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Sample records for adenovirus

  1. Adenovirus structure.

    Science.gov (United States)

    Rux, John J; Burnett, Roger M

    2004-12-01

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

  2. Adenovirus DNA Replication

    OpenAIRE

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

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

  3. Initiation of adenovirus DNA replication.

    OpenAIRE

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

    1980-01-01

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

  4. Structure of Human Adenovirus

    OpenAIRE

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

    2012-01-01

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

  5. 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.

  6. Adenovirus Type 11 Uses CD46 as a Cellular Receptor

    OpenAIRE

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

    2003-01-01

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

  7. Oncolytic Adenoviruses in Cancer Treatment

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    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.

  8. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K.; Pearson, G D

    1985-01-01

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

  9. Replication-Defective Vector Based on a Chimpanzee Adenovirus

    OpenAIRE

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

    2001-01-01

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

  10. Anti-Viral Drugs for Human Adenoviruses

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    Chor Wing Sing

    2010-10-01

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

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

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    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

  12. Regulation of human adenovirus replication by RNA interference

    OpenAIRE

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

    2015-01-01

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

  13. 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

  14. Structure, Function and Dynamics in Adenovirus Maturation

    OpenAIRE

    Mangel, Walter F.; Carmen San Martín

    2014-01-01

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

  15. Predicted structure of two adenovirus tumor antigens.

    OpenAIRE

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

    1980-01-01

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

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

    OpenAIRE

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

    2012-01-01

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

  17. Structure and Uncoating of Immature Adenovirus

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-09-18

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

  18. 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.

  19. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    Science.gov (United States)

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

    2017-02-01

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

  20. 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

  1. Latest Insights on Adenovirus Structure and Assembly

    OpenAIRE

    Carmen San Martín

    2012-01-01

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

  2. Chromatin structure of adenovirus DNA throughout infection

    OpenAIRE

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

    2011-01-01

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

  3. Enhanced structural stability of adenovirus nanocapsule

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  4. 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).

  5. 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.

  6. 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.

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

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    Sook-Young Lee

    2014-05-01

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

  8. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

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

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

    Science.gov (United States)

    Thomson, Darelle; Meers, Joanne; Harrach, Balázs

    2002-02-26

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

  10. 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.

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

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    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.

  12. 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.

  13. 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.

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

    OpenAIRE

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

    1981-01-01

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

  15. 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.

  16. Adenovirus-based vaccine against Listeria monocytogenes

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  17. 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.

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

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    Annasara Lenman

    2015-02-01

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

  19. 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.

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2003-11-01

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

  2. Latest insights on adenovirus structure and assembly.

    Science.gov (United States)

    San Martín, Carmen

    2012-05-01

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

  3. 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.

  4. Increasing the Efficacy of Oncolytic Adenovirus Vectors

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    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.

  5. 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.

  6. Inhibition of adenovirus replication in vitro by trifluridine.

    Science.gov (United States)

    Lennette, D A; Eiferman, R A

    1978-09-01

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

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

    OpenAIRE

    Jiang, Shaokai; Caffrey, Michael

    2007-01-01

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

  8. 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

  9. Adenovirus respiratory tract infections in Peru.

    Directory of Open Access Journals (Sweden)

    Julia S Ampuero

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

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    Kellam Paul

    2009-02-01

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

  13. Bioaccumulation of animal adenoviruses in the pink shrimp

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    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.

  14. 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

  15. Bioaccumulation of animal adenoviruses in the pink shrimp.

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    Feduchi, E; Carrasco, L

    1987-04-06

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

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

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    Eric A Weaver

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

  18. 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

  19. 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.

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

    Science.gov (United States)

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

    2001-08-01

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

  1. 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;...

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

    Science.gov (United States)

    2014-10-01

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

  3. 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...

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

    Science.gov (United States)

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

    2009-06-01

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

  5. 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.

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

    Science.gov (United States)

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

    1989-06-15

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

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

    Science.gov (United States)

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

    1971-11-01

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

  8. 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...

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

    Science.gov (United States)

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

    2010-07-01

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

  10. 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.

  11. 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

  12. 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.

  13. Replication-Uncoupled Histone Deposition during Adenovirus DNA Replication

    OpenAIRE

    Komatsu, Tetsuro; Nagata, Kyosuke

    2012-01-01

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

  14. The Dual Nature of Nek9 in Adenovirus Replication

    OpenAIRE

    Jung, Richard; Radko, Sandi; Pelka, Peter

    2016-01-01

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

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

    OpenAIRE

    Mysiak, Monika Elzbieta

    2004-01-01

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

  16. 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.

  17. Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

    Science.gov (United States)

    2016-04-12

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

  18. Effect of CD4 gene expression on adenovirus replication.

    Science.gov (United States)

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

    1994-11-01

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

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Eric J. Kremer

    2010-09-01

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

  1. 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.

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

    Science.gov (United States)

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

    2013-08-30

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

  3. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Science.gov (United States)

    2010-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    1996-03-01

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

  6. 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.

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

  9. 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

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

    NARCIS (Netherlands)

    Gras, Jan Cornelis Emile

    2007-01-01

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

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

    Science.gov (United States)

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

    2017-02-15

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

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

    OpenAIRE

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

    2008-01-01

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

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

    Science.gov (United States)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2005-11-15

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

  15. 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.

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

    Science.gov (United States)

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

    2010-11-05

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

  17. 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.

  18. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    Science.gov (United States)

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

    2016-01-01

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

  19. 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.

  20. 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.

  1. Fluctuating expression of microRNAs in adenovirus infected cells.

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    1999-09-09

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

  3. 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

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

    Science.gov (United States)

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

    1979-01-01

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

  5. [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.

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

    OpenAIRE

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Science.gov (United States)

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

    2010-02-01

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

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

    OpenAIRE

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

    2015-01-01

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

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

    OpenAIRE

    2013-01-01

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

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

    OpenAIRE

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

    2005-01-01

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

  12. 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...

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

    OpenAIRE

    1987-01-01

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

  14. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-01-01

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

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

    Science.gov (United States)

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

    2015-08-21

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

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

    Science.gov (United States)

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

    2015-12-01

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

  18. Adenovirus-mediated gene transfer to tumor cells.

    Science.gov (United States)

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

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

  19. Use of cidofovir in pediatric patients with adenovirus infection

    Science.gov (United States)

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

    2016-01-01

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

  20. Role of preterminal protein processing in adenovirus replication.

    Science.gov (United States)

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

    1997-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-15

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    Science.gov (United States)

    Li, Wenyan; Shen, Jun

    2016-01-01

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

  4. 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.

  5. 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.

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

    OpenAIRE

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

    1998-01-01

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

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Julian Scherer

    2011-08-01

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

  10. 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.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2001-08-10

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

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

    Directory of Open Access Journals (Sweden)

    Johan Rebetz

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

  14. 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. 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

  16. 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.

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

    Science.gov (United States)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2006-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Shakti Singh

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. 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.

  2. Adenovirus-mediated nitric oxide synthase gene transfer.

    Science.gov (United States)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ravi Parasuraman

    2013-01-01

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

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

    Science.gov (United States)

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

    2000-06-01

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

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

    Science.gov (United States)

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

    2007-06-01

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

  6. 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.

  7. 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.

  8. 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.

  9. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

    Directory of Open Access Journals (Sweden)

    Akseli Hemminki

    2010-09-01

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

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

    NARCIS (Netherlands)

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

    2000-01-01

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

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

    NARCIS (Netherlands)

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

    1989-01-01

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

  12. 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.

  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. 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

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

    Science.gov (United States)

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

    2013-01-01

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

  16. 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.

  17. 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.

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

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

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

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Manjunatha Hiremath

    2014-06-01

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

  1. 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.

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

    Science.gov (United States)

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

    2016-08-31

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

  3. 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.

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

    Science.gov (United States)

    Wigand, R

    1979-01-01

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

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

    Science.gov (United States)

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yabe,Yoshiro

    1988-02-01

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

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

    Science.gov (United States)

    Liu, Li

    2005-06-20

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

  8. 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...

  9. 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

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

    Directory of Open Access Journals (Sweden)

    Andrea Amalfitano

    2010-09-01

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

  11. 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)

  12. 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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  16. 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.

  17. Life-Threatening Adenovirus Infections in the Setting of the Immunocompromised Allogeneic Stem Cell Transplant Patients

    Directory of Open Access Journals (Sweden)

    Cedar J. Fowler

    2010-01-01

    Full Text Available A single institution case series of adenovirus infections after allogeneic hematopoietic stem cell transplantation is presented to highlight the consideration for adenovirus infections as an etiology in patients with rapid hepatic or other sudden organ deterioration in the setting of apparent GVHD stabilization. The series also highlights that survival is limited with these infections often due in part to concomitant opportunistic infections. In addition, the pathophysiological events, such as GVHD and hepatic dysfunction, may complicate the clinical picture and delay therapy of an opportunistic infection. This is particularly true for adenoviral infections as they also have a distinct clinical picture in immunocompromised patients when compared to immune competent patients. Adenovirus infections also have the additional challenge that its treatment, cidofovir, has associated toxicities that can delay its administration. Recent developments has yielded an assay that can be used in the early detection and for serial determinations of adenovirus in patients with advanced GVHD, as well as a new therapeutic agent currently undergoing clinical trials.

  18. 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

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

    Directory of Open Access Journals (Sweden)

    Sarah R Klein

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

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

    Science.gov (United States)

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

    2015-03-23

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

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

    NARCIS (Netherlands)

    Breukelen, B. van

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Elenice Stroparo

    2010-12-01

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

  3. 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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  6. 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.

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

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    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.

  8. 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

  9. 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

  10. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

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

  11. 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.

  12. 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.

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

    Science.gov (United States)

    2002-03-01

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

  14. 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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Science.gov (United States)

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

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    H. Saderi

    2005-01-01

    Full Text Available Baekgrouund & purpose: In recent years, the role of adenoviruses in infection and disease in recipients of bone marrow transplantation (BMT has been studied. It suppose that adenoviral infections are prevalant in these patients Due to using medicines for preventing transplant rejection. This study was performed to compare the incidence of adenoviruses in urine samples taken before and after BMT from individuals undergoing BMT. In addition, The correlation between age, sex, etiology and kind of transplantation and adenovirus infection was studied.Materials and Methods: From 11 November 2002 to 12 June 2003, 91 patients received BMT in Hematology, Oncology and Bone Marrow Transplantation center of Tehran University of Medical Sciences. From 72 patients, 2 urine samples were taken before and 4 weeks after transplantation The DNA samples were examined by PCR using primers that detect 134 bp sequence in Hexon gene shared in human adenovirus DNA was extracted using Phenol-Chloroform method and concentrated by sodium acetate and ethanol.In Faculty of Medicine, Shahed University.Results: Adenovirus DNA was found in 39 patients (54.2% before transplantation and in 37 patients (51.4% after that. Both before and after BMT samples were negative for adenoviruses in 21 patients and positive in 25 patients. In 14 patients only before BMT and in 12 patients only after BMT urine samples were positive. No statistical difference between before and after BMT viruria was shown by McNemar’s test. Also, no statistical difference was shown between mean age of infected and non-infected patients by t-test. In spite of higher frequency of males, malignant and allogeneic transplant among studied patients, there was no statistical difference between two mentioned patients (P>0.05.Conclusion: In the present study, increase of prevalence of viruria in patients after bone marrow transplantation was not seen. In adition, there was no correlation between patients with various

  19. 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

  20. [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.

  1. p53/E1b58kDa complex regulates adenovirus replication.

    Science.gov (United States)

    Ridgway, P J; Hall, A R; Myers, C J; Braithwaite, A W

    1997-10-27

    We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.

  2. 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.

  3. 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.

  4. Experimental cross-species infection of common marmosets by titi monkey adenovirus.

    Directory of Open Access Journals (Sweden)

    Guixia Yu

    Full Text Available Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV, as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus. Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

  5. Coxsackie and adenovirus receptor is a modifier of cardiac conduction and arrhythmia vulnerability in the setting of myocardial ischemia. : Coxsackie and adenovirus receptor is a modifier of cardiac conduction and arrhythmia vulnerability in the setting of myocardial ischemia.

    NARCIS (Netherlands)

    Marsman, Roos F; Bezzina, Connie R; Freiberg, Fabian; Verkerk, Arie O; Adriaens, Michiel E; Podliesna, Svitlana; Chen, Chen; Purfürst, Bettina; Spallek, Bastian; Koopmann, Tamara T; Baczko, Istvan; Dos Remedios, Cristobal G; George, Alfred L; Bishopric, Nanette H; Lodder, Elisabeth M; de Bakker, Jacques M; Fischer, Robert; Coronel, Ruben; Wilde, Arthur A; Gotthardt, Michael; Remme, Carol A

    2014-01-01

    The aim of this study was to investigate the modulatory effect of the coxsackie and adenovirus receptor (CAR) on ventricular conduction and arrhythmia vulnerability in the setting of myocardial ischemia.

  6. 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...

  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. 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.

  9. Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

    Science.gov (United States)

    Gonzalez, R; Vereecque, R; Wickham, T J; Vanrumbeke, M; Kovesdi, I; Bauters, F; Fenaux, P; Quesnel, B

    1999-03-01

    Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

  10. 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.

  11. Regeneration of adenovirus specific T-cells after allogeneic, hematopietic stem cell transplantation in children and adolescents

    OpenAIRE

    2010-01-01

    Adenovirus is a significant cause of morbidity and mortality in pediatric allogeneic hematopoietic stem cell transplant recipients, and control of infection seems to require antigen-specific T cells. Aim of this Work was to estimate the Regeneration of Adenovirus-specific T-cells In 26 children (Age 8 months -25 years) over 6 months after HSCT and to investigate the effects of the transplantations parameter, Virus reactivation and the Transplantations complications on the adv-specific cell...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

    PURPOSE: Multiple viruses have been detected in cardiac tissue, but their role in causing myocarditis remains controversial. Viral diagnostics are increasingly used in forensic medicine, but the interpretation of the results can sometimes be challenging. In this study, we examined the prevalence...... 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...... was made by the serological determination of PVB-specific immunoglobulins M and G. RESULTS: PVB was detected in 33 of 112 (29 %) myocarditis cases and 37 of 84 (44 %) control cases. All of the samples were negative for the presence of adenovirus and enterovirus. Serological evidence of an acute PVB...

  17. Identification of a novel adenovirus in a cotton-top tamarin (Saguinus oedipus).

    Science.gov (United States)

    Hall, Natalie H; Archer, Linda L; Childress, April L; Wellehan, James F X

    2012-03-01

    A novel adenovirus was identified in a cotton-top tamarin (Saguinus oedipus) with diarrhea by negative-staining electron microscopy of feces, consensus polymerase chain reaction, and sequencing. Partial sequences were obtained from the DNA-dependent DNA polymerase, the p52k gene, and the hexon. Bayesian and maximum likelihood phylogenetic analyses indicated that the virus is a member of the genus Mastadenovirus, and is herein termed Saguinus siadenovirus 1. The phylogeny of the mastadenoviruses is similar to that of their hosts, supporting coevolution. Support for this was strongest in the analysis of the predicted hexon protein. The obtained sequences were GC-rich, which may suggest a lack of recent host jumps. The diversity and evolution of the adenoviruses of platyrrhine primates merits further investigation. Additional study of the association of this virus with diarrhea is indicated.

  18. 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.

  19. 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.

  20. 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.

  1. Chronic Activation of Innate Immunity Correlates With Poor Prognosis in Cancer Patients Treated With Oncolytic Adenovirus.

    Science.gov (United States)

    Taipale, Kristian; Liikanen, Ilkka; Juhila, Juuso; Turkki, Riku; Tähtinen, Siri; Kankainen, Matti; Vassilev, Lotta; Ristimäki, Ari; Koski, Anniina; Kanerva, Anna; Diaconu, Iulia; Cerullo, Vincenzo; Vähä-Koskela, Markus; Oksanen, Minna; Linder, Nina; Joensuu, Timo; Lundin, Johan; Hemminki, Akseli

    2016-02-01

    Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

  2. Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses.

    OpenAIRE

    Everett, R D; Dunlop, M

    1984-01-01

    This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA is...

  3. Differential Innate Immune Stimulation Elicited by Adenovirus and Poxvirus Vaccine Vectors

    OpenAIRE

    Teigler, Jeffrey Edward

    2014-01-01

    Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited b...

  4. 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.

  5. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Directory of Open Access Journals (Sweden)

    Ling Zhang

    Full Text Available Recombinant interferon-γ (IFNγ production in cultured lentivirus (LV was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5. The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041, and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032, which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  6. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Science.gov (United States)

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  7. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Directory of Open Access Journals (Sweden)

    Erkko Ylösmäki

    Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  8. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Science.gov (United States)

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  9. Interaction between mouse adenovirus type 1 and cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Liesbeth Lenaerts

    Full Text Available Application of human adenovirus type 5 (Ad5 derived vectors for cancer gene therapy has been limited by the poor cell surface expression, on some tumor cell types, of the primary Ad5 receptor, the coxsackie-adenovirus-receptor (CAR, as well as the accumulation of Ad5 in the liver following interaction with blood coagulation factor X (FX and subsequent tethering of the FX-Ad5 complex to heparan sulfate proteoglycan (HSPG on liver cells. As an alternative vector, mouse adenovirus type 1 (MAV-1 is particularly attractive, since this non-human adenovirus displays pronounced endothelial cell tropism and does not use CAR as a cellular attachment receptor. We here demonstrate that MAV-1 uses cell surface heparan sulfate proteoglycans (HSPGs as primary cellular attachment receptor. Direct binding of MAV-1 to heparan sulfate-coated plates proved to be markedly more efficient compared to that of Ad5. Experiments with modified heparins revealed that the interaction of MAV-1 to HSPGs depends on their N-sulfation and, to a lesser extent, 6-O-sulfation rate. Whereas the interaction between Ad5 and HSPGs was enhanced by FX, this was not the case for MAV-1. A slot blot assay demonstrated the ability of MAV-1 to directly interact with FX, although the amount of FX complexed to MAV-1 was much lower than observed for Ad5. Analysis of the binding of MAV-1 and Ad5 to the NCI-60 panel of different human tumor cell lines revealed the preference of MAV-1 for ovarian carcinoma cells. Together, the data presented here enlarge our insight into the HSPG receptor usage of MAV-1 and support the development of an MAV-1-derived gene vector for human cancer therapy.

  10. Functional role of the Coxsackie and Adenovirus Receptor (CAR) in colorectal carcinomas

    OpenAIRE

    Küster, Katrin

    2009-01-01

    Der Coxsackie- und Adenovirus Rezeptor (CAR) ist als Bestandteil von Tight Junctions (TJ) an interzellulären Adhäsionsprozessen beteiligt und scheint eine wichtige Rolle in der Karzinogenese zu spielen. Diese ist jedoch insbesondere bei Entstehung von Darmkrebs weitgehend unklar. Ziel der Arbeit war es daher, die funktionelle Bedeutung, mögliche Interaktionspartner sowie die Expressionsregulation von CAR im kolorektalen Karzinom zu analysieren. In den Zelllinien CaCo2, Colo205, DLD1, HCT116, ...

  11. Murine adenovirus infection of SCID mice induces hepatic lesions that resemble human Reye syndrome.

    OpenAIRE

    Pirofski, L.; Horwitz, M S; Scharff, M. D.; Factor, S. M.

    1991-01-01

    Murine adenovirus type 1 (MAV-1) infection of CB-17 SCID mice (which are homozygous for the severe combined immunodeficiency mutation) induces hepatic histopathologic and ultrastructural features that are strikingly similar to human Reye syndrome. Gross pathologic examination of MAV-1-infected mice revealed only pale yellow liver tissue. Histopathologic studies of tissue from MAV-1-infected mice revealed diffuse hepatic injury manifested by microvesicular fatty degenerative changes of hepatoc...

  12. Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus ▿

    OpenAIRE

    2009-01-01

    Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infe...

  13. 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.

  14. 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.

  15. 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

  16. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    OpenAIRE

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping

    2013-01-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...

  17. 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.

  18. 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.``

  19. 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.

  20. 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.

  1. Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

    Science.gov (United States)

    Li, Shun; Ou, Mengting; Wang, Guixue; Tang, Liling

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future.

  2. 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.

  3. Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

    Energy Technology Data Exchange (ETDEWEB)

    Han Jianfeng; Zhao Dong; Zhong Zhirong; Zhang Zhirong; Gong Tao; Sun Xun, E-mail: xunsun22@gmail.com [Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, 610041 (China)

    2010-03-12

    Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

  4. Clinical data analysis of 19 cases of community-acquired adenovirus pneumonia in immunocompetent adults.

    Science.gov (United States)

    Yu, Hong-Xia; Zhao, Mao-Mao; Pu, Zeng-Hui; Wang, Yun-Qiang; Liu, Yan

    2015-01-01

    The aim of this study was to investigate the characteristics of clinical manifestations, laboratory tests and imaging changes of community-acquired adenovirus pneumonia in immunocompetent adults. A retrospective study was performed on 19 adult community-acquired adenovirus pneumonia cases in Yantai, whereby the clinical data were collected and analyzed. Of 19 cases, 14 (73.68%) had fever and 17 (89.47%) had cough symptoms. Moreover, 14 cases (73.68%) had normal white blood cell counts, while 11 cases (57.89%) exhibited a reduction in lymphocyte proportion. Among the 19 cases, 17 cases exhibited lesions in a single lung, while 2 cases involved bilateral lungs. The lesions predominantly exhibited ground glass-like changes. The clinical manifestations of adult community-acquired adenovirus pneumonia patients with normal immune functions were mild, with such presenting symptoms as fever, cough, and sputum; most patients did not exhibit high levels of white blood cells or low lymphocyte counts, and the imaging features (ground glass-like effusion) were indicative of single-lung involvement.

  5. Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

    Science.gov (United States)

    Doucas, V; Ishov, A M; Romo, A; Juguilon, H; Weitzman, M D; Evans, R M; Maul, G G

    1996-01-15

    Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.

  6. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    Science.gov (United States)

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  7. 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.

  8. CAR chasing: canine adenovirus vectors-all bite and no bark?

    Science.gov (United States)

    Kremer, Eric J

    2004-02-01

    This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (DeltaE1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues.

  9. Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation.

    Science.gov (United States)

    Bounty, Sarah; Rodriguez, Roberto A; Linden, Karl G

    2012-12-01

    Adenovirus has consistently been observed to be the most resistant known pathogen to disinfection by ultraviolet light. This has had an impact on regulations set by the United States Environmental Protection Agency regarding the use of UV disinfection for virus inactivation in groundwater and surface water. In this study, enhancement of UV inactivation of adenovirus was evaluated when hydrogen peroxide was added to create an advanced oxidation process (AOP). While 4 log reduction of adenovirus was determined to require a UV dose (UV fluence) of about 200 mJ/cm(2) from a low pressure (LP) UV source (emitting at 253.7 nm), addition of 10 mg/L H(2)O(2) achieved 4 log inactivation at a dose of 120 mJ/cm(2). DNA damage was assessed using a novel nested PCR approach, and similar levels of DNA damage between the two different treatments were noted, suggesting the AOP enhancement in inactivation was not due to additional DNA damage. Hydroxyl radicals produced in the advanced oxidation process are likely able to damage parts of the virus not targeted by LPUV, such as attachment proteins, enhancing the UV-induced inactivation. The AOP-enhanced inactivation potential was modeled in three natural waters. This research sheds light on the inactivation mechanisms of viruses with ultraviolet light and in the presence of hydroxyl radicals and provides a practical means to enhance inactivation of this UV-resistant virus.

  10. 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.

  11. 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.

  12. 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.

  13. Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

    Science.gov (United States)

    Lemiale, Franck; Haddada, Hedi; Nabel, Gary J; Brough, Douglas E; King, C Richter; Gall, Jason G D

    2007-03-01

    Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.

  14. 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.

  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. The Serological and Virological Investigation of Canine Adenovirus Infection on the Dogs

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    Oya Bulut

    2013-01-01

    Full Text Available Two types of Canine Adenovirus (CAVs, Canine Adenovirus type 1 (CAV-1, the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2, which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188–54.7% blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188–45.2% blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs.

  17. 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.

  18. 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.

  19. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Science.gov (United States)

    Ibanes, Sandy; Kremer, Eric J

    2013-01-01

    When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  20. 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.

  1. Prevalence of Rotavirus, Adenovirus, Norovirus, and Astrovirus Infections and Coinfections among Hospitalized Children in Northern France▿

    Science.gov (United States)

    Tran, Adissa; Talmud, Déborah; Lejeune, Benoît; Jovenin, Nicolas; Renois, Fanny; Payan, Christopher; Leveque, Nicolas; Andreoletti, Laurent

    2010-01-01

    From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (P = 0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments. PMID:20305010

  2. Adenovirus-mediated eNOS expression augments liver injury after ischemia/reperfusion in mice.

    Directory of Open Access Journals (Sweden)

    Arun P Palanisamy

    Full Text Available Hepatic ischemia/reperfusion (l/R injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.

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

    Science.gov (United States)

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

    2014-02-01

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

  4. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    Science.gov (United States)

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-06-02

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.

  5. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

    Directory of Open Access Journals (Sweden)

    Jason S Richardson

    Full Text Available BACKGROUND: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP. The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. METHODOLOGY/PRINCIPAL FINDINGS: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP. Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. CONCLUSIONS/SIGