin allograft survival

    Institute of Scientific and Technical Information of China (English)

    LI Zhao-lun; TIAN Pu-xun; XUE Wu-jun; WU Jun

    2006-01-01

    Objective: To investigate the role of simultaneous blockade of CD40/CD40L and B7/CD28 pathways in the immune tolerance via co-expression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus. Methods: Ad-sCD40LIgIRES2-CTLA4Ig, replication-defective adenovirus co-expressing sCD40LIg and CTLA4Ig, was constructed and identified. The co-expression of sCD40LIg and CTLA4Ig was evaluated with confocal laser scanning microscope and Western blotting. Skin transplantations of C57BL/6 to BALB/c mice were performed. PBS, Ad-Shuttle-CMV and Ad-sCD40LIg-IRES2-CTLA4Ig were administered. Skin graft survival was monitored and the mRNA expression of both genes was evaluated in the skin allografts.Results: Ad-sCD40LIg-IRES2-CTLA4Ig was constructed successfully and identified. The co-expression of sCD40LIg and CTLA4Ig was identified with confocal laser scanning microscopy and Western blotting. Compared to the skin graft mean survival time (MST) of non-treated group ((5.75±0.71) d) or Ad-Shuttle-CMV-treated group ((5.50±0.53) d), the skin graft MST was dramatically prolonged in the Ad-sCD40LIg-IRES2-CTLA4Ig-treated group (( 16.38± 1.19) d, P<0.001). The mRNA expression of both genes was detected. Conclusion: Ad-sCD40LIg-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding sCD40LIg and CTLA4Ig, was constructed. Simultaneous blockade of CD40/CD40L and B7/CD28 costimulatory pathway mediated by replication-defective adenovirus significantly prolonged skin allograft survival in mice.

  2. Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep.

    Directory of Open Access Journals (Sweden)

    José M Rojas

    Full Text Available Peste des petits ruminants (PPR is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV. Two recombinant replication-defective human adenoviruses serotype 5 (Ad5 expressing either the highly immunogenic fusion protein (F or hemagglutinin protein (H from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

  3. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    International Nuclear Information System (INIS)

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- XTM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-XTM genome, which was transformed into E. coli to get recombinant Adeno-XTM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 1011 TCID50/ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-XTM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

  4. The arms race control

    International Nuclear Information System (INIS)

    Written in 1961, this paper presents the content of a book entitled 'The arms race control' where the author outlined the difference between disarmament and arms control, described the economic and moral role of arms race, the importance of force balance for international security. He wandered whether arms control could ensure this balance and whether nuclear balance meant force balance. Force balance then appears to be a precarious and unsteady component of international security. He commented the challenges of disarmament, recalled some arguments for a nuclear disarmament. Then he discussed what would be an arms control with or without disarmament (either nuclear or conventional)

  5. An oncolytic adenovirus in combination with radiation treatment (RT) for nasopharyngeal carcinoma (NPC)

    International Nuclear Information System (INIS)

    We have succeeded in regulating gene expression in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein Barr virus (EBV) genome in the cancer cells. When the latently expressed EBV-gene product, EBNA-1, binds to a family of repeats (FR) sequence, located in the oriP of the EBV genome (oriP-FR), this results in transcriptional activation of downstream genes. Based on these observations we designed a transcriptional targeting strategy that now allows for the construction of a conditionally replicating adenovirus under the control of the oriP-FR promoter. Results: Cloning the E1A transcriptional unit downstream of the EBV-responsive, oriP-FR promoter sequence generated the novel adenovirus adv.oriP.E1A. E1A protein expression using Western blot analysis demonstrated a time-dependent increase in expression in the EBV-positive C666-1 cells, in contrast to minimal expression in EBV-negative NPC cells. The MTT assay showed a dose-dependent cytotoxic effect of adv.oriP.E1A in the C666-1 cells, and the combination with 6 Gy of ionizing radiation resulted in an additive cytotoxic effect. In contrast, no evidence of cytotoxicity was observed in a panel of four other human cell lines after adv.oriP.E1A treatment. Western blotting for adenoviral fiber knob protein demonstrated a time-dependent increase in expression, consistent with viral replication; this was confirmed using quantitative real-time PCR. Ex-vivo infection of C666-1 cells with adv.oriP.E1A inhibited tumor formation for at least 100 days after intramuscular injection as compared to control cells that established palpable tumors one week after injection. Treatment of established C666-1 tumours with six intratumoral injections of 2 x 109 pfu adv.oriP.E1A in combination with 2 x 4 Gy radiation therapy (RT) resulted in complete regression for 3-weeks. Conclusion: We have successfully constructed a novel selectively oncolytic virus for EBV-positive NPC cells, demonstrating significant

  6. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  7. Replication Research and Special Education

    Science.gov (United States)

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  8. Dendritic cells serve as a “Trojan horse” for oncolytic adenovirus delivery in the treatment of mouse prostate cancer

    Science.gov (United States)

    Li, Zhao-lun; Liang, Xuan; Li, He-cheng; Wang, Zi-ming; Chong, Tie

    2016-01-01

    Aim: Adenovirus-mediated gene therapy is a novel therapeutic approach for the treatment of cancer, in which replication of the virus itself is the anticancer method. However, the success of this novel therapy is limited due to inefficient delivery of the virus to the target sites. In this study, we used dendritic cells (DCs) as carriers for conditionally replicating adenoviruses (CRAds) in targeting prostate carcinoma (PCa). Methods: Four types of CRAds, including Ad-PC (without PCa-specific promoter and a recombinant human tumor necrosis factor, rmhTNF, sequence), Ad-PC-rmhTNF (without PCa-specific promoter), Ad-PPC-NCS (without an rmhTNF sequence) and Ad-PPC-rmhTNF, were constructed. The androgen-insensitive mouse PCa RM-1 cells were co-cultured with CRAd-loading DCs, and the viability of RM-1 cells was examined using MTT assay. The in vivo effects of CRAd-loading DCs on PCa were evaluated in RM-1 xenograft mouse model. Results: Two PCa-specific CRAds (Ad-PPC-NCS, Ad-PPC-rmhTNF) exhibited more potent suppression on the viability of RM-1 cells in vitro than the PCa-non-specific CRAds (Ad-PC, Ad-PC-rmhTNF). In PCa-bearing mice, intravenous injection of the PCa-specific CRAd-loading DCs significantly inhibited the growth of xenografted tumors, extended the survival time, and induced T-cell activation. Additionally, the rmhTNF-containing CRAds exhibited greater tumor killing ability than CRAds without rmhTNF. Conclusion: DCs may be an effective vector for the delivery of CRAds in the treatment of PCa. PMID:27345628

  9. Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers.

    Science.gov (United States)

    Hoyos, Valentina; Del Bufalo, Francesca; Yagyu, Shigeki; Ando, Miki; Dotti, Gianpietro; Suzuki, Masataka; Bouchier-Hayes, Lisa; Alemany, Ramon; Brenner, Malcolm K

    2015-09-01

    Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC. PMID:26084970

  10. Genomic variation of adenovirus type 5 isolates recovered from bone marrow transplant recipients.

    OpenAIRE

    Webb, D H; Shields, A. F.; Fife, K H

    1987-01-01

    We characterized the genomic variation of adenovirus type 5 isolates recovered from bone marrow transplant recipients in Seattle between 1976 and 1982. By restriction endonuclease analysis, we identified three new adenovirus genomic variants, each associated with a single invasive adenovirus infection. In addition, we were able to obtain suggestive evidence for a nosocomial spread of a particular group of isolates within this population. This study demonstrates that the technique of restricti...

  11. Incidence of enteric adenoviruses among children in Thailand and the significance of these viruses in gastroenteritis.

    OpenAIRE

    Herrmann, J E; Blacklow, N R; Perron-Henry, D M; Clements, E; Taylor, D N; Echeverria, P

    1988-01-01

    In countries with temperate climates, enteric adenoviruses have been shown to be a substantial cause of pediatric gastroenteritis. To determine the incidence of adenovirus infection in a tropical climate, stools were collected from children under age 7 during a 1-year period at an outpatient clinic in Bangkok, Thailand. Stools from 1,114 children with gastroenteritis and from 947 children without gastroenteritis were tested. Each stool was tested for adenovirus group antigen and for specific ...

  12. The frequency of rotavirus and enteric adenovirus in children with acute gastroenteritis in Mardin

    OpenAIRE

    Alicem Tekin

    2010-01-01

    Objectives: Infectious gastroenteritis is one of most important causes of morbidity and mortality in children. Rotavirus and enteric adenoviruses are the most important agents of infectious gastroenteritis. Little is known about the epide-miology of rotavirus and enteric adenovirus gastroenteritis in our region. This study was aimed to determine the fre-quency of rotavirus and enteric adenovirus gastroenteritis in pediatric patients admitted to our hospital.Materials and Methods: Fresh stool ...

  13. TheQ1 Influence of Innate and Pre-Existing Immunity on Adenovirus Therapy

    OpenAIRE

    Zaiss, Anne K.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2009-01-01

    Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed;...

  14. Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

    OpenAIRE

    Roy, Soumitra; Vandenberghe, Luk H.; Kryazhimskiy, Sergey; Grant, Rebecca; Calcedo, Roberto; Yuan, Xin; Keough, Martin; Sandhu, Arbans; Wang, Qiang; Medina-Jaszek, C. Angelica; Plotkin, Joshua B.; Wilson, James M.

    2009-01-01

    Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and m...

  15. Isolation and characterization of adenoviruses persistently shed from the gastrointestinal tract of non-human primates.

    OpenAIRE

    Soumitra Roy; Vandenberghe, Luk H.; Sergey Kryazhimskiy; Rebecca Grant; Roberto Calcedo; Xin Yuan; Martin Keough; Arbans Sandhu; Qiang Wang; C Angelica Medina-Jaszek; Plotkin, Joshua B.; Wilson, James M.

    2009-01-01

    Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and m...

  16. Host Cell Autophagy Modulates Early Stages of Adenovirus Infections in Airway Epithelial Cells

    OpenAIRE

    Zeng, Xuehuo; Carlin, Cathleen R

    2013-01-01

    Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lys...

  17. Replication studies in longevity

    DEFF Research Database (Denmark)

    Varcasia, O; Garasto, S; Rizza, T;

    2001-01-01

    In Danes we replicated the 3'APOB-VNTR gene/longevity association study previously carried out in Italians, by which the Small alleles (less than 35 repeats) had been identified as frailty alleles for longevity. In Danes, neither genotype nor allele frequencies differed between centenarians and 20......-sex interaction relevant to Long alleles (more than 37 repeats). The different findings in Denmark and Italy suggest that gene/longevity associations are population-specific, and heavily affected by the population-specific genetic and environmental history....

  18. First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

    Science.gov (United States)

    Lima, Francisco Esmaile de Sales; Cibulski, Samuel Paulo; Elesbao, Felipe; Carnieli Junior, Pedro; Batista, Helena Beatriz de Carvalho Ruthner; Roehe, Paulo Michel; Franco, Ana Cláudia

    2013-10-01

    This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses. PMID:23828618

  19. 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.

  20. 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...

  1. 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.

  2. Novel bat adenoviruses with an extremely large E3 gene.

    Science.gov (United States)

    Tan, Bing; Yang, Xing-Lou; Ge, Xing-Yi; Peng, Cheng; Zhang, Yun-Zhi; Zhang, Li-Biao; Shi, Zheng-Li

    2016-07-01

    Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs. PMID:27032099

  3. 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

  4. 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.

  5. 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

  6. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

    Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

  7. 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.

  8. 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.

  9. The Adenovirus Genome Contributes to the Structural Stability of the Virion

    Directory of Open Access Journals (Sweden)

    Bratati Saha

    2014-09-01

    Full Text Available Adenovirus (Ad vectors are currently the most commonly used platform for therapeutic gene delivery in human gene therapy clinical trials. Although these vectors are effective, many researchers seek to further improve the safety and efficacy of Ad-based vectors through detailed characterization of basic Ad biology relevant to its function as a vector system. Most Ad vectors are deleted of key, or all, viral protein coding sequences, which functions to not only prevent virus replication but also increase the cloning capacity of the vector for foreign DNA. However, radical modifications to the genome size significantly decreases virion stability, suggesting that the virus genome plays a role in maintaining the physical stability of the Ad virion. Indeed, a similar relationship between genome size and virion stability has been noted for many viruses. This review discusses the impact of the genome size on Ad virion stability and emphasizes the need to consider this aspect of virus biology in Ad-based vector design.

  10. Pathogenesis of a Chinese strain of bovine adenovirus type 3 infection in albino guinea pigs.

    Science.gov (United States)

    Shi, Hong-Fei; Zhu, Yuan-Mao; Yan, Hao; Ma, Lei; Wang, Xue-Zhi; Xue, Fei

    2014-12-01

    Bovine adenovirus type 3 (BAV-3) is considered one of the most important respiratory tract agents of cattle and is widespread among cattle around the world. A BAV-3 strain was isolated from a bovine nasal swab for the first time in China in 2009 and named HLJ0955. Subsequently, BAV-3 has frequently been isolated from calves with respiratory diseases in China. To date, only limited study on the pathogenesis of BAV-3 infection in cotton rats has been conducted, and the pathogenesis of BAV-3 infection in guinea pigs has not been reported. Therefore, sixteen albino guinea pigs were inoculated intranasally with HLJ0955. All of the infected guinea pigs had apparently elevated rectal temperatures (39.2 °C-39.9 °C) at 2-7 days post-inoculation (PI). Consolidation and petechial hemorrhage were also observed in guinea pigs experimentally infected with HLJ0955. Viral replication was detectable by virus isolation and titration and by immunohistochemistry in the lungs of guinea pigs as early as 24 h PI. Viral DNA was detectable in the lungs of infected guinea pigs during 11 days of observation by real-time PCR. Virus-neutralizing antibodies against BAV-3 were detectable from 11 days PI and reached a peak titer at 15 days PI. Histopathological changes mainly occurred in the lungs of infected guinea pigs and were characterized by thickening of alveolar septa, mononuclear cell infiltration, hemorrhage and alveolar epithelial necrosis. These results indicate that HLJ0955 can replicate in the lungs of guinea pigs and cause fever and gross and histological lesions. The guinea pig infection model of BAV-3 would serve as a useful system for monitoring the infection process and pathogenesis of the Chinese BAV-3 strain HLJ0955, as well as immune responses to BAV-3 vaccines.

  11. Replicated Spectrographs in Astronomy

    CERN Document Server

    Hill, Gary J

    2014-01-01

    As telescope apertures increase, the challenge of scaling spectrographic astronomical instruments becomes acute. The next generation of extremely large telescopes (ELTs) strain the availability of glass blanks for optics and engineering to provide sufficient mechanical stability. While breaking the relationship between telescope diameter and instrument pupil size by adaptive optics is a clear path for small fields of view, survey instruments exploiting multiplex advantages will be pressed to find cost-effective solutions. In this review we argue that exploiting the full potential of ELTs will require the barrier of the cost and engineering difficulty of monolithic instruments to be broken by the use of large-scale replication of spectrographs. The first steps in this direction have already been taken with the soon to be commissioned MUSE and VIRUS instruments for the Very Large Telescope and the Hobby-Eberly Telescope, respectively. MUSE employs 24 spectrograph channels, while VIRUS has 150 channels. We compa...

  12. SUMO and KSHV Replication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: hkung@nhri.org.tw [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)

    2014-09-29

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  13. Managing new arms races

    International Nuclear Information System (INIS)

    The management of new arms races in the region of Asia-Pacific includes considerations of weapons trade and transfer in the region, with an emphasis on nuclear weapons proliferation. It deals with the problem of controlling the arms trade and the efforts to control conventional weapons and underlines the possible role and influence of Conference on Cooperation and Security in Europe (CSCE)

  14. ARM Mentor Selection Process

    Energy Technology Data Exchange (ETDEWEB)

    Sisterson, D. L. [Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-10-01

    The Atmospheric Radiation Measurement (ARM) Program was created in 1989 with funding from the U.S. Department of Energy (DOE) to develop several highly instrumented ground stations to study cloud formation processes and their influence on radiative transfer. In 2003, the ARM Program became a national scientific user facility, known as the ARM Climate Research Facility. This scientific infrastructure provides for fixed sites, mobile facilities, an aerial facility, and a data archive available for use by scientists worldwide through the ARM Climate Research Facility—a scientific user facility. The ARM Climate Research Facility currently operates more than 300 instrument systems that provide ground-based observations of the atmospheric column. To keep ARM at the forefront of climate observations, the ARM infrastructure depends heavily on instrument scientists and engineers, also known as lead mentors. Lead mentors must have an excellent understanding of in situ and remote-sensing instrumentation theory and operation and have comprehensive knowledge of critical scale-dependent atmospheric processes. They must also possess the technical and analytical skills to develop new data retrievals that provide innovative approaches for creating research-quality data sets. The ARM Climate Research Facility is seeking the best overall qualified candidate who can fulfill lead mentor requirements in a timely manner.

  15. 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.

  16. 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 lin

  17. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 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 cell... hepatitis, the test is inconclusive and may be repeated. (B) If at least 19 of the 20 vaccinates do...

  18. Efficient usage of Adabas replication

    CERN Document Server

    Storr, Dieter W

    2011-01-01

    In today's IT organization replication becomes more and more an essential technology. This makes Software AG's Event Replicator for Adabas an important part of your data processing. Setting the right parameters and establishing the best network communication, as well as selecting efficient target components, is essential for successfully implementing replication. This book provides comprehensive information and unique best-practice experience in the field of Event Replicator for Adabas. It also includes sample codes and configurations making your start very easy. It describes all components ne

  19. A randomized clinical trial comparing advanced pneumatic truncal, chest, and arm treatment to arm treatment only in self-care of arm lymphedema.

    Science.gov (United States)

    Ridner, Sheila H; Murphy, Barbara; Deng, Jie; Kidd, Nancy; Galford, Emily; Bonner, Candace; Bond, Stewart M; Dietrich, Mary S

    2012-01-01

    Treatment of the truncal lymphatics prior to treatment of the lymphedematous arm is an accepted, although not empirically tested, therapeutic intervention delivered during decongestive lymphatic therapy (DLT). Breast cancer survivors with arm lymphedema are encouraged to use these techniques when performing simple lymphatic drainage as part of their life-long lymphedema self-care. Self-massage is at times difficult and pneumatic compression devices are used by many patients to assist with self-care. One such device, the Flexitouch(®) System, replicates the techniques used during DLT; however, the need for application of pneumatic compression in unaffected truncal areas to improve self-care outcomes in arm only lymphedema is not established. The objective of this study was to compare the therapeutic benefit of truncal/chest/arm advanced pneumatic compression therapy (experimental group) verses arm only pneumatic compression (control group) in self-care for arm lymphedema without truncal involvement using the Flexitouch(®) System. Outcomes of interest were self-reported symptoms, function, arm impedance ratios, circumference, volume, and trunk circumference. Forty-two breast cancer survivors, (21 per group), with Stage II lymphedema completed 30 days of home self-care using the Flexitouch(®) System. Findings revealed a statistically significant reduction in both the number of symptoms and overall symptom burden within each group; however, there were no statistically significant differences in these outcomes between the groups. There was no statistically significant overall change or differential pattern of change between the groups in function. A statistically significant reduction in bioelectrical impedance and arm circumference within both of the groups was achieved; however, there was no statistically significant difference in reduction between groups. These findings indicate that both configurations are effective, but that there may be no added benefit to

  20. Nonspecific Arm Pain

    Directory of Open Access Journals (Sweden)

    Ali Moradi

    2013-12-01

    Full Text Available   Nonspecific activity-related arm pain is characterized by an absence of objective physical findings and symptoms that do not correspond with objective pathophysiology. Arm pain without strict diagnosis is often related to activity, work-related activity in particular, and is often seen in patients with physically demanding work. Psychological factors such as catastrophic thinking, symptoms of depression, and heightened illness concern determine a substantial percentage of the disability associated with puzzling hand and arm pains. Ergonomic modifications can help to control symptoms, but optimal health may require collaborative management incorporating psychosocial and psychological elements of illness.

  1. Nonspecific Arm Pain

    Directory of Open Access Journals (Sweden)

    Ali Moradi

    2013-12-01

    Full Text Available Nonspecific activity-related arm pain is characterized by an absence of objective physical findings and symptoms that do not correspond with objective pathophysiology. Arm pain without strict diagnosis is often related to activity, work-related activity in particular, and is often seen in patients with physically demanding work. Psychological factors such as catastrophic thinking, symptoms of depression, and heightened illness concern determine a substantial percentage of the disability associated with puzzling hand and arm pains. Ergonomic modifications can help to control symptoms, but optimal health may require collaborative management incorporating psychosocial and psychological elements of illness.

  2. Charter School Replication. Policy Guide

    Science.gov (United States)

    Rhim, Lauren Morando

    2009-01-01

    "Replication" is the practice of a single charter school board or management organization opening several more schools that are each based on the same school model. The most rapid strategy to increase the number of new high-quality charter schools available to children is to encourage the replication of existing quality schools. This policy guide…

  3. DATABASE REPLICATION IN HETEROGENOUS PLATFORM

    Directory of Open Access Journals (Sweden)

    Hendro Nindito

    2014-01-01

    Full Text Available The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MySQL running on Linux as the destination. The method applied in this research is prototyping in which the processes of development and testing can be done interactively and repeatedly. The key result of this research is that the replication technology applied, which is called Oracle GoldenGate, can successfully manage to do its task in replicating data in real-time and heterogeneous platforms.

  4. An Elastica Arm Scale

    CERN Document Server

    Bosi, F; Corso, F Dal; Bigoni, D

    2015-01-01

    The concept of 'deformable arm scale' (completely different from a traditional rigid arm balance) is theoretically introduced and experimentally validated. The idea is not intuitive, but is the result of nonlinear equilibrium kinematics of rods inducing configurational forces, so that deflection of the arms becomes necessary for the equilibrium, which would be impossible for a rigid system. In particular, the rigid arms of usual scales are replaced by a flexible elastic lamina, free of sliding in a frictionless and inclined sliding sleeve, which can reach a unique equilibrium configuration when two vertical dead loads are applied. Prototypes realized to demonstrate the feasibility of the system show a high accuracy in the measure of load within a certain range of use. It is finally shown that the presented results are strongly related to snaking of confined beams, with implications on locomotion of serpents, plumbing, and smart oil drilling.

  5. ARM7-kehityskortti

    OpenAIRE

    Kukkonen, Henri

    2006-01-01

    Tämän opinnäytetyön tarkoituksena oli suunnitella ja toteuttaa ARM-mikro-ohjain pohjainen kehityskortti. Kortin tuli olla soveltuva ARM-ohjelmoinnin opettamiseen. Työssä myös selvitettiin ARM-mikro-ohjaimen ohjelmointiympäristön käyttöönotto. Teoriaosassa käsitellään ARM-arkkitehtuuria, työssä käytettyjen Atmelin AT91R40008-mikro-ohjaimen sekä Philipsin LPC2105-mikro-ohjaimen ominaisuuksia. Erityisesti työssä keskitytään kehityskorttien suunnitteluun. Kehityskortin vaatimuksina oli, että se ...

  6. Species D human adenovirus type 9 exhibits better virus-spread ability for antitumor efficacy among alternative serotypes.

    Directory of Open Access Journals (Sweden)

    Junji Uchino

    Full Text Available Species C human adenovirus serotype 5 (HAdV-C5 is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR, HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.

  7. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    Science.gov (United States)

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí

    2015-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

  8. Hello to Arms

    Science.gov (United States)

    2005-01-01

    This image highlights the hidden spiral arms (blue) that were discovered around the nearby galaxy NGC 4625 by the ultraviolet eyes of NASA's Galaxy Evolution Explorer. The image is composed of ultraviolet and visible-light data, from the Galaxy Evolution Explorer and the California Institute of Technology's Digitized Sky Survey, respectively. Near-ultraviolet light is colored green; far-ultraviolet light is colored blue; and optical light is colored red. As the image demonstrates, the lengthy spiral arms are nearly invisible when viewed in optical light while bright in ultraviolet. This is because they are bustling with hot, newborn stars that radiate primarily ultraviolet light. The youthful arms are also very long, stretching out to a distance four times the size of the galaxy's core. They are part of the largest ultraviolet galactic disk discovered so far. Located 31 million light-years away in the constellation Canes Venatici, NGC 4625 is the closest galaxy ever seen with such a young halo of arms. It is slightly smaller than our Milky Way, both in size and mass. However, the fact that this galaxy's disk is forming stars very actively suggests that it might evolve into a more massive and mature galaxy resembling our own. The armless companion galaxy seen below NGC 4625 is called NGC 4618. Astronomers do not know why it lacks arms but speculate that it may have triggered the development of arms in NGC 4625.

  9. Segregation of chromosome arms in growing and non-growing Escherichia coli cells

    Directory of Open Access Journals (Sweden)

    Conrad L. Woldringh

    2015-05-01

    Full Text Available In slow-growing Escherichia coli cells the chromosome is organized with its left (L and right (R arms lying separated in opposite halves of the nucleoid and with the origin (O in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three coloured loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances and between duplicated loci (LL- or RR-distances as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not accordingto the doughnut model.

  10. Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

    Directory of Open Access Journals (Sweden)

    Stefania Piersanti

    Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

  11. Adenovirus vaccine vectors expressing hepatitis B surface antigen: importance of regulatory elements in the adenovirus major late intron.

    Science.gov (United States)

    Mason, B B; Davis, A R; Bhat, B M; Chengalvala, M; Lubeck, M D; Zandle, G; Kostek, B; Cholodofsky, S; Dheer, S; Molnar-Kimber, K

    1990-08-01

    Adenovirus types 4 and 7 are currently used as live oral vaccines for prevention of acute respiratory disease caused by these adenovirus serotypes. To investigate the concept of producing live recombinant vaccines using these serotypes, adenovirus types 4 (Ad4) and 7 (Ad7) were constructed that produce HBsAg upon infection of cell cultures. Ad4 recombinants were constructed that express HBsAg from a cassette inserted 135 bp from the right-hand terminus of the viral genome. The cassette contained the Ad4 major late promoter followed by leader 1 of the tripartite leader, the first intervening sequence between leaders 1 and 2, leaders 2 and 3, the HBsAg gene, and tandem polyadenylation signals from the Ad4 E3B and hexon genes. Using this same cassette, a series of Ad4 recombinants expressing HBsAg were constructed with deletions in the intervening sequence between leaders 1 and 2 to evaluate the contribution of the downstream control elements more precisely. Inclusion of regions located between +82 and +148 as well as +148 and +232 resulted in increases in expression levels of HBsAg in A549-infected cells by 22-fold and 44-fold, respectively, over the levels attained by an adenovirus recombinant retaining only sequences from +1 to +82, showing the importance of these elements in the activation of the major late promoter during the course of a natural Ad4 viral infection. Parallel increases were also observed in steady-state levels of cytoplasmic HBsAg-specific mRNA. When similar Ad7 recombinant viruses were constructed, these viruses also expressed 20-fold more HBsAg due to the presence of the intron. All Ad4 and Ad7 recombinants produced HBsAg particles containing gp27 and p24 which were secreted in the medium. When dogs were immunized intratracheally with one of these Ad7 recombinants, they seroconverted to both Ad7 and HBsAg to a high level. PMID:2371766

  12. 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.

  13. Enhanced immune responses against Japanese encephalitis virus using recombinant adenoviruses coexpressing Japanese encephalitis virus envelope and porcine interleukin-6 proteins in mice.

    Science.gov (United States)

    Liu, Hanyang; Wu, Rui; Liu, Kai; Yuan, Lei; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Yan, Qigui; Zhao, Qin; Wen, Xintian; Cao, Sanjie

    2016-08-15

    Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Previous studies have demonstrated that recombinant adenovirus serotype 5 (Ad5) may be a potential vaccine candidate because it can express JEV envelope epitopes and induce immune responses against JEV. Still, it will be necessary to develop an adjuvant that can enhance both humoral and cellular immune responses to the recombinant antigen delivered by non-replicating Ad5. In this study, we investigated the systemic immune responses of BALB/c mice immunized with recombinant adenovirus expressing JEV envelope epitopes in combination with porcine interleukin-6 (rAdE-IL-6).The rAdE-IL-6 immunized group had the highest titers of anti-JEV antibody as detected by an enzyme-linked immunosorbent assay (ELISA), as well as the highest levels of neutralizing antibody (1:75) as detected by a serum neutralization test. Similarly, higher concentrations of interferon-gamma (834.7pg/ml) and interleukin-6 (IL-6) (229.7pg/ml) were detected in the rAdE-IL-6 group using an ELISA assay. These data indicate that immunized BALB/c induce a strong cellular response against rAdE-IL-6. Furthermore, after challenge with the virulent JEV SCYA201201 strain, the rAdE-IL-6 group generated an immune protective response 70% greater than that of the control group, indicating that rAdE-IL-6 induced a protective immune response against JEV challenge in mice. The results from this study demonstrated that IL-6 is a strong adjuvant that can enhance both humoral and cellular immune responses in mice. Furthermore, a recombinant adenovirus coexpressing JEV envelope epitopes and porcine IL-6 protein may be an effective vaccine in animals. PMID:27235810

  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. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    Science.gov (United States)

    Gahéry-Ségard, H; Molinier-Frenkel, V; Le Boulaire, C; Saulnier, P; Opolon, P; Lengagne, R; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A; Schatz, C; Courtney, M; Le Chevalier, T; Tursz, T; Guillet, J G; Farace, F

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein. PMID:9410899

  16. 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.

  17. 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.

  18. 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.

  19. Regulation of Replication Recovery and Genome Integrity

    DEFF Research Database (Denmark)

    Colding, Camilla Skettrup

    Preserving genome integrity is essential for cell survival. To this end, mechanisms that supervise DNA replication and respond to replication perturbations have evolved. One such mechanism is the replication checkpoint, which responds to DNA replication stress and acts to ensure replication pausing...

  20. Molecular detection of two adenoviruses associated with disease in Australian lizards.

    Science.gov (United States)

    Hyndman, T; Shilton, C M

    2011-06-01

    We give the first published description of the pathology and molecular findings associated with adenovirus infection in lizards in Australia. A central netted dragon (Ctenophorus nuchalis) exhibited severe necrotising hepatitis with abundant intranuclear inclusion bodies within hepatocytes and rarely within intestinal epithelial cells. Polymerase chain reaction (PCR) using pooled tissues yielded an amplicon that shared strong nucleotide identity with an agamid adenovirus (EU914203). PCR on the liver of a bearded dragon (Pogona minor minor) with illthrift, coccidiosis, nematodiasis and hepatic lipidosis yielded an amplicon with strong nucleotide identity to a helodermatid adenovirus (EU914207). PMID:21595645

  1. 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

  2. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul

    2011-01-01

    Business organizations may expand internationally by replicating a part of their value chain, such as a sales and marketing format, in other countries. However, little is known regarding how such “international replicators” build a format for replication, or how they can adjust it in order to adapt...... to local environments and under the impact of new learning. To illuminate these issues, we draw on a longitudinal in-depth study of Swedish home furnishing giant IKEA, involving more than 70 interviews. We find that IKEA has developed organizational mechanisms that support an ongoing learning process aimed...... at frequent modification of the format for replication. Another finding is that IKEA treats replication as hierarchical: lower-level features (marketing efforts, pricing, etc.) are allowed to vary across IKEA stores in response to market-based learning, while higher-level features (fundamental values, vision...

  3. 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.

  4. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  5. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  6. Phoenix Stretches its Arm

    Science.gov (United States)

    2008-01-01

    [figure removed for brevity, see original site] Click on image for animation The Phoenix spacecraft is scheduled to begin raising its robotic arm up and out of its stowed configuration on the third Martian day, or Sol 3 (May 28, 2008) of the mission. This artist's animation, based on engineering models, shows how Phoenix will accomplish this task. First, its wrist actuator will rotate, releasing its launch-restraint pin. Next, the forearm moves up, releasing the elbow launch-restraint pin. The elbow will then move up and over in small steps, a process referred to as 'staircasing.' This ensures that the arm's protective biobarrier wrap, now unpeeled and lying to the side of the arm, will not get in the way of the arm's deployment. The arm is scheduled to straighten all the way out on Sol 4 (May 29, 2008), after engineers have reviewed images and telemetry data from the spacecraft showing that the biobarrier material has been cleared. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  7. Therapeutic efficacy of an oncolytic adenovirus containing RGD ligand in minor capsid protein IX and Fiber, Δ24DoubleRGD, in an ovarian cancer model

    Directory of Open Access Journals (Sweden)

    Anton V Borovjagin

    2012-02-01

    Full Text Available Ovarian cancer is the leading cause of gynecological disease death despite advances in medicine. Therefore, novel strategies are required for ovarian cancer therapy. Conditionally replicative adenoviruses (CRAds, genetically modified as anti-cancer therapeutics, are one of the most attractive candidate agents for cancer therapy. However, a paucity of coxsackie B virus and adenovirus receptor (CAR expression on the surface of ovarian cancer cells has impeded treatment of ovarian cancer using this approach.This study sought to engineer a CRAd with enhanced oncolytic ability in ovarian cancer cells, “Δ24DoubleRGD.” Δ24DoubleRGD carries an arginine-glycine-aspartate (RGD motif incorporated into both fiber and capsid protein IX (pIX and its oncolytic efficacy was evaluated in ovarian cancer. In vitro analysis of cell viability showed that infection of ovarian cancer cells with Δ24DoubleRGD leads to increased cell killing relative to the control CRAds. Data from this study suggested that not only an increase in number of RGD motifs on the CRAd capsid, but also a change in the repertoir of targeted integrins could lead to enhanced oncolytic potency of Δ24DoubleRGD in ovarian cancer cells in vitro. In an intraperitoneal model of ovarian cancer, mice injected with Δ24DoubleRGD showed, however, a similar survival rate as mice treated with control CRAds.

  8. Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus

    Indian Academy of Sciences (India)

    Helin Li; Pengbo Ning; Zhi Lin; Wulong Liang; Kai Kang; Lei He; Yanming Zhang

    2015-03-01

    The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×106 TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

  9. Where does DNA replication start in archaea?

    OpenAIRE

    Vas, Amit; Leatherwood, Janet

    2000-01-01

    Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

  10. Control of foot-and-mouth disease by using replication-defective human adenoviruses to deliver vaccines and biotherapeutics

    Science.gov (United States)

    Foot-and-mouth disease (FMD) is one of the most contagious viral diseases that can affect cloven-hoofed livestock and wild animals. Outbreaks of FMD have caused devastating economic losses and the slaughter of millions of animals in many regions of the world affecting the food chain and global devel...

  11. Arm To Arm Interface Using Embedded C

    Directory of Open Access Journals (Sweden)

    Mohanraj.C

    2013-02-01

    Full Text Available Embedded systems are the most emerging field in these recent years. In this paper a different number of ARM processors (LPC2148 and LPC2378 are interconnected using C for distributed services. N numbers of processors are connected as the network and each processing devices are interlinked with each other, so that the each data that is processed by the devices and it can be used by the other device to activate their entire process. All the processed data’s are communicated to other device through Xbee interface card. LPC2148 and LPC2378 ARM processors are used in this prototype and winXtalk is used as a software terminal window. In this paper, the ultimate benefits of multiple processor interactions related to the embedded applications and design issues of processor interconnection are discussed. The features of multiple processor interaction in inter process communication and executions of embedded multitasking are also discussed. In modern embedded computing platform, embedded processor used in various applications like home automation, industrial control, medical system, access control, etc. In this paper, using embedded processor interactions, the several data communication is established.

  12. Children In Armed Conflicts

    OpenAIRE

    Ranjan, Tejaswini

    2013-01-01

    This is a descriptive study. A child soldier is a child under the age of 18 that is recruited into the armed forces and engages in political violence. Child Soldiers are recruited by a state or non-state armed group and used as fighters, cooks, suicide bombers, human shields, messengers, spies, or for sexual purposes. This work of research describes the plight of child soldiers taking in context the scenario of different nations. The International mechanisms to combat this problem have also b...

  13. PHENIX Muon Arms

    International Nuclear Information System (INIS)

    The PHENIX Muon Arms detect muons at rapidities of |y|=(1.2-2.4) with full azimuthal acceptance. Each muon arm must track and identify muons and provide good rejection of pions and kaons (∼10-3). In order to accomplish this we employ a radial field magnetic spectrometer with precision tracking (Muon Tracker) followed by a stack of absorber/low resolution tracking layers (Muon Identifier). The design, construction, testing and expected run parameters of both the muon tracker and the muon identifier are described

  14. PHENIX Muon Arms

    Energy Technology Data Exchange (ETDEWEB)

    Akikawa, H.; Al-Jamel, A.; Archuleta, J.B.; Archuleta, J.R.; Armendariz, R.; Armijo, V.; Awes, T.C.; Baldisseri, A.; Barker, A.B.; Barnes, P.D.; Bassalleck, B.; Batsouli, S.; Behrendt, J.; Bellaiche, F.G.; Bland, A.W.; Bobrek, M.; Boissevain, J.G.; Borel, H.; Brooks, M.L.; Brown, A.W.; Brown, D.S.; Bruner, N.; Cafferty, M.M.; Carey, T.A.; Chai, J.-S.; Chavez, L.L.; Chollet, S.; Choudhury, R.K.; Chung, M.S.; Cianciolo, V.; Clark, D.J.; Cobigo, Y.; Dabrowski, C.M.; Debraine, A.; DeMoss, J.; Dinesh, B.V.; Drachenberg, J.L.; Drapier, O.; Echave, M.A.; Efremenko, Y.V.; En' yo, H.; Fields, D.E.; Fleuret, F.; Fried, J.; Fujisawa, E.; Funahashi, H.; Gadrat, S.; Gastaldi, F.; Gee, T.F.; Glenn, A.; Gogiberidze, G.; Gonin, M.; Gosset, J.; Goto, Y.; Granier de Cassagnac, R.; Hance, R.H.; Hart, G.W.; Hayashi, N.; Held, S.; Hicks, J.S.; Hill, J.C.; Hoade, R.; Hong, B.; Hoover, A.; Horaguchi, T.; Hunter, C.T.; Hurst, D.E.; Ichihara, T.; Imai, K.; Isenhower, L.D.L. Davis; Isenhower, L.D.L. Donald; Ishihara, M.; Jang, W.Y.; Johnson, J.; Jouan, D.; Kamihara, N.; Kamyshkov, Y.; Kang, J.H.; Kapoor, S.S.; Kim, D.J.; Kim, D.-W.; Kim, G.-B.; Kinnison, W.W.; Klinksiek, S.; Kluberg, L.; Kobayashi, H.; Koehler, D.; Kotchenda, L.; Kuberg, C.H.; Kurita, K.; Kweon, M.J.; Kwon, Y.; Kyle, G.S.; LaBounty, J.J.; Lajoie, J.G.; Lee, D.M.; Lee, S.; Leitch, M.J.; Li, Z.; Liu, M.X.; Liu, X.; Liu, Y.; Lockner, E.; Lopez, J.D.; Mao, Y.; Martinez, X.B.; McCain, M.C.; McGaughey, P.L.; Mioduszewski, S.; Mischke, R.E.; Mohanty, A.K.; Montoya, B.C.; Moss, J.M.; Murata, J.; Murray, M.M.; Nagle, J.L.; Nakada, Y.; Newby, J.; Obenshain, F.; Palounek, A.P.T.; Papavassiliou, V.; Pate, S.F.; Plasil, F.; Pope, K.; Qualls, J.M.; Rao, G.; Read, K.F. E-mail: readkf@ornl.gov; Robinson, S.H.; Roche, G.; Romana, A.; Rosnet, P.; Roth, R.; Saito, N.; Sakuma, T.; Sandhoff, W.F.; Sanfratello, L.; Sato, H.D.; Savino, R.; Sekimoto, M.; Shaw, M.R.; Shibata, T.-A.; Sim, K.S.; Skank, H.D.; Smith, D.E.; Smith, G.D. [and others

    2003-03-01

    The PHENIX Muon Arms detect muons at rapidities of |y|=(1.2-2.4) with full azimuthal acceptance. Each muon arm must track and identify muons and provide good rejection of pions and kaons ({approx}10{sup -3}). In order to accomplish this we employ a radial field magnetic spectrometer with precision tracking (Muon Tracker) followed by a stack of absorber/low resolution tracking layers (Muon Identifier). The design, construction, testing and expected run parameters of both the muon tracker and the muon identifier are described.

  15. 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 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...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T...

  16. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.

    OpenAIRE

    Ketner, G; Spencer, F; Tugendreich, S; C. Connelly; Hieter, P

    1994-01-01

    A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the Y...

  17. Neoplastic transformation of chimpanzee cells induced by adenovirus type 12--simian virus 40 hybrid virus.

    OpenAIRE

    Rhim, J S; Trimmer, R; Arnstein, P; Huebner, R J

    1981-01-01

    The adenovirus 12--simian virus 40 hybrid virus produced neoplastic transformation of chimpanzee skin fibroblasts in vitro. The transformed fibroblasts showed morphological alteration and became permanent lines. The transformed cells contained both adenovirus 12 and simian virus 40 large tumor antigens and were virus producers. However at passage 9, one line (WES) was found to be a nonproducer, producing neither infectious virus nor virus-specific antigen detectable by the complement fixation...

  18. 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.

  19. Frequency of Rotavirus and Adenovirus Gastroenteritis Among Children in Shiraz, Iran

    OpenAIRE

    Motamedifar, Mohammad; Amini, Elham; Talezadeh Shirazi, Pedram

    2013-01-01

    Background Viral pathogens are the main cause of acute gastroenteritis in developed and developing countries. Rotavirus and adenovirus are the two important agents associated with hospitalization for diarrhea especially in children. Limitation and control of diarrhea as a costly disease must be considered in national health programs. Objectives Epidemiological studies on viral diarrhea and collecting data for rotavirus and adenovirus prevalence, as two important viral agents of gastroenteriti...

  20. A retrospective investigation of canine adenovirus (CAV) infection in adult dogs in Turkey : article

    OpenAIRE

    Gur, S; A. Acar

    2009-01-01

    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 Es...

  1. Mutation in fiber of adenovirus serotype 5 gene therapy vector decreases liver tropism

    OpenAIRE

    Wang, Zhen; Wang, Baoming; Lou, Junfang; Yan, Jingyi; Gao, Lei; Geng, Ranshen; Yu, Bin

    2014-01-01

    Recombinant adenovirus (Ad) vectors are widely used for both in vitro and in vivo gene transfer. However, intravenous administration of Ad vectors results mainly in hepatocyte transduction and subsequent hepatotoxicity. Coxsackie-adenovirus receptor (CAR) and αvβ integrins, which are functional receptors for the fiber and penton proteins, respectively, are the tropism determinants of Ad type 5 (Ad5). We previously developed a system for rapid construction of fiber-modified Ad5 vectors. We als...

  2. Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes.

    OpenAIRE

    Hyypiä, T

    1985-01-01

    The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an...

  3. Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses

    OpenAIRE

    Mück-Häusl, Martin; Solanki, Manish; Zhang, Wenli; Ruzsics, Zsolt; Ehrhardt, Anja

    2015-01-01

    Recombinant adenoviruses containing a double-stranded DNA genome of 26–45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we ...

  4. 3D modeling of human cancer: A PEG-fibrin hydrogel system to study the role of tumor microenvironment and recapitulate the in vivo effect of oncolytic adenovirus.

    Science.gov (United States)

    Del Bufalo, Francesca; Manzo, Teresa; Hoyos, Valentina; Yagyu, Shigeki; Caruana, Ignazio; Jacot, Jeffrey; Benavides, Omar; Rosen, Daniel; Brenner, Malcolm K

    2016-04-01

    Interactions between malignant and stromal cells and the 3D spatial architecture of the tumor both substantially modify tumor behavior, including the responses to small molecule drugs and biological therapies. Conventional 2D culture systems cannot replicate this complexity. To overcome these limitations and more accurately model solid tumors, we developed a highly versatile 3D PEG-fibrin hydrogel model of human lung adenocarcinoma. Our model relevantly recapitulates the effect of oncolytic adenovirus; tumor responses in this setting nearly reproduce those observed in vivo. We have also validated the use of this model for complex, long-term, 3D cultures of cancer cells and their stroma (fibroblasts and endothelial cells). Both tumor proliferation and invasiveness were enhanced in the presence of stromal components. These results validate our 3D hydrogel model as a relevant platform to study cancer biology and tumor responses to biological treatments. PMID:26826297

  5. Modernization of African Armed Forces

    DEFF Research Database (Denmark)

    Mandrup, Thomas

    2015-01-01

    Concept paper framing the debate at the Dakar Forum Workshop on Modernization of Armed forces in Africa.......Concept paper framing the debate at the Dakar Forum Workshop on Modernization of Armed forces in Africa....

  6. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    Science.gov (United States)

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  7. 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.

  8. 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.

  9. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question.

    Science.gov (United States)

    Koonin, Eugene V; Starokadomskyy, Petro

    2016-10-01

    The question whether or not "viruses are alive" has caused considerable debate over many years. Yet, the question is effectively without substance because the answer depends entirely on the definition of life or the state of "being alive" that is bound to be arbitrary. In contrast, the status of viruses among biological entities is readily defined within the replicator paradigm. All biological replicators form a continuum along the selfishness-cooperativity axis, from the completely selfish to fully cooperative forms. Within this range, typical, lytic viruses represent the selfish extreme whereas temperate viruses and various mobile elements occupy positions closer to the middle of the range. Selfish replicators not only belong to the biological realm but are intrinsic to any evolving system of replicators. No such system can evolve without the emergence of parasites, and moreover, parasites drive the evolution of biological complexity at multiple levels. The history of life is a story of parasite-host coevolution that includes both the incessant arms race and various forms of cooperation. All organisms are communities of interacting, coevolving replicators of different classes. A complete theory of replicator coevolution remains to be developed, but it appears likely that not only the differentiation between selfish and cooperative replicators but the emergence of the entire range of replication strategies, from selfish to cooperative, is intrinsic to biological evolution.

  10. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  11. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease.

  12. Mimiviruses: Replication, Purification, and Quantification.

    Science.gov (United States)

    Abrahão, Jônatas Santos; Oliveira, Graziele Pereira; Ferreira da Silva, Lorena Christine; Dos Santos Silva, Ludmila Karen; Kroon, Erna Geessien; La Scola, Bernard

    2016-01-01

    The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc. PMID:27153385

  13. Van Yöresinde Gözlenen Gastroenteritlerde Rotavirus ve Adenovirus Sıklığı

    OpenAIRE

    Gültepe, Bilge; Güdücüoğlu, Hüseyin; Çıkman, Aytekin; PARLAK, Mehmet; Berktaş, Mustafa

    2013-01-01

    Objectives: Infectious gastroenteritis is one of most important causes of morbidity and mortality in children. Rotavirus and enteric adenoviruses are the most important agents of infectious gastroenteritis. The epidemiology of rotavirus and enteric adenovirus gastroenteritis is not well known in our region. This study was aimed to determine the frequency of rotavirus and enteric adenovirus gastroenteritis in pediatric patients admitted to our hospital.Materials and Methods: Fresh stool spec...

  14. Design and Implementation of Prosthetic Arm using Gear Motor Control Technique with Appropriate Testing

    CERN Document Server

    Neogi, Biswarup; Ghosal, Soumya; Das, Achintya; Tibarewala, D N

    2011-01-01

    Any part of the human body replication procedure commences the prosthetic control science. This paper highlights the hardware design technique of a prosthetic arm with implementation of gear motor control aspect. The prosthetic control arm movement has been demonstrated in this paper applying processor programming and with the successful testing of the designed prosthetic model. The architectural design of the prosthetic arm here has been replaced by lighter material instead of heavy metal, as well as the traditional EMG (electro myographic) signal has been replaced by the muscle strain.

  15. Up-regulation of integrin β3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy

    International Nuclear Information System (INIS)

    Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P<0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. (author)

  16. 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.

  17. Cellular factors required for papillomavirus DNA replication.

    OpenAIRE

    Melendy, T; Sedman, J; Stenlund, A

    1995-01-01

    In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a ...

  18. Optimal Placement of Origins for DNA Replication

    OpenAIRE

    Karschau, Jens; Blow, J. Julian; de Moura, Alessandro P. S.

    2012-01-01

    DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyse these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to ...

  19. 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

  20. Vector-mediated expression of interferon gamma inhibits replication of hepatitis B virus in vitro.

    Science.gov (United States)

    Kan, Q C; Li, D L; Yu, Z J

    2013-01-01

    Despite the existence of efficient vaccines against hepatitis B virus (HBV) infections, these still represent a serious threat to human health worldwide. Acute HBV infections often become chronic, marked by liver cirrhosis and hepatocellular carcinoma. Promising results with interferons alpha or gamma (IFN-α, γ) or nucleoside/nucleotide analogs in inhibiting HBV replication in vitro have led to therapeutic applications to chronic HBV patients, however, their results so far have not been satisfactory. The treatments were either not effective in all patients or had adverse effects. Certain progress was expected from expression of interferons targeted to liver by adenovirus vectors, however, this approach turned out to be limited by undesired expression of toxic viral genes and high production costs. Therefore, in this study, we attempted to inhibit HBV replication in HepG2.2.15 cells by human IFN-γ expressed through a non-viral vector, an eukaryotic plasmid. The results demonstrated that IFN-γ, targeted to HBV-replicating cells, significantly inhibited the virus growth without inducing apoptosis and indicated that local expression of this kind of cytokine may be a promising strategy of gene therapy. PMID:24294955

  1. The replication-transcription conflict

    OpenAIRE

    Soultanas, Panos

    2011-01-01

    In response to environmental and nutritional stimuli, a whole array of proteins remodel genome architecture, activate or transcribe genes, suppress genes, repair lesions and base-modifications, faithfully replicate and safely separate the parental and daughter genomes during cell division. Negotiating and resolving conflicts of genome trafficking is essential for genome stability.

  2. Covert Reinforcement: A Partial Replication.

    Science.gov (United States)

    Ripstra, Constance C.; And Others

    A partial replication of an investigation of the effect of covert reinforcement on a perceptual estimation task is described. The study was extended to include an extinction phase. There were five treatment groups: covert reinforcement, neutral scene reinforcement, noncontingent covert reinforcement, and two control groups. Each subject estimated…

  3. Cellular Responses to Replication Problems

    NARCIS (Netherlands)

    M. Budzowska (Magdalena)

    2008-01-01

    textabstractDuring every S-phase cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. It is a tremendous task, given the large sizes of mammalian genomes and the required precision of DNA replication. A major threat to the accuracy and eff

  4. Hyperthermia stimulates HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Ferdinand Roesch

    Full Text Available HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C and Heat Shock Proteins (HSPs modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

  5. Biomarkers of replicative senescence revisited

    DEFF Research Database (Denmark)

    Nehlin, Jan

    2016-01-01

    of telomere length and associated damage, and the accompanying changes that take place elicit signals that have an impact on a number of molecules and downstream events. Precise measurements of replicative senescence biomarkers in biological samples from individuals could be clinically associated...

  6. Crinivirus replication and host interactions

    Directory of Open Access Journals (Sweden)

    Zsofia A Kiss

    2013-05-01

    Full Text Available Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense ssRNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was available. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as BYV-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP, CPm, Hsp70h, and p59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5’ end of RNA 2 as ORF1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the

  7. Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

    International Nuclear Information System (INIS)

    Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors

  8. Oncolytic adenovirus and doxorubicin-based chemotherapy results in synergistic antitumor activity against soft-tissue sarcoma.

    Science.gov (United States)

    Siurala, Mikko; Bramante, Simona; Vassilev, Lotta; Hirvinen, Mari; Parviainen, Suvi; Tähtinen, Siri; Guse, Kilian; Cerullo, Vincenzo; Kanerva, Anna; Kipar, Anja; Vähä-Koskela, Markus; Hemminki, Akseli

    2015-02-15

    Despite originating from several different tissues, soft-tissue sarcomas (STS) are often grouped together as they share mesenchymal origin and treatment guidelines. Also, with some exceptions, a common denominator is that when the tumor cannot be cured with surgery, the efficacy of current therapies is poor and new treatment modalities are thus needed. We have studied the combination of a capsid-modified oncolytic adenovirus CGTG-102 (Ad5/3-D24-GMCSF) with doxorubicin, with or without ifosfamide, the preferred first-line chemotherapeutic options for most types of STS. We show that CGTG-102 and doxorubicin plus ifosfamide together are able to increase cell killing of Syrian hamster STS cells over single agents, as well as upregulate immunogenic cell death markers. When tested in vivo against established STS tumors in fully immunocompetent Syrian hamsters, the combination was highly effective. CGTG-102 and doxorubicin (without ifosfamide) resulted in synergistic antitumor efficacy against human STS xenografts in comparison with single agent treatments. Doxorubicin increased adenoviral replication in human and hamster STS cells, potentially contributing to the observed therapeutic synergy. In conclusion, the preclinical data generated here support clinical translation of the combination of CGTG-102 and doxorubicin, or doxorubicin plus ifosfamide, for the treatment of STS, and provide clues on the mechanisms of synergy.

  9. ARM User Survey Report

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, LR

    2010-06-22

    The objective of this survey was to obtain user feedback to, among other things, determine how to organize the exponentially growing data within the Atmospheric Radiation Measurement (ARM) Climate Research Facility, and identify users’ preferred data analysis system. The survey findings appear to have met this objective, having received approximately 300 responses that give insight into the type of work users perform, usage of the data, percentage of data analysis users might perform on an ARM-hosted computing resource, downloading volume level where users begin having reservations, opinion about usage if given more powerful computing resources (including ability to manipulate data), types of tools that would be most beneficial to them, preferred programming language and data analysis system, level of importance for certain types of capabilities, and finally, level of interest in participating in a code-sharing community.

  10. Robot arm apparatus

    Science.gov (United States)

    Nachbar, Henry D.

    1992-01-01

    A robot arm apparatus is provided for inspecting and/or maintaining an interior of a steam generator which has an outside wall and a port for accessing the interior of the steam generator. The robot arm apparatus includes a flexible movable conduit for conveying inspection and/or maintenance apparatus from outside the steam generator to the interior of the steam generator. The flexible conduit has a terminal working end which is translated into and around the interior of the steam generator. Three motors located outside the steam generator are employed for moving the terminal working end inside the steam generator in "x", "y", and "z" directions, respectively. Commonly conducted inspection and maintenance operations include visual inspection for damaged areas, water jet lancing for cleaning sludge deposits, core boring for obtaining sludge deposits, and scrubbing of internal parts.

  11. Kiikuv maja / Anu Arm

    Index Scriptorium Estoniae

    Arm, Anu

    2006-01-01

    Eesti Kunstiakadeemia esimese kursuse arhitektuuriüliõpilaste II semestri töö. Juhendaja arhitekt Andres Alver, ehitamise Pedaspeale organiseeris suvepraktika juhendaja arhitekt Jaan Tiidemann. Autor Anu Arm, kaasa töötasid ja valmis ehitasid: Ott Alver, Maarja Elm, Mari Hunt, Alvin Järving, Marten Kaevats, Riho Kerge, Reedik Poopuu, Anu Põime, Helen Rebane, Kaisa Saarva, Martin Tago, Reet Volt. Valmis: 19. VIII 2006

  12. Early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children.

    Science.gov (United States)

    Legrand, F; Berrebi, D; Houhou, N; Freymuth, F; Faye, A; Duval, M; Mougenot, J F; Peuchmaur, M; Vilmer, E

    2001-03-01

    Adenovirus infection remains an important cause of mortality after bone marrow transplantation (BMT). Currently no efficient antiviral treatment is known. Thus, testing new modalities of early diagnosis and treatment is a crucial objective. Adenovirus infection is defined by the combination of symptoms and the isolation of virus from the source of clinical symptoms. The involvement of two or more organs and the presence of virus in blood cultures define disseminated disease. Seven children with a median age of 7 years received bone marrow transplantation for leukemia. All received an unrelated graft without T cell depletion. Adenovirus was sought in blood, urine and biopsy specimens using PCR and culture. Analysis of biopsy specimens included systematic immunohistochemistry. Cidofovir treatment was initiated as soon as biopsy revealed the histopathological signs of adenovirus. Cidofovir was given at 5 mg/kg once weekly for 3 weeks then every 2 weeks. Six patients had diarrhoea and one patient had cystitis. Adenovirus infection and disseminated disease were diagnosed in four cases and three cases, respectively. In six cases, serotype A31 was isolated from gastrointestinal biopsy and in two cases serotypes B2 and C6 were detected in blood and urine. Cidofovir treatment was associated with clinical improvement of diarrhoea, cystitis and fever in five patients, in whom the virus became undetectable in cultures and PCR analyses despite the persistence of immunodeficiency. The median follow-up was 360 days after BMT (240-570). One child died of invasive aspergillosis and another of disseminated adenovirus after interruption of cidofovir therapy. Further studies in immunocompromised patients will be needed to extend these promising results concerning the role of cidofovir in adenovirus infection.

  13. 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

  14. 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.

  15. Compaction and transport properties of newly replicated Caulobacter crescentus DNA.

    Science.gov (United States)

    Hong, Sun-Hae; McAdams, Harley H

    2011-12-01

    Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ΔHU1ΔHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.

  16. Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

    OpenAIRE

    Wellehan, James F. X.; Johnson, April J.; Harrach, Balázs; Benkö, Mária; Pessier, Allan P.; Johnson, Calvin M.; Garner, Michael M.; Childress, April; Jacobson, Elliott R.

    2004-01-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phyloge...

  17. Genomic and Bioinformatics Analysis of HAdV-4, a Human Adenovirus Causing Acute Respiratory Disease: Implications for Gene Therapy and Vaccine Vector Development

    OpenAIRE

    Purkayastha, Anjan; Ditty, Susan E.; Su, Jing; McGraw, John; Hadfield, Ted L.; Tibbetts, Clark; Seto, Donald

    2005-01-01

    Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and...

  18. Replication Timing of Human Telomeres is Conserved during Immortalization and Influenced by Respective Subtelomeres.

    Science.gov (United States)

    Piqueret-Stephan, Laure; Ricoul, Michelle; Hempel, William M; Sabatier, Laure

    2016-01-01

    Telomeres are specific structures that protect chromosome ends and act as a biological clock, preventing normal cells from replicating indefinitely. Mammalian telomeres are replicated throughout S-phase in a predetermined order. However, the mechanism of this regulation is still unknown. We wished to investigate this phenomenon under physiological conditions in a changing environment, such as the immortalization process to better understand the mechanism for its control. We thus examined the timing of human telomere replication in normal and SV40 immortalized cells, which are cytogenetically very similar to cancer cells. We found that the timing of telomere replication was globally conserved under different conditions during the immortalization process. The timing of telomere replication was conserved despite changes in telomere length due to endogenous telomerase reactivation, in duplicated homologous chromosomes, and in rearranged chromosomes. Importantly, translocated telomeres, possessing their initial subtelomere, retained the replication timing of their homolog, independently of the proportion of the translocated arm, even when the remaining flanking DNA is restricted to its subtelomere, the closest chromosome-specific sequences (inferior to 500 kb). Our observations support the notion that subtelomere regions strongly influence the replication timing of the associated telomere. PMID:27587191

  19. Comparative study of adenoviruses with monoclonal antibodies Estudo comparativo de diferentes tipos de adenovirus através de anticorpos monoclonais

    Directory of Open Access Journals (Sweden)

    Terezinha Maria de Paiva

    1992-02-01

    Full Text Available The obtainment of monoclonal antibodies for adenovirus species 4(Ad4 is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA, in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C, 4 (subgenus E, 7 and 16 (subgenus B and 9 (subgenus D. Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII were obtained.O estudo relata a obtenção de anticorpos monoclonais para o adenovirus tipo (espécie 4. A especificidade dos anticorpos monoclonais, selecionados nesse experimento, foi determinada testando-os frente às diferentes espécies de adenovírus: 1, 2, 5 (subgênero C, 4 (subgênero E, 7 e 16 (subgênero B e 9 (subgênero D, pelas técnicas de neutralização em cultura de células, imunofluorescência e ensaio imunoenzimático (ELISA. Os resultados demonstram a obtenção de anticorpos monoclonais que reagiram de maneira específica para o adenovírus 4 (1CIII e 3DIII e anticorpo monoclonal apresentando reação cruzada com as espécies de adenovírus 4 e 7 (2HIII.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Expression of adenovirus-mediated neurotrophin-3 gene in Schwann cells of sciatic nerve in rats

    Institute of Scientific and Technical Information of China (English)

    朱锦宇; 黄耀添; 朱庆生; 吕荣

    2003-01-01

    Objective: To investigate the expression of neurotrophin-3 (NT-3) gene in Schwann cells of rat sciatic nerve introduced by an adenovirus vector in vivo. Methods: A recombinant adenovirus vector for NT-3 (Ad-NT-3) was propagated in 293 packaging cells and titered with tissue culture infectious dose50 (TCID50). Ad-NT-3 was injected directly into the rat sciatic nerve after transection and immediate repair. Immunohistochemical staining was employed to determine the expression of NT-3 in Schwann cells in rat sciatic nerve and the expressive intensity of the tissue slices of the sciatic nerve was measured with LEICA M550 image analysis system. Results: On the 2nd day after injection of Ad-NT-3, positive stain in the Schwann cells was apparent in the vicinity of anastomosis. NT-3 expression increased significantly on the 7th day (P0.05). Compared with the 2nd day group, the 14th and 28th day groups still maintained a relatively high level of NT-3 (P<0.01). Intact and repaired nerves, which were injected with adenovirus encoding LacZ genes (Ad-LacZ) or physiological saline served as controls, showed no NT-3-positive Schwann cells. Conclusions: An adenovirus vector can be used to induce efficiently the expression of NT-3 gene in Schwann cells of rat peripheral nerves following nerve injury and repair, which suggests that neurotrophic factors can be introduced into Schwann cells with an adenovirus vector to promote peripheral nerve regeneration.

  2. Technical aspects of using human adenovirus as a viral water quality indicator.

    Science.gov (United States)

    Rames, Emily; Roiko, Anne; Stratton, Helen; Macdonald, Joanne

    2016-06-01

    Despite dramatic improvements in water treatment technologies in developed countries, waterborne viruses are still associated with many of cases of illness each year. These illnesses include gastroenteritis, meningitis, encephalitis, and respiratory infections. Importantly, outbreaks of viral disease from waters deemed compliant from bacterial indicator testing still occur, which highlights the need to monitor the virological quality of water. Human adenoviruses are often used as a viral indicator of water quality (faecal contamination), as this pathogen has high UV-resistance and is prevalent in untreated domestic wastewater all year round, unlike enteroviruses and noroviruses that are often only detected in certain seasons. Standard methods for recovering and measuring adenovirus numbers in water are lacking, and there are many variations in published methods. Since viral numbers are likely under-estimated when optimal methods are not used, a comprehensive review of these methods is both timely and important. This review critically evaluates how estimates of adenovirus numbers in water are impacted by technical manipulations, such as during adenovirus concentration and detection (including culturing and polymerase-chain reaction). An understanding of the implications of these issues is fundamental to obtaining reliable estimation of adenovirus numbers in water. Reliable estimation of HAdV numbers is critical to enable improved monitoring of the efficacy of water treatment processes, accurate quantitative microbial risk assessment, and to ensure microbiological safety of water. PMID:27065054

  3. Nitrogen Gas Plasma Generated by a Static Induction Thyristor as a Pulsed Power Supply Inactivates Adenovirus.

    Science.gov (United States)

    Sakudo, Akikazu; Toyokawa, Yoichi; Imanishi, Yuichiro

    2016-01-01

    Adenovirus is one of the most important causative agents of iatrogenic infections derived from contaminated medical devices or finger contact. In this study, we investigated whether nitrogen gas plasma, generated by applying a short high-voltage pulse to nitrogen using a static induction thyristor power supply (1.5 kilo pulse per second), exhibited a virucidal effect against adenoviruses. Viral titer was reduced by one log within 0.94 min. Results from detection of viral capsid proteins, hexon and penton, by Western blotting and immunochromatography were unaffected by the plasma treatment. In contrast, analysis using the polymerase chain reaction suggested that plasma treatment damages the viral genomic DNA. Reactive chemical products (hydrogen peroxide, nitrate, and nitrite), ultraviolet light (UV-A) and slight temperature elevations were observed during the operation of the gas plasma device. Viral titer versus intensity of each potential virucidal factor were used to identify the primary mechanism of disinfection of adenovirus. Although exposure to equivalent levels of UV-A or heat treatment did not inactivate adenovirus, treatment with a relatively low concentration of hydrogen peroxide efficiently inactivated the virus. Our results suggest the nitrogen gas plasma generates reactive chemical products that inactivate adenovirus by damaging the viral genomic DNA.

  4. Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation.

    Science.gov (United States)

    Yang, Xiaoshan; Xu, Rong; Lin, Yajun; Zhen, Yongzhan; Wei, Jie; Hu, Gang; Sun, Hongfan

    2016-01-01

    The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes, and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene, and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy. PMID:27530145

  5. Nitrogen Gas Plasma Generated by a Static Induction Thyristor as a Pulsed Power Supply Inactivates Adenovirus.

    Science.gov (United States)

    Sakudo, Akikazu; Toyokawa, Yoichi; Imanishi, Yuichiro

    2016-01-01

    Adenovirus is one of the most important causative agents of iatrogenic infections derived from contaminated medical devices or finger contact. In this study, we investigated whether nitrogen gas plasma, generated by applying a short high-voltage pulse to nitrogen using a static induction thyristor power supply (1.5 kilo pulse per second), exhibited a virucidal effect against adenoviruses. Viral titer was reduced by one log within 0.94 min. Results from detection of viral capsid proteins, hexon and penton, by Western blotting and immunochromatography were unaffected by the plasma treatment. In contrast, analysis using the polymerase chain reaction suggested that plasma treatment damages the viral genomic DNA. Reactive chemical products (hydrogen peroxide, nitrate, and nitrite), ultraviolet light (UV-A) and slight temperature elevations were observed during the operation of the gas plasma device. Viral titer versus intensity of each potential virucidal factor were used to identify the primary mechanism of disinfection of adenovirus. Although exposure to equivalent levels of UV-A or heat treatment did not inactivate adenovirus, treatment with a relatively low concentration of hydrogen peroxide efficiently inactivated the virus. Our results suggest the nitrogen gas plasma generates reactive chemical products that inactivate adenovirus by damaging the viral genomic DNA. PMID:27322066

  6. 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.

  7. Hyperthermia stimulates HIV-1 replication.

    OpenAIRE

    Ferdinand Roesch; Oussama Meziane; Anna Kula; Sébastien Nisole; Françoise Porrot; Ian Anderson; Fabrizio Mammano; Ariberto Fassati; Alessandro Marcello; Monsef Benkirane; Olivier Schwartz

    2012-01-01

    International audience HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C) on HIV-1 infection has not been extensively inve...

  8. Dynamic replication of Web contents

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The phenomenal growth of the World Wide Web has brought huge increase in the traffic to the popular web sites.Long delays and denial of service experienced by the end-users,especially during the peak hours,continues to be the common problem while accessing popular sites.Replicating some of the objects at multiple sites in a distributed web-server environment is one of the possible solutions to improve the response time/Iatency. The decision of what and where to replicate requires solving a constraint optimization problem,which is NP-complete in general.In this paper, we consider the problem of placing copies of objects in a distributed web server system to minimize the cost of serving read and write requests when the web servers have Iimited storage capacity.We formulate the problem as a 0-1 optimization problem and present a polynomial time greedy algorithm with backtracking to dynamically replicate objects at the appropriate sites to minimize a cost function.To reduce the solution search space,we present necessary condi tions for a site to have a replica of an object jn order to minimize the cost function We present simulation resuIts for a variety of problems to illustrate the accuracy and efficiency of the proposed algorithms and compare them with those of some well-known algorithms.The simulation resuIts demonstrate the superiority of the proposed algorithms.

  9. 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 (Ab); 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

  10. 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

  11. The photobinding of 5,7-dimethoxycoumarin to adenovirus type-2 DNA. A method for in vitro mutagenesis

    International Nuclear Information System (INIS)

    The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNAsub(p): 5,7-dimethyoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na+. The maximum photobinding of 5,7-[3H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited 'library' of mutations in each of the five early gene regions of the adenovirus genome. (Auth.)

  12. Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

    OpenAIRE

    Gao, Dan-Dan; Fu, Jia; Qin, Bo; Huang, Wen-Xiang; Yang, Chun; Jia, Bei

    2016-01-01

    AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6 (Hyper-IL-6, HIL-6) and hepatocyte growth factor (HGF) (Ad-HGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF (Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure (ACLF).

  13. 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

  14. 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. PMID:25163614

  15. 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.

  16. 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.

  17. Magnesium-Dependent Interaction of PKR with Adenovirus VAI

    Energy Technology Data Exchange (ETDEWEB)

    K Launer -Felty; C Wong; A Wahid; G Conn; J Cole

    2011-12-31

    Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the {alpha}-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade the antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg{sup 2+}. Two PKR monomers bind in the absence of Mg{sup 2+}, but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

  18. Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

    Directory of Open Access Journals (Sweden)

    Anil Babu Korrapati

    Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

  19. Evaluating replicability of laboratory experiments in economics.

    Science.gov (United States)

    Camerer, Colin F; Dreber, Anna; Forsell, Eskil; Ho, Teck-Hua; Huber, Jürgen; Johannesson, Magnus; Kirchler, Michael; Almenberg, Johan; Altmejd, Adam; Chan, Taizan; Heikensten, Emma; Holzmeister, Felix; Imai, Taisuke; Isaksson, Siri; Nave, Gideon; Pfeiffer, Thomas; Razen, Michael; Wu, Hang

    2016-03-25

    The replicability of some scientific findings has recently been called into question. To contribute data about replicability in economics, we replicated 18 studies published in the American Economic Review and the Quarterly Journal of Economics between 2011 and 2014. All of these replications followed predefined analysis plans that were made publicly available beforehand, and they all have a statistical power of at least 90% to detect the original effect size at the 5% significance level. We found a significant effect in the same direction as in the original study for 11 replications (61%); on average, the replicated effect size is 66% of the original. The replicability rate varies between 67% and 78% for four additional replicability indicators, including a prediction market measure of peer beliefs. PMID:26940865

  20. Replication of micro and nano surface geometries

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Hocken, R.J.; Tosello, Guido

    2011-01-01

    The paper describes the state-of-the-art in replication of surface texture and topography at micro and nano scale. The description includes replication of surfaces in polymers, metals and glass. Three different main technological areas enabled by surface replication processes are presented......: manufacture of net-shape micro/nano surfaces, tooling (i.e. master making), and surface quality control (metrology, inspection). Replication processes and methods as well as the metrology of surfaces to determine the degree of replication are presented and classified. Examples from various application areas...... are given including replication for surface texture measurements, surface roughness standards, manufacture of micro and nano structured functional surfaces, replicated surfaces for optical applications (e.g. optical gratings), and process chains based on combinations of repeated surface replication steps....

  1. In search of the holy replicator

    OpenAIRE

    Gilbert, David M.

    2004-01-01

    After 40 years of searching for the eukaryotic replicator sequence, it is time to abandon the concept of ‘the’ replicator as a single genetic entity. Here I propose a ‘relaxed replicon model’ in which a positive initiator–replicator interaction is facilitated by a combination of several complex features of chromatin. An important question for the future is whether the positions of replication origins are simply a passive result of local chromatin structure or are actively localized to coordin...

  2. Mutator phenotypes due to DNA replication infidelity

    OpenAIRE

    Arana, Mercedes E.; Kunkel, Thomas A.

    2010-01-01

    This article considers the fidelity of DNA replication performed by eukaryotic DNA polymerases involved in replicating the nuclear genome. DNA replication fidelity can vary widely depending on the DNA polymerase, the composition of the error, the flanking sequence, the presence of DNA damage and the ability to correct errors. As a consequence, defects in processes that determine DNA replication fidelity can confer strong mutator phenotypes whose specificity can help determine the molecular na...

  3. Possible applications for replicating HIV 1 vectors

    OpenAIRE

    Das, Atze T.; Jeeninga, Rienk E.; Berkhout, Ben

    2010-01-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV base...

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

    International Nuclear Information System (INIS)

    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

  5. 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.

  6. Crystal Structure of Human Adenovirus at 3.5 Å Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Vijay S.; Natchiar, S. Kundhavai; Stewart, Phoebe L.; Nemerow, Glen R. (Scripps); (Vanderbilt)

    2010-09-27

    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 angstrom 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 protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a substantial advance toward understanding the assembly and cell entry mechanisms of a large double-stranded DNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.

  7. 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.

  8. Genetic and Antigenic Analysis of Adenovirus Type 3 Strains Showing Intermediate Behavior in Standard Seroneutralization Test

    Directory of Open Access Journals (Sweden)

    Márcia TB Moraes

    1998-03-01

    Full Text Available During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites

  9. DNA Replication Reaches the Breaking Point

    OpenAIRE

    Petrini, John H.J.

    2009-01-01

    DNA strand breaks that result in stalled or damaged replication forks can be detrimental to the DNA replication process. In this issue, Doksani et al. (2009) examine the impact of a single double-stranded DNA break on replication in the budding yeast, Saccharomyces cerevisiae.

  10. Exploiting replicative stress to treat cancer

    DEFF Research Database (Denmark)

    Dobbelstein, Matthias; Sørensen, Claus Storgaard

    2015-01-01

    DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...

  11. Efficient construction of recombinant adenovirus expression vector of the Qinchuan cattle LYRM1 gene.

    Science.gov (United States)

    Li, Y K; Fu, C Z; Zhang, Y R; Zan, L S

    2015-01-01

    In this study, we cloned the coding DNA sequence (CDS) region of Qinchuan cattle LYR motif-containing 1 (LYRM1) and constructed a recombinant adenovirus expression vector to examine the function of LYRM1 on the cellular level. Total RNA was extracted from the adipose tissue of Qinchuan cattle, cDNA was obtained by reverse transcription, and polymerase chain reaction was used to amplify the CDS region of the LYRM1 gene. The CDS-containing fragment was inserted into the shuttle vector pAdTrack-CMV to construct pAdTrack-CMV-LYRM1 vector. After linearization of pAdTrack-CMV-LYRM1 and the negative control vector pAdTrack-CMV by restriction endonuclease PmeI, the vectors were transformed into Escherichia coli BJ5183 containing pAdEasy-1 to obtain the recombinant adenovirus vector pAd-LYRM1 and pAd-CMV through homologous recombination. pAd-LYRM1 and pAd-CMV were then digested by PacI and transfected into the 293A cell line. The recombinant adenovirus Ad-LYRM1 and Ad-CMV was obtained at a concentration of 7 x 108 and 1.3 x 109 green fluorescent units/mL, respectively. Preadipocytes derived from Qinchuan cattle were separately infected with Ad-LYRM1 and Ad- CMV. Quantitative real-time polymerase chain reaction demonstrated that the expression of LYRM1 was increased by approximate 28,000-folds after the infection with recombinant adenovirus for 48 h. In conclusion, we successfully cloned the CDS region of the Qinchuan cattle LYRM1 gene, constructed the recombinant adenovirus expression vector, and obtained the adenovirus with high titer, providing valuable materials for studying the function of LYRM1 at the cellular level. PMID:26345880

  12. 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.

  13. Detection and Analysis of Six Lizard Adenoviruses by Consensus Primer PCR Provides Further Evidence of a Reptilian Origin for the Atadenoviruses

    Science.gov (United States)

    Wellehan, James F. X.; Johnson, April J.; Harrach, Balázs; Benkö, Mária; Pessier, Allan P.; Johnson, Calvin M.; Garner, Michael M.; Childress, April; Jacobson, Elliott R.

    2004-01-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses. PMID:15542689

  14. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses.

    Science.gov (United States)

    Wellehan, James F X; Johnson, April J; Harrach, Balázs; Benkö, Mária; Pessier, Allan P; Johnson, Calvin M; Garner, Michael M; Childress, April; Jacobson, Elliott R

    2004-12-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses. PMID:15542689

  15. 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.

  16. Lipid Membranes in Poxvirus Replication

    Directory of Open Access Journals (Sweden)

    Jason P. Laliberte

    2010-04-01

    Full Text Available Poxviruses replicate in the cytoplasm, where they acquire multiple lipoprotein membranes. Although a proposal that the initial membrane arises de novo has not been substantiated, there is no accepted explanation for its formation from cellular membranes. A subsequent membrane-wrapping step involving modified trans-Golgi or endosomal cisternae results in a particle with three membranes. These wrapped virions traverse the cytoplasm on microtubules; the outermost membrane is lost during exocytosis, the middle one is lost just prior to cell entry, and the remaining membrane fuses with the cell to allow the virus core to enter the cytoplasm and initiate a new infection.

  17. LISA Long-Arm Interferometry

    Science.gov (United States)

    Thorpe, James I.

    2009-01-01

    An overview of LISA Long-Arm Interferometry is presented. The contents include: 1) LISA Interferometry; 2) Constellation Design; 3) Telescope Design; 4) Constellation Acquisition; 5) Mechanisms; 6) Optical Bench Design; 7) Phase Measurement Subsystem; 8) Phasemeter Demonstration; 9) Time Delay Interferometry; 10) TDI Limitations; 11) Active Frequency Stabilization; 12) Spacecraft Level Stabilization; 13) Arm-Locking; and 14) Embarassment of Riches.

  18. 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

  19. A novel recombinant Peste des petits ruminants-canine adenovirus vaccine elicits long-lasting neutralizing antibody response against PPR in goats.

    Directory of Open Access Journals (Sweden)

    Junling Qin

    Full Text Available BACKGROUND: Peste des petits ruminants (PPR is a highly contagious infectious disease of goats, sheep and small wild ruminant species with high morbidity and mortality rates. The Peste des petits ruminants virus (PPRV expresses a hemagglutinin (H glycoprotein on its outer envelope that is crucial for viral attachment to host cells and represents a key antigen for inducing the host immune response. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether H can be exploited to generate an effective PPRV vaccine, a replication-competent recombinant canine adenovirus type-2 (CAV-2 expressing the H gene of PPRV (China/Tibet strain was constructed by the in vitro ligation method. The H expression cassette, including the human cytomegalovirus (hCMV promoter/enhancer and the BGH early mRNA polyadenylation signal, was inserted into the SspI site of the E3 region, which is not essential for proliferation of CAV-2. Infectious recombinant rCAV-2-PPRV-H virus was generated in transfected MDCK cells and used to immunize goats. All vaccinated animals produced antibodies upon primary injection that were effective in neutralizing PPRV in vitro. Higher antibody titer was obtained following booster inoculation, and the antibody was detectable in goats for at least seven months. No serious recombinant virus-related adverse effect was observed in immunized animals and no adenovirus could be isolated from the urine or feces of vaccinated animals. Results showed that the recombinant virus was safe and could stimulate a long-lasting immune response in goats. CONCLUSIONS/SIGNIFICANCE: This strategy not only provides an effective PPR vaccine candidate for goats but may be a valuable mean by which to differentiate infected from vaccinated animals (the so-called DIVA approach.

  20. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells: not as bad as their reputation.

    Science.gov (United States)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang; Jensen, Benjamin Anderschou Holbech; Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2013-06-01

    It has been reported that adenovirus (Ad)-primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells by directly comparing these cells to CD8 T cells induced through infection with lymphocytic choriomeningitis virus (LCMV). We found that localized immunization with intermediate doses of Ad vector induces a moderate number of functional CD8 T cells which qualitatively match those found in LCMV-infected mice. The numbers of these cells may be efficiently increased by additional adenoviral boosting, and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad vaccination led to even higher numbers of memory cells. In this case, the vaccination led to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression. These memory CD8 T cells were capable of proliferating in response to viral challenge and protecting against infection with live virus. Furthermore, viral challenge was followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells did not appear to have been driven toward exhaustive differentiation. Based on these findings, we suggest that adenovirus-based prime-boost regimens (including Ad serotype 5 [Ad5] and Ad5-like vectors) represent an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells.

  1. An evolutionary conserved early replicating segment on the sex chromosomes of man and the great apes.

    Science.gov (United States)

    Weber, B; Schempp, W; Wiesner, H

    1986-01-01

    Replication studies on prometaphase chromosomes of man, the chimpanzee, the pygmy chimpanzee, the gorilla, and the orangutan reveal great interspecific homologies between the autosomes. The early replicating X chromosomes clearly show a high degree of conservation of both the pattern and the time course of replication. An early replicating segment on the short arm of the X chromosomes of man (Xp22.3) which escapes inactivation can be found on the X chromosomes of the great apes as well. Furthermore, the most early replicating segment on the Y chromosomes of all species tested appears to be homologous to this segment on the X chromosomes. Therefore, these early replicating segments in the great apes may correspond to the pseudoautosomal segment proposed to exist in man. From further cytogenetic characterization of the Y chromosomes it is evident that structural alterations have resulted in an extreme divergence in both the euchromatic and heterochromatic parts. It is assumed, therefore, that, in contrast to the X chromosomes, the Y chromosomes have undergone a rapid evolution within the higher primates. PMID:3096642

  2. Robotic Arm Biobarrier Cable

    Science.gov (United States)

    2008-01-01

    This image, taken by the Surface Stereo Imager on NASA's Phoenix Mars Lander on the 14th Martian day of the mission (June 7, 2008), shows the cable that held the Robotic Arm's biobarrier in place during flight has snapped. The cable's springs retracted to release the biobarrier right after landing. To the lower right of the image a spring is visible. Extending from that spring is a length of cable that snapped during the biobarrier's release. A second spring separated from the cable when it snapped and has been photographed on the ground under the lander near one of the legs. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  3. Self-replication of DNA rings

    Science.gov (United States)

    Kim, Junghoon; Lee, Junwye; Hamada, Shogo; Murata, Satoshi; Ha Park, Sung

    2015-06-01

    Biology provides numerous examples of self-replicating machines, but artificially engineering such complex systems remains a formidable challenge. In particular, although simple artificial self-replicating systems including wooden blocks, magnetic systems, modular robots and synthetic molecular systems have been devised, such kinematic self-replicators are rare compared with examples of theoretical cellular self-replication. One of the principal reasons for this is the amount of complexity that arises when you try to incorporate self-replication into a physical medium. In this regard, DNA is a prime candidate material for constructing self-replicating systems due to its ability to self-assemble through molecular recognition. Here, we show that DNA T-motifs, which self-assemble into ring structures, can be designed to self-replicate through toehold-mediated strand displacement reactions. The inherent design of these rings allows the population dynamics of the systems to be controlled. We also analyse the replication scheme within a universal framework of self-replication and derive a quantitative metric of the self-replicability of the rings.

  4. Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses.

    Science.gov (United States)

    Mück-Häusl, Martin; Solanki, Manish; Zhang, Wenli; Ruzsics, Zsolt; Ehrhardt, Anja

    2015-04-30

    Recombinant adenoviruses containing a double-stranded DNA genome of 26-45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies. PMID:25609697

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

    OpenAIRE

    Jatin M Vyas; Marasco, Wayne A.

    2012-01-01

    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.

  6. 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

  7. 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...

  8. 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

  9. [The complexes of adenovirus and anionic liposomes: preparation and in vitro characterization].

    Science.gov (United States)

    Zhong, Zhi-Rong; Wan, Yu; Shi, San-Jun; Zhang, Zhi-Rong; Sun, Xun

    2012-01-01

    This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5. PMID:22493816

  10. 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)...

  11. 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.

  12. 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

  13. 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.

  14. 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)

  15. 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.

  16. 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

  17. 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

  18. DNA Replication via Entanglement Swapping

    CERN Document Server

    Pusuluk, Onur

    2010-01-01

    Quantum effects are mainly used for the determination of molecular shapes in molecular biology, but quantum information theory may be a more useful tool to understand the physics of life. Molecular biology assumes that function is explained by structure, the complementary geometries of molecules and weak intermolecular hydrogen bonds. However, both this assumption and its converse are possible if organic molecules and quantum circuits/protocols are considered as hardware and software of living systems that are co-optimized during evolution. In this paper, we try to model DNA replication as a multiparticle entanglement swapping with a reliable qubit representation of nucleotides. In the model, molecular recognition of a nucleotide triggers an intrabase entanglement corresponding to a superposition state of different tautomer forms. Then, base pairing occurs by swapping intrabase entanglements with interbase entanglements.

  19. 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.

  20. 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.

  1. Therapeutic targeting of replicative immortality.

    Science.gov (United States)

    Yaswen, Paul; MacKenzie, Karen L; Keith, W Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I; Azmi, Asfar S; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-12-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

  2. ARM Lead Mentor Selection Process

    Energy Technology Data Exchange (ETDEWEB)

    Sisterson, DL

    2013-03-13

    The ARM Climate Research Facility currently operates more than 300 instrument systems that provide ground-based observations of the atmospheric column. To keep ARM at the forefront of climate observations, the ARM infrastructure depends heavily on instrument scientists and engineers, also known as Instrument Mentors. Instrument Mentors must have an excellent understanding of in situ and remote-sensing instrumentation theory and operation and have comprehensive knowledge of critical scale-dependent atmospheric processes. They also possess the technical and analytical skills to develop new data retrievals that provide innovative approaches for creating research-quality data sets.

  3. 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

  4. 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.

  5. Arménie

    Directory of Open Access Journals (Sweden)

    François Verdier

    2012-04-01

    Full Text Available L’Arménie est une petite république du Caucase, à la limite sud–est de l’Europe, qui a gagné son autonomie en 1990 après l’ouverture du bloc soviétique. Le nouveau Ministère du Patrimoine a sollicité la coopération de la France pour mettre en place une nouvelle politique culturelle. Tout d’abord, une évaluation sur place de la situation dans les domaines des monuments historiques, de l’archéologie et de l’Inventaire a permis d’envisager les réponses à proposer. Pour la demande d’informatisation des dossiers d’inventaire déjà réalisés sous l’autorité de l’Académie de Saint–Petersbourg, nous avons proposé de former des chercheurs arméniens aux méthodes et techniques de l’Inventaire général. L’accueil d’une stagiaire pendant trois mois au service régional de l’Inventaire de Haute–Normandie a été suivi par la mise en place d’un équipement informatique à Yérévan, puis par l’accueil et la formation de techniciens informaticiens et photographes arméniens. De retour dans leur pays ils ont commencé à remettre en place un service d’inventaire dont le programme comprend la création d’une base de données patrimoniales, le recensement de la ville de Yérévan, la numérisation d’images pour la publication d’un indicateur du patrimoine et la préparation de dossiers de protection au titre du patrimoine mondial.The Armenian heritage comprises both archaeological remains of towns destroyed by never–ending wars and a number of old churches from the eleventh and twelfth centuries. Yerevan, the capital of Armenia, was founded three thousand years ago and is one of Europe’s oldest capitals. From 1925 it has developed according to an ambitious urban planning project. After the major political upheavals of 1991, a special ministry was created to look after the architectural and movable heritage of the country and to promote the Armenian national identity. A mission in Yerevan was

  6. 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

  7. Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

    Institute of Scientific and Technical Information of China (English)

    Dongxu Feng; Haopeng Li; Siyue Xu; Yu Liu; Xiaofei Hou

    2011-01-01

    Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA (shRNA) for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments (shRNA) were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector (carrying shRNA) and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells (PC12). Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49±0.68%, 95.08±1.79% and 96.60±1.14% at the mRNA level, and 72.89±2.17%, 58.83±1.15% and 59.88±0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

  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. 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.

  10. Federated SPARQL Queries Processing with Replicated Fragments

    OpenAIRE

    Montoya, Gabriela; Skaf-Molli, Hala; Molli, Pascal; Vidal, Maria-Esther

    2015-01-01

    Federated query engines allow to consume linked data from SPARQL endpoints. Replicating data fragments from different sources allows to reorganize data to better fit federated query processing of data consumers. However, existing federated query engines poorly support replication. In this paper, we propose a replication-aware federated query engine that extends state-of-art federated query engine ANAPSID and FedX with Fedra, a source selection strategy that approximates the source selection p...

  11. Replicating organizational knowledge: principles or templates?

    OpenAIRE

    Baden-Fuller, Charles; Winter, Sidney G.

    2005-01-01

    We discuss how firms can replicate practices and knowledge embedded in practices by following principles, with no direct reference to an extant working example (template). Definitions are provided for the key concepts of templates, principles, and background knowledge. We address the challenges of providing operational measures for successful replication, and for comparing the efficacy of principles and templates. By using two longitudinal case studies of replication across the units of two m...

  12. Do transcriptional enhancers also augment DNA replication?

    OpenAIRE

    O'Connor, D T; Subramani, S

    1988-01-01

    Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (S...

  13. Controlled rereplication at DNA replication origins

    OpenAIRE

    Gómez, María

    2008-01-01

    No-more-than-once per cell cycle initiation of DNA replication is a golden rule to maintain genome stability and guarantee cell survival. In our recent work we discovered that small genome fragments of about 200 bp are repeatedly synthesised at human DNA replication origins at the time of origin firing during S phase in normal cells. Rereplicated DNA fragments coincide physical and temporarily with replication origin activity, implying that their generation is intimately associated with the i...

  14. Replication licensing and the DNA damage checkpoint

    OpenAIRE

    Cook, Jeanette Gowen

    2009-01-01

    Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings sug...

  15. Persistent HIV-1 replication during antiretroviral therapy

    OpenAIRE

    Martinez-Picado, Javier; Deeks, Steven G

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on ef...

  16. Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Vinay Kumar Srivastava; Dharani Dhar Dubey

    2007-08-01

    Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.

  17. Regulation of chromosomal replication in Caulobacter crescentus.

    Science.gov (United States)

    Collier, Justine

    2012-03-01

    The alpha-proteobacterium Caulobacter crescentus is characterized by its asymmetric cell division, which gives rise to a replicating stalked cell and a non-replicating swarmer cell. Thus, the initiation of chromosomal replication is tightly regulated, temporally and spatially, to ensure that it is coordinated with cell differentiation and cell cycle progression. Waves of DnaA and CtrA activities control when and where the initiation of DNA replication will take place in C. crescentus cells. The conserved DnaA protein initiates chromosomal replication by directly binding to sites within the chromosomal origin (Cori), ensuring that DNA replication starts once and only once per cell cycle. The CtrA response regulator represses the initiation of DNA replication in swarmer cells and in the swarmer compartment of pre-divisional cells, probably by competing with DnaA for binding to Cori. CtrA and DnaA are controlled by multiple redundant regulatory pathways that include DNA methylation-dependent transcriptional regulation, temporally regulated proteolysis and the targeting of regulators to specific locations within the cell. Besides being critical regulators of chromosomal replication, CtrA and DnaA are also master transcriptional regulators that control the expression of many genes, thus connecting DNA replication with other events of the C. crescentus cell cycle.

  18. Comparison of three replication strategies in complex multicellular organisms: Asexual replication, sexual replication with identical gametes, and sexual replication with distinct sperm and egg gametes

    Science.gov (United States)

    Tannenbaum, Emmanuel

    2008-01-01

    This paper studies the mutation-selection balance in three simplified replication models. The first model considers a population of organisms replicating via the production of asexual spores. The second model considers a sexually replicating population that produces identical gametes. The third model considers a sexually replicating population that produces distinct sperm and egg gametes. All models assume diploid organisms whose genomes consist of two chromosomes, each of which is taken to be functional if equal to some master sequence, and defective otherwise. In the asexual population, the asexual diploid spores develop directly into adult organisms. In the sexual populations, the haploid gametes enter a haploid pool, where they may fuse with other haploids. The resulting immature diploid organisms then proceed to develop into mature organisms. Based on an analysis of all three models, we find that, as organism size increases, a sexually replicating population can only outcompete an asexually replicating population if the adult organisms produce distinct sperm and egg gametes. A sexual replication strategy that is based on the production of large numbers of sperm cells to fertilize a small number of eggs is found to be necessary in order to maintain a sufficiently low cost for sex for the strategy to be selected for over a purely asexual strategy. We discuss the usefulness of this model in understanding the evolution and maintenance of sexual replication as the preferred replication strategy in complex, multicellular organisms.

  19. Cells and prions: a license to replicate.

    Science.gov (United States)

    Nuvolone, Mario; Aguzzi, Adriano; Heikenwalder, Mathias

    2009-08-20

    Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular prion protein (PrP(C)). The infectious agent - termed prion - is mainly composed of misfolded PrP(Sc). In addition to the central nervous system prions can colonize secondary lymphoid organs and inflammatory foci. Follicular dendritic cells are important extraneural sites of prion replication. However, recent data point to a broader range of cell types that can replicate prions. Here, we review the state of the art in regards to peripheral prion replication, neuroinvasion and the determinants of prion replication competence. PMID:19527722

  20. Ideas are Not Replicators but Minds Are

    OpenAIRE

    Gabora, Liane M

    2004-01-01

    An idea is not a replicator because it does not consist of coded self-assembly instructions. It may retain structure as it passes from one individual to another, but does not replicate it. The cultural replicator is not an idea but an associatively-structured network of them that together form an internal model of the world, or worldview. A worldview is a primitive, uncoded replicator, like the autocatalytic sets of polymers widely believed to be the earliest form of life. Primitive replicato...

  1. Arms control: misplaced focus

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, W.A.; Derber, C.

    1986-03-01

    Most of the nuclear debate consists of arguments about which weapons systems should be built, controlled, canceled, frozen, or retired. Short of virtually complete, multilateral nuclear disarmament, however, no change in the pace, balance, or even the direction of the arms race can make much difference in the risk of nuclear war, the damage should one occur, or the division of international political power. This includes Star Wars, the nuclear freeze, and even large cuts in or stabilization of offensive nuclear arsenals. A better starting point for nuclear politics would be the insight that nuclear weapons have completely changed the logic of power as it has been handed down through the ages. Military force, perfected to its highest level, has invalidated itself - for in a nuclearized world, any resort to force by a nuclear power risks escalation to its ultimate level, and thus to oblivion for all. Trying to rationalize and control the ultimate force is far less realistic and important than limiting the provocation of conflict and the use of force at lower, non-nuclear levels - by the United States, it clients, and, to the extent possible, its adversaries. 13 references.

  2. ARM Standards Policy Committee Report

    Energy Technology Data Exchange (ETDEWEB)

    Cialella, A; Jensen, M; Koontz, A; McFarlane, S; McCoy, R; Monroe, J; Palanisamy, G; Perez, R; Sivaraman, C

    2012-09-19

    Data and metadata standards promote the consistent recording of information and are necessary to ensure the stability and high quality of Atmospheric Radiation Measurement (ARM) Climate Research Facility data products for scientific users. Standards also enable automated routines to be developed to examine data, which leads to more efficient operations and assessment of data quality. Although ARM Infrastructure agrees on the utility of data and metadata standards, there is significant confusion over the existing standards and the process for allowing the release of new data products with exceptions to the standards. The ARM Standards Policy Committee was initiated in March 2012 to develop a set of policies and best practices for ARM data and metadata standards.

  3. [Apothecaries' arms of Voltaire's family].

    Science.gov (United States)

    Chaigneau, M

    1998-01-01

    Apothecaries of Voltaire's family can be divided into two groups. Marceton Family: Claude Marceton, Hierosme and Pierre Testefolle, from Thouars. Arouet family: Jehan Arouet, Pierre Rochard and Jean Gougeard. Five of those apothecaries beand arms. PMID:11625327

  4. Biodistribution and Toxicological Safety of Adenovirus Type 5 and Type 35 Vectored Vaccines Against Human Immunodeficiency Virus-1 (HIV-1), Ebola, or Marburg Are Similar Despite Differing Adenovirus Serotype Vector, Manufacturer's Construct, or Gene Inserts

    OpenAIRE

    Sheets, Rebecca L.; Stein, Judith; Bailer, Robert T.; Koup, Richard A.; Andrews, Charla; Nason, Martha; He, Bin; Koo, Edward; Trotter, Holly; Duffy, Chris; Manetz, T. Scott; Gomez, Phillip

    2008-01-01

    The Vaccine Research Center has developed vaccine candidates for different diseases/infectious agents (including HIV-1, Ebola, and Marburg viruses) built on an adenovirus vector platform, based on adenovirus type 5 or 35. To support clinical development of each vaccine candidate, pre-clinical studies were performed in rabbits to determine where in the body they biodistribute and how rapidly they clear, and to screen for potential toxicities (intrinsic and immunotoxicities). The vaccines biodi...

  5. Bistable Head Positioning Arm Latch

    Science.gov (United States)

    Wasson, Ken; Endo, Juro; Mita, Masahiro; Abelein, Nathan

    A simple, low cost, yet effective device has been developed for immobilizing the head-arm assembly in a disk drive or similar mechanism during power-off conditions. The latching scheme also provides a consistent means of releasing the head-arm assembly from the immobilized position upon power up of the disk drive. The latch uses no electrical power in either immobilized or released state. This design is immune to extreme torque and linear shock forces applied to the disk drive case. The latch system can use the energy stored in the spinning disks to drive the head-arm assembly toward a safe position while simultaneously arming the latch mechanism to secure the head-arm assembly in the safe position upon arrival. A low energy five msec pulse of current drives the latch from one state to the other. Solenoids as presently used in latch mechanisms are bulky, expensive, have variable force characteristics, and often generate contaminants. The latch described in this paper is expected to replace such solenoids. It may also replace small magnet latches, which have limited latch force and apply unwanted torque to a proximate head positioning arm.

  6. 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.

  7. 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...

  8. 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...

  9. Anti-tumor effect of adenovirus-mediated suicide gene therapy under control of tumor-specific and radio-inducible chimeric promoter in combination with γ-ray irradiation in vivo

    International Nuclear Information System (INIS)

    Objective: To detect the selective inhibitory effects of irradiation plus adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) suicide gene system using tumor-specific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse. Methods: Recombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radio-inducible CArG elements was constructed. A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA. The change of tumor volume and tumor growth inhibiting rate, the survival time of nude mice, as well as histopathology of xenograft tumor and normal tissues were evaluated. Results: Thirty one days after the treatment, the relative tumor volumes in the negative, adenovirus therapy, irradiation, and combination groups were 49.23±4.55, 27.71±7.74, 28.53±10.48 and 11.58±3.23, respectively.There was a significantly statistical difference among them (F=16.288, P<0.01).The inhibition effect in the combination group was strongest as compared with that in other groups, and its inhibition ratio was 76.5%. The survival period extended to 43 d in the combination group, which showed a significantly difference with that in the control group (χ2=18.307, P<0.01). The area of tumors necrosis in the combination group was larger than that in the other groups, and the normal tissues showed no treatment-related toxic effect in all groups. However, multiple hepatocellular carcinoma metastases were observed in the liver in the control group, there were a few metastases in the monotherapy groups and no metastasis in the combination group. Conclusions: Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong median

  10. In vivo study on the effect of adenovirus mediating Smad 7 gene expression regulated by radiation via Egr-1 promoter in C57BL mice implanted with lewis lung cancer

    International Nuclear Information System (INIS)

    Objective: Objective To study the effect of adenovirus mediating Smad 7 gene regulated by radiation via Egr-1 on the primary tumor and lung metastasis in C57BL mice implanted with Lewis lung cancer. Methods: The radio-inducible elements from the Egr-1 gene promoter were inserted upstream to a cDNA encoding Smad 7 and integrated into a replication-defective adenovirus to generate recombinant adenovirus (AD. Egr-Smad 7). 270 mice implanted with Lewis lung cancer in the hind legs were used and the experiment was started when the transplanted tumor diameter reached 0.8 to l.0 cm. Then three investigations were undertaken, each demanding 90 mice implanted with Lewis lung cancer respectively. To each group, 90 mice models were randomized into 3 groups: the normal control group; the NS control group; and the implanted AD. Egr-Smad 7 group. Every 6 mice in each group were irradiated by different single close to study the following: 1. The maximal and minimal diameters of the tumor were recorded to observe the tumor growth tendency, the tumor growth delay and the mice survival time, 2. The incidence of lung metastasis two weeks after the radiation was recorded. 3. The incidence of lung metastasis when the tumor volume was four times as large as that at the beginning of radiation was recorded. Results: The adenovirus mediating Smad 7 gene expression regulated by irradiation via Egr-1 in C57BL mice implanted with Lewis lung cancer was able to inhibit the progression of the primary tumor and prolong the survival of the mice significantly as compared with the control group (P 0.05). Conclusions: The gene expression of AD. Egr-Smad 7 regulated by radiation is not risky in promoting the local progression and distant metastasis of Lewis lung cancer in mice. On the other hand, the gene expression of AD. Egr-Smad 7 regulated by radiation could inhibit the progression of the primary tumor and prolong the survival time of the mice significantly. It is safe, to some extent, of using AD

  11. Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus.

    OpenAIRE

    Lubeck, M D; Davis, A R; Chengalvala, M; Natuk, R J; Morin, J E; Molnar-Kimber, K; Mason, B. B.; Bhat, B M; Mizutani, S; Hung, P P

    1989-01-01

    As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems world-wide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitis B infection. Although considerable progress has been made in the construction of recombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and ...

  12. 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.

  13. 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. PMID:27290717

  14. Epidemiology and transmission characteristics of human adenovirus type 7 caused acute respiratory disease outbreak in military trainees in East China

    OpenAIRE

    Cheng, Jun; Qi, Xiaoping; Chen, Dawei; Xu, Xujian; Wang, Guozheng; Dai, Yuzhu; Cui, Dawei; Chen, Qingyong; Fan, Ping; Ni, Liuda; Liu, Miao; Zhu, Feiyan; Yang, Mei; Wang, Changjun; Li, Yuexi

    2016-01-01

    Background: Human adenovirus type 7 (HAdV7) is globally attracting great concern as its high morbidity and severity in respiratory diseases, especially in Asia. Objective: To investigate the clinical and epidemiologic characteristics of HAdV7 infection outbreak in East China. Methods: The clinical samples were collected from the patients of an ARD outbreak in East Chinafor the detection of causative pathogens by multiplex PCR. The molecular type of human adenovirus isolates were identified by...

  15. Combined transductional untargeting/retargeting and transcriptional restriction enhance adenovirus gene targeting and therapy for hepatic colorectal cancer tumors

    OpenAIRE

    Li, Hua-Jung; Everts, Maaike; Yamamoto, Masato; Curiel, David T.; Herschman, Harvey R.

    2009-01-01

    Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors, and other epithelial tumors, often express both cyclooxygenase 2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus vectors infect liver and lack tumor tropism, they cannot be utilized for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional adenovirus hepatic untargeting and tumor retargeting by a bispecifi...

  16. Depletion of Cellular Pre-Replication Complex Factors Results in Increased Human Cytomegalovirus DNA Replication

    OpenAIRE

    Tamara Evans Braun; Emma Poole; John Sinclair

    2012-01-01

    Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recog...

  17. Replication and Robustness in Developmental Research

    Science.gov (United States)

    Duncan, Greg J.; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J.

    2014-01-01

    Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key…

  18. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

    Energy Technology Data Exchange (ETDEWEB)

    Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se [Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, 75123 Uppsala (Sweden); Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se [Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, 75123 Uppsala (Sweden); Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se [Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, 75123 Uppsala (Sweden)

    2012-11-25

    The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.

  19. Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications

    Science.gov (United States)

    Coyne, Michael D.; Cook, Bryan G.; Therrien, William J.

    2016-01-01

    Special education researchers conduct studies that can be considered replications. However, they do not often refer to them as replication studies. The purpose of this article is to consider the potential benefits of conceptualizing special education intervention research within a framework of systematic, conceptual replication. Specifically, we…

  20. 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.

  1. 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.

  2. 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.

  3. Continuous neuronal ensemble control of simulated arm reaching by a human with tetraplegia

    Science.gov (United States)

    Chadwick, E. K.; Blana, D.; Simeral, J. D.; Lambrecht, J.; Kim, S. P.; Cornwell, A. S.; Taylor, D. M.; Hochberg, L. R.; Donoghue, J. P.; Kirsch, R. F.

    2011-06-01

    Functional electrical stimulation (FES), the coordinated electrical activation of multiple muscles, has been used to restore arm and hand function in people with paralysis. User interfaces for such systems typically derive commands from mechanically unrelated parts of the body with retained volitional control, and are unnatural and unable to simultaneously command the various joints of the arm. Neural interface systems, based on spiking intracortical signals recorded from the arm area of motor cortex, have shown the ability to control computer cursors, robotic arms and individual muscles in intact non-human primates. Such neural interface systems may thus offer a more natural source of commands for restoring dexterous movements via FES. However, the ability to use decoded neural signals to control the complex mechanical dynamics of a reanimated human limb, rather than the kinematics of a computer mouse, has not been demonstrated. This study demonstrates the ability of an individual with long-standing tetraplegia to use cortical neuron recordings to command the real-time movements of a simulated dynamic arm. This virtual arm replicates the dynamics associated with arm mass and muscle contractile properties, as well as those of an FES feedback controller that converts user commands into the required muscle activation patterns. An individual with long-standing tetraplegia was thus able to control a virtual, two-joint, dynamic arm in real time using commands derived from an existing human intracortical interface technology. These results show the feasibility of combining such an intracortical interface with existing FES systems to provide a high-performance, natural system for restoring arm and hand function in individuals with extensive paralysis. This paper was originally submitted for the special issue containing contributions from the Fourth International Brain-Computer Interface Meeting.

  4. Persistent HIV-1 replication during antiretroviral therapy

    Science.gov (United States)

    Martinez-Picado, Javier; Deeks, Steven G.

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on efficiently targeting these three aspects. The degree to which HIV replicates during ART remains controversial. Most studies have failed to find any evidence of HIV evolution in blood, even with samples collected over many years, although a recent very intensive study of three individuals suggested that the virus population does shift, at least during the first few months of therapy. Stronger but still not definitive evidence for replication comes from a series of studies in which standard regimens were intensified with an integration inhibitor, resulting in changes in episomal DNA (blood) and cell-associated RNA (tissue). Limited drug penetration within tissues and the presence of immune sanctuaries have been argued as potential mechanisms allowing HIV to spread during ART. Mathematical models suggest that HIV replication and evolution is possible even without the selection of fully drug-resistant variants. As persistent HIV replication could have clinical consequences and might limit the efficacy of curative interventions, determining if HIV replicates during ART and why, should remain a key focus of the HIV research community. Summary Residual viral replication likely persists in lymphoid tissues, at least in a subset of individuals. Abnormal levels of immune activation might contribute to sustain virus replication. PMID:27078619

  5. A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

    OpenAIRE

    To, Kelvin K. W.; Herman Tse; Wan-Mui Chan; Choi, Garnet K. Y.; Zhang, Anna J. X.; Siddharth Sridhar; Wong, Sally C. Y.; Chan, Jasper F. W.; Chan, Andy S. F.; Woo, Patrick C. Y.; Susanna K P Lau; Lo, Janice Y. C.; Kwok-Hung Chan; Cheng, Vincent C. C.; Kwok-Yung Yuen

    2014-01-01

    Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Du...

  6. Design of Robotic Arm Control System Mimics Human Arm Motion

    Directory of Open Access Journals (Sweden)

    A. Salam Al-Ammri

    2013-01-01

    Full Text Available This paper presents a control system to make the robotic hand mimic human hand motion in real time and offline mode. The human hand tracking system is a wearable sensing arm (potentiometers used to determine the position in space and to sense the grasping task of human hand. The maskable sensing arm was designed with same geometrical arrangement of robotic hand that needs to be controlled. The control software of a robot was implemented using Visual Basic and supported with graphical user interface (GUI. The control algorithm depends on joint to joint mapping method to match between the motions at each joint of portable sensing arm with corresponding joint of a robot in order to make the robot mimic the motion.

  7. Inferring Where and When Replication Initiates from Genome-Wide Replication Timing Data

    Science.gov (United States)

    Baker, A.; Audit, B.; Yang, S. C.-H.; Bechhoefer, J.; Arneodo, A.

    2012-06-01

    Based on an analogy between DNA replication and one dimensional nucleation-and-growth processes, various attempts to infer the local initiation rate I(x,t) of DNA replication origins from replication timing data have been developed in the framework of phase transition kinetics theories. These works have all used curve-fit strategies to estimate I(x,t) from genome-wide replication timing data. Here, we show how to invert analytically the Kolmogorov-Johnson-Mehl-Avrami model and extract I(x,t) directly. Tests on both simulated and experimental budding-yeast data confirm the location and firing-time distribution of replication origins.

  8. Data from Investigating Variation in Replicability: A “Many Labs” Replication Project

    Directory of Open Access Journals (Sweden)

    Richard A. Klein

    2014-04-01

    Full Text Available This dataset is from the Many Labs Replication Project in which 13 effects were replicated across 36 samples and over 6,000 participants. Data from the replications are included, along with demographic variables about the participants and contextual information about the environment in which the replication was conducted. Data were collected in-lab and online through a standardized procedure administered via an online link. The dataset is stored on the Open Science Framework website. These data could be used to further investigate the results of the included 13 effects or to study replication and generalizability more broadly.

  9. NASA's Asteroid Redirect Mission (ARM)

    Science.gov (United States)

    Abell, Paul; Mazanek, Dan; Reeves, David; Naasz, Bo; Cichy, Benjamin

    2015-11-01

    The National Aeronautics and Space Administration (NASA) is developing a robotic mission to visit a large near-Earth asteroid (NEA), collect a multi-ton boulder from its surface, and redirect it into a stable orbit around the Moon. Once returned to cislunar space in the mid-2020s, astronauts will explore the boulder and return to Earth with samples. This Asteroid Redirect Mission (ARM) is part of NASA’s plan to advance the technologies, capabilities, and spaceflight experience needed for a human mission to the Martian system in the 2030s. Subsequent human and robotic missions to the asteroidal material would also be facilitated by its return to cislunar space. Although ARM is primarily a capability demonstration mission (i.e., technologies and associated operations), there exist significant opportunities to advance our knowledge of small bodies in the synergistic areas of science, planetary defense, asteroidal resources and in-situ resource utilization (ISRU), and capability and technology demonstrations. In order to maximize the knowledge return from the mission, NASA is organizing an ARM Investigation Team, which is being preceded by the Formulation Assessment and Support Team. These teams will be comprised of scientists, technologists, and other qualified and interested individuals to help plan the implementation and execution of ARM. An overview of robotic and crewed segments of ARM, including the mission requirements, NEA targets, and mission operations, will be provided along with a discussion of the potential opportunities associated with the mission.

  10. 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.

  11. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    Science.gov (United States)

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

  12. 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. PMID:24569225

  13. Acute respiratory distress syndrome in adenovirus type 4 pneumonia: A case report.

    Science.gov (United States)

    Narra, R; Bono, P; Zoccoli, A; Orlandi, A; Piconi, S; Grasselli, G; Crotti, S; Girello, A; Piralla, A; Baldanti, F; Lunghi, G

    2016-08-01

    Human adenoviruses (HAdVs) cause a wide spectrum of clinical syndromes, depending on species and types, from mild respiratory infections to deadly pneumonia: in particular, severe infections occur in immunocompromised patients. In this report, we describe the case of a 36 years-old woman admitted to our intensive care unit (ICU) with severe respiratory distress syndrome caused by adenovirus pneumonia, that required invasive respiratory support (mechanical ventilation and extracorporeal membrane oxygenation). Molecular assays detected the virus in respiratory and plasma specimen and sequencing procedure identified HAdV type 4. Patient improved after cidofovir administration. Leukopenia and subsequent bacterial infection occurred, but the patient recovered completely and was discharged from the hospital after 54days. PMID:27354307

  14. 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.

  15. 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.

  16. 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.

  17. 猴腺病毒研究进展%The Research Progress of Simian Adenovirus

    Institute of Scientific and Technical Information of China (English)

    张荣建; 贺争鸣

    2011-01-01

    Simian adenovirus( SAdV), as common viral agent persistent in respiratory and gastrointestinal tract of non-human primates, can cause pneumonia, gastroenteritis and conjunctivitis and spread mainly by Fecal-oral way. SadV can infect macaques, chimpanzees, baboons, etal belonging to genus Mastadenovirus . Some resarchers agured that SadV had strict species specificity. Other reports suggested that cross species transmission may be a common occurance and is zoonotic transmission. No report says SadV can infect human, but found a novel adenovirus serotype that may be recombination with a human adenovirus. SadV and HadV are close, casuing similar clinical symptoms, which make it a good model for adenovirus research. To date, the goal of developing a non-human primate adenovirus model has been made to study the mechanisms of infection and promote progress on gene therapies and genetic vaccines with adenovirus vector. However,little is known about the prevalence of adenovirus infection in non-human primates. This article reviewed discovery and classification, biological characterization, and detection of SAdV so as to provide some useful insights for researchers.%猴腺病毒(simian adenovirus,SAdV)是猴类呼吸道和消化道的常在病毒之一,可引起肺炎、咽炎、肠胃炎、结膜炎等以粪口途径传播为主,主要感染猕猴、非洲绿猴、黑猩猩和狒狒等[1]属腺病毒科,哺乳动物腺病毒属.有学者认为SAdV感染有严格的种属特异性[1],也有报道认为其种间交叉传播在非人灵长类中可能普遍发生,是动物传染病似的感染[2,3].SAdV是否感染人类尚未见报道,但已从猴的分泌物中发现了可能和人腺病毒(human adenovius,HAdV)发生重组的新型腺病毒[4].SAdV与HAdV亲缘关系很近,感染引起的临床症状也相似,是人腺病毒研究的理想模型.目前已经制定了研发猴腺病毒模型的目标,用于研究感染机制,促进腺病毒基因治

  18. 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.

  19. A retrospective investigation of canine adenovirus (CAV) infection in adult dogs in Turkey.

    Science.gov (United States)

    Gür, S; Acar, A

    2009-06-01

    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, serum samples were collected from native pure-bred Kangal(n = 11), and Akbash dogs (n = 17) and Turkish Greyhounds (n = 15) in Eskişehir and Konya provinces. None of the 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. PMID:19831268

  20. Segregation of chromosome arms in growing and non-growing Escherichia coli cells

    DEFF Research Database (Denmark)

    Woldringh, Conrad L.; Hansen, Flemming G.; Vischer, Norbert O. E.;

    2015-01-01

    In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (0) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same...... organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from...... that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized...

  1. Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

    Directory of Open Access Journals (Sweden)

    Clerzius Guerline

    2005-10-01

    Full Text Available Abstract Increasing evidence indicates that RNA interference (RNAi may be used to provide antiviral immunity in mammalian cells. Human micro (miRNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN-induced protein kinase R (PKR but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si and micro (miRNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

  2. Replication landscape of the human genome.

    Science.gov (United States)

    Petryk, Nataliya; Kahli, Malik; d'Aubenton-Carafa, Yves; Jaszczyszyn, Yan; Shen, Yimin; Silvain, Maud; Thermes, Claude; Chen, Chun-Long; Hyrien, Olivier

    2016-01-01

    Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border 'topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs. PMID:26751768

  3. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2016-08-01

    Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

  4. Targeting DNA Replication Stress for Cancer Therapy

    Science.gov (United States)

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-01-01

    The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress. PMID:27548226

  5. Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

    OpenAIRE

    Okada, T; Ramsey, W J; Munir, J; Wildner, O.; Blaese, R M

    1998-01-01

    We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promo...

  6. Triple-controlled oncolytic adenovirus expressing melittin to exert inhibitory efficacy on hepatocellular carcinoma

    OpenAIRE

    Qian, Chun-Yu; Wang, Kai-Li; Fang, Fan-Fu; Gu, Wei; Huang, Feng; Wang, Fu-Zhe; Li, Bai; Wang, Li-Na

    2015-01-01

    Hepatocellular carcinoma (HCC) is a highly malignant disease, and its outcome of routine therapies is poor. Comprehensive treatment including gene therapy is an important way to improve patients’ prognosis and survival. In this study, we successfully constructed a triple-controlled cancer-selective oncolytic adenovirus, QG511-HA-Melittin, carrying melittin gene, in which the hybrid promoter, hypoxia-response element (HRE)-AFP promoter, was used to control viral E1a expression targeting AFP-po...

  7. 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.

  8. Frequency of Rotavirus and Enteric Adenoviruses among children with acute gastroenteritis in a district hospital

    OpenAIRE

    Özer, Türkan Toka; Yula, Erkan; Deveci, Özcan; Tekin, Alicem; Durmaz, Süleyman; Gülenç, Mustafa; Yanık, Keramettin

    2011-01-01

    Objective: Rotavirus and Enteric Adenoviruses (EA) are most important viral enteric agents which cause acute infectious gastroenteritis. Little is known about the epidemiology of Rotavirus and EA gastroenteritis in our city. In this study, it was purposed to determine of the frequency of Rotavirus, EA, and to detect of the seasonal distribution among pediatric patients with acute gastroenteritis in Kiziltepe General Hospital, Mardin-Turkey. Materials and methods: The records of acute infe...

  9. Investigation of rotavirus and adenovirus antigens in patients with acute gastroenteritis

    OpenAIRE

    Hatice Türk Dağı,; Duygu Fındık

    2014-01-01

    Objective: Nowadays, viruses are the most common agents of acute gastroenteritis all over the world. Acute gastroenteritis, especially in children, is an important cause of mortality and morbidity. Both the lack of effective treatments as well as due to the unnecessary use of antibiotics, detection of viral agents in stool is important in terms of the epidemiology and monitoring of the disease. The purpose of this study is to determine the frequency of rotavirus and adenovirus in patients wit...

  10. Genetic Diversity of Human Adenovirus in Children with Acute Gastroenteritis, Albania, 2013–2015

    OpenAIRE

    La Rosa, G.; Della Libera, S.; S. Petricca; M. Iaconelli; D. Donia; Saccucci, P.; Cenko, F.; Xhelilaj, G.; Divizia, M.

    2015-01-01

    The objectives of the present study were to assess the occurrence of human adenoviruses (HAdVs) in paediatric patients with gastroenteritis in Albania and to characterize HAdV strains. Faecal specimens from children admitted with acute gastroenteritis to the Paediatric Hospital in Tirana were screened for HAdV, using broad-range primers targeting the hexon gene, in combination with species-specific primers targeting the fiber gene. Phylogenetic analysis was then performed to assess the geneti...

  11. Frequency of Rotavirus and Enteric Adenoviruses among children with acute gastroenteritis in a district hospital

    OpenAIRE

    Süleyman Durmaz; Mustafa Gülenç; Alicem Tekin; Keramettin Yanık; Türkan Toka Özer; Özcan Deveci; Erkan Yula

    2011-01-01

    Objective: Rotavirus and Enteric Adenoviruses (EA) are most important viral enteric agents which cause acute infectious gastroenteritis. Little is known about the epidemiology of Rotavirus and EA gastroenteritis in our city. In this study, it was purposed to determine of the frequency of Rotavirus, EA, and to detect of the seasonal distribution among pediatric patients with acute gastroenteritis in Kiziltepe General Hospital, Mardin-Turkey.Materials and methods: The records of acute infectiou...

  12. Prevalence of Rotavirus, Adenovirus, and Astrovirus Infections Among Patients with Acute Gastroenteritis in, Northern Iran

    OpenAIRE

    R Savad-Koohi; A Pakfetrat; AA Poor-Babaei; S Vaziri; L Adibi; Jalilvand, S; K Nourijelyani; Noroozi, M.; Y Yahyapour; Hamkar, R

    2010-01-01

    Background: The aim of the study was to determine the incidence of non-bacterial acute gastroenteritis associated with diarrheal diseases in Mazandaran Province, northern Iran. Methods: A total of 400 symptomatic cases from patients with acute gastroenteritis from Mazandaran Province in Iran were screened using EIA method for the presence of rotavirus, adenovirus and astrovirus during 2005–2006. Chi-square tests were used for testing relationships between different variables. Results: Rotavir...

  13. 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.

  14. Anti-Cocaine Vaccine Based on Coupling a Cocaine Analog to a Disrupted Adenovirus

    OpenAIRE

    Koob, George; Hicks, Martin J.; Wee, Sunmee; Rosenberg, Jonathan B; De, Bishnu P.; Kaminksy, Stephen M.; Moreno, Amira; Kim D. Janda; Crystal, Ronald G.

    2011-01-01

    The challenge in developing an anti-cocaine vaccine is that cocaine is a small molecule, invisible to the immune system. Leveraging the knowledge that adenovirus (Ad) capsid proteins are highly immunogenic in humans, we hypothesized that linking a cocaine hapten to Ad capsid proteins would elicit high-affinity, high-titer antibodies against cocaine, sufficient to sequester systemically administered cocaine and prevent access to the brain, thus suppressing cocaine-induced behaviors. Based on t...

  15. Novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions.

    OpenAIRE

    Wevers, Diana; Metzger, Sonja; Babweteera, Fred; Bieberbach, Marc; Boesch, Christophe; Cameron, Kenneth; Couacy-Hymann, Emmanuel; Cranfield, Mike; Gray, Maryke; Harris, Laurie A.; Head, Josephine; Jeffery, Kathryn; Knauf, Sascha; Lankester, Felix; Siv Aina J. Leendertz

    2011-01-01

    Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly...

  16. Association of Human Adenovirus-36 With Dyslipidemia in Tehranian Children and Adolescent; TLGS

    OpenAIRE

    Zarkesh; Sadat Daneshpour; Ehsandar; Bandehpour; Alfadhli; Azizi; Hedayati

    2015-01-01

    Background The human adenovirus-36 (Adv36) has been associated with obesity and lipid disorders in some countries. The primary dyslipidemia related to obesity is characterized by increased TG, decreased HDL levels and abnormal LDL composition. Childhood obesity is a major public health problem in most developing countries. Objectives The aim of this study was to investigate the association between Adv36 and lipid disorders in Tehr...

  17. Binding of Adenovirus Capsid to Dipalmitoyl Phosphatidylcholine Provides a Novel Pathway for Virus Entry

    OpenAIRE

    Balakireva, Larissa; Schoehn, Guy; Thouvenin, Eric; Chroboczek, Jadwiga

    2003-01-01

    Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-...

  18. Enzyme immunoassay for direct detection of influenza type A and adenovirus antigens in clinical specimens.

    OpenAIRE

    Harmon, M W; Pawlik, K M

    1982-01-01

    Detection of viral antigens in specimens without prior cultivation in cell culture provides the most rapid method for specific viral diagnosis. A solid-phase, double-antibody enzyme immunoassay was developed for this purpose and tested with clinical specimens containing influenza type A and adenovirus. Polystyrene microtiter wells were the solid phase and were coated with virus-specific guinea pig immunoglobulins. Specimens were added, and bound viral antigens were detected by addition of vir...

  19. Scanning ARM Cloud Radar Handbook

    Energy Technology Data Exchange (ETDEWEB)

    Widener, K; Bharadwaj, N; Johnson, K

    2012-06-18

    The scanning ARM cloud radar (SACR) is a polarimetric Doppler radar consisting of three different radar designs based on operating frequency. These are designated as follows: (1) X-band SACR (X-SACR); (2) Ka-band SACR (Ka-SACR); and (3) W-band SACR (W-SACR). There are two SACRs on a single pedestal at each site where SACRs are deployed. The selection of the operating frequencies at each deployed site is predominantly determined by atmospheric attenuation at the site. Because RF attenuation increases with atmospheric water vapor content, ARM's Tropical Western Pacific (TWP) sites use the X-/Ka-band frequency pair. The Southern Great Plains (SGP) and North Slope of Alaska (NSA) sites field the Ka-/W-band frequency pair. One ARM Mobile Facility (AMF1) has a Ka/W-SACR and the other (AMF2) has a X/Ka-SACR.

  20. Study on the micro-replication of shark skin

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Direct replication of creatural scarfskins to form biomimetic surfaces with relatively vivid morphology is a new attempt of the bio-replicated forming technology at animal body. Taking shark skins as the replication templates, and the micro-embossing and micro-molding as the material forming methods, the micro-replicating technology of the outward morphology on shark skins was demonstrated. The preliminary analysis on replication precision indicates that the bio-replicated forming technology can replicate the outward morphology of the shark scales with good precision, which validates the application of the bio-replicated forming technology in the direct morphology replication of the firm creatural scarfskins.

  1. Nuclear arms: ethics, strategy, politics

    Energy Technology Data Exchange (ETDEWEB)

    Woolsey, R.J. (ed.)

    1984-01-01

    The range of debate over strategy and arms control today is broader than it was in the late 1950s and early 1960s. In part this is because the early 1980s debate has involved questioning of the fundamental notion of deterrence - by no less than the American Catholic bishops. Today's debate has also seen a national nuclear freeze campaign that, although its congressional supporters have held firmly to a bilateral approach, was tilted perceptibly towards unilateralism by noncongressional leaders at their national conference in February 1983. In one way or another the authors wrestle with different aspects of one central question: could the beginning steps for any consensus be possible, in today's climate, on strategic issues. Ethical issues are addressed in the first 3 papers: Charles Krauthammer, On Nuclear Morality; Patrick Glynn, The Moral Case for the Arms Buildup; and Michael Quinlarc, Thinking Deterrence Through. Three chapters on strategic considerations include: Brent Snowcroft, Understanding the US Strategic Arsenal; William J. Perry, Technological Prospects for US Strategic Forces; and Richard Burt, The Strategic and Political Lessons of INF. Arms control and politics is treated in chapters by: Walter B. Slocombe, Arms Control: Prospects; and Colin S. Gray, Arms Control: Problems. The nonnuclear dimensions of strategy are discussed in chapters by Amory B. and L. Hunter Lovins, Reducing Vulnerability - The Energy Jugular; and Robert Kupperman, Vulnerable America. The chapters on space and defense are: Hans Mark, Arms Control and Space Technology; and Newt Gingrich and John Madison, Space and National Defense. The concluding chapters are by Sen. Sam Nunn, The Need to Reshape Military Stategy; and the editor, R. James Woolsey, The Politics of Vulnerability, 1980-1983.

  2. Replicated Data Management for Mobile Computing

    CERN Document Server

    Douglas, Terry

    2008-01-01

    Managing data in a mobile computing environment invariably involves caching or replication. In many cases, a mobile device has access only to data that is stored locally, and much of that data arrives via replication from other devices, PCs, and services. Given portable devices with limited resources, weak or intermittent connectivity, and security vulnerabilities, data replication serves to increase availability, reduce communication costs, foster sharing, and enhance survivability of critical information. Mobile systems have employed a variety of distributed architectures from client-server

  3. Replication and Analysis of Ebbinghaus' Forgetting Curve.

    Directory of Open Access Journals (Sweden)

    Jaap M J Murre

    Full Text Available We present a successful replication of Ebbinghaus' classic forgetting curve from 1880 based on the method of savings. One subject spent 70 hours learning lists and relearning them after 20 min, 1 hour, 9 hours, 1 day, 2 days, or 31 days. The results are similar to Ebbinghaus' original data. We analyze the effects of serial position on forgetting and investigate what mathematical equations present a good fit to the Ebbinghaus forgetting curve and its replications. We conclude that the Ebbinghaus forgetting curve has indeed been replicated and that it is not completely smooth but most probably shows a jump upwards starting at the 24 hour data point.

  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. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Nucleotide sequence determination of the region in adenovirus 5 DNA involved in cell transformation

    International Nuclear Information System (INIS)

    A description is given of investigations into the primary structure of the transforming region of adenovirus type 5 DNA. The phenomenon of cell transformation is discussed in general terms and the principles of a number of fairly recent techniques, which have been in use for DNA sequence determination since 1975 are dealt with. A few of the author's own techniques are described which deal both with nucleotide sequence analysis and with the determination of DNA cleavage sites of restriction endonucleases. The results are given of the mapping of cleavage sites in the HpaI-E fragment of adenovirus DNA of HpaII, HaeIII, AluI, HinfI and TaqI and of the determination of the nucleotide sequence in the transforming region of adenovirus type 5 DNA. The results of the sequence determination of the Ad5 HindIII-G fragment are discussed in relation with the investigation on the transforming proteins isolated from in vitro and in vivo synthesizing systems. Labelling procedures of DNA are described including the exonuclease III/DNA polymerase 1 method and TA polynucleotide kinase labelling of DNA fragments. (Auth.)

  11. Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry.

    Science.gov (United States)

    Li, Xiaoxin; Bangari, Dinesh S; Sharma, Anurag; Mittal, Suresh K

    2009-09-30

    Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin-Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized alpha(2,3)-linked and alpha(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.

  12. 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.

  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. 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.``

  15. Adenovirus E4-ORF3-dependent relocalization of TIF1{alpha} and TIF1{gamma} relies on access to the Coiled-Coil motif

    Energy Technology Data Exchange (ETDEWEB)

    Vink, Elizabeth I. [Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794 (United States); Yondola, Mark A. [Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029 (United States); Wu, Kai [Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794 (United States); Hearing, Patrick, E-mail: phearing@ms.cc.sunysb.edu [Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794 (United States)

    2012-01-20

    The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1{alpha}), but not homologous TIF1{beta}. Here, we introduce TIF1{gamma} as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1{gamma} in virus-infected cells in vivo and binds to TIF1{gamma} in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1{alpha}, TIF1{beta}, and TIF1{gamma}, and that the C-terminal half of TIF1{beta} interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

  16. Determination of the minimum fully protective dose of adenovirus-based DIVA vaccine against peste des petits ruminants virus challenge in East African goats.

    Science.gov (United States)

    Holzer, Barbara; Taylor, Geraldine; Rajko-Nenow, Paulina; Hodgson, Sophia; Okoth, Edward; Herbert, Rebecca; Toye, Philip; Baron, Michael D

    2016-01-01

    Peste des petits ruminants virus (PPRV) causes an economically important disease of sheep and goats, primarily in developing countries. It is becoming the object of intensive international control efforts. Current vaccines do not allow vaccinated and infected animals to be distinguished (no DIVA capability). We have previously shown that recombinant, replication-defective, adenovirus expressing the PPRV H glycoprotein (AdH) gives full protection against wild type PPRV challenge. We have now tested lower doses of the vaccine, as well as AdH in combination with a similar construct expressing the PPRV F glycoprotein (AdF). We show here that, in a local breed of goat in a country where PPR disease is common (Kenya), as little as 10(7) pfu of AdH gives significant protection against PPRV challenge, while a vaccine consisting of 10(8) pfu of each of AdH and AdF gives apparently sterile protection. These findings underline the utility of these constructs as DIVA vaccines for use in PPR control. PMID:26796101

  17. Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

    International Nuclear Information System (INIS)

    Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8+ T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.

  18. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    International Nuclear Information System (INIS)

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus

  19. Hemagglutinin-targeting Artificial MicroRNAs Expressed by Adenovirus Protect Mice From Different Clades of H5N1 Infection.

    Science.gov (United States)

    Tang, Xinying; Zhang, Hongbo; Song, Yufeng; Zhou, Dongming; Wang, Jieru

    2016-01-01

    Influenza virus (IV) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality. Re-emergence of human infection with avian influenza virus H5N1 poses extra challenge to IV control. Artificial microRNA (amiRNA)-mediated RNA interference has become a powerful antiviral approach due to its high specificity and rapid effect. Here, we designed several amiRNAs targeting the hemagglutinin gene of H5N1, a major determinant of pathogenicity. Expression and delivery efficiency were enhanced by presenting functional amiRNA with chimpanzee adenovirus serotype 68 (AdC68). One amiRNA, HA-1405, significantly limited H5N1 replication in vitro and inhibited 96.7% of clade 2.3.2 replication. AdC68-conjugated HA-1405 treatment remarkably decreased different clades of H5N1 plaque formation in Madin-Darby canine kidney cells. Moreover, prophylactic administration with rAd(HA-1405) markedly alleviated clinical symptoms and reduced ~3- to 40-folds of lung viral RNA copies against four clades of H5N1 in Institute of Cancer Research (ICR) mice. Our results further showed that rAd(HA-1405) conferred 70 and 40% immediate protection against lethal clade 2.3.2 and clade 2.3.4 H5N1 challenge, respectively. In conclusion, these data provided information that HA-targeting amiRNA delivered by AdC68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention. PMID:27093169

  20. What Should Researchers Expect When They Replicate Studies? A Statistical View of Replicability in Psychological Science.

    Science.gov (United States)

    Patil, Prasad; Peng, Roger D; Leek, Jeffrey T

    2016-07-01

    A recent study of the replicability of key psychological findings is a major contribution toward understanding the human side of the scientific process. Despite the careful and nuanced analysis reported, the simple narrative disseminated by the mass, social, and scientific media was that in only 36% of the studies were the original results replicated. In the current study, however, we showed that 77% of the replication effect sizes reported were within a 95% prediction interval calculated using the original effect size. Our analysis suggests two critical issues in understanding replication of psychological studies. First, researchers' intuitive expectations for what a replication should show do not always match with statistical estimates of replication. Second, when the results of original studies are very imprecise, they create wide prediction intervals-and a broad range of replication effects that are consistent with the original estimates. This may lead to effects that replicate successfully, in that replication results are consistent with statistical expectations, but do not provide much information about the size (or existence) of the true effect. In this light, the results of the Reproducibility Project: Psychology can be viewed as statistically consistent with what one might expect when performing a large-scale replication experiment. PMID:27474140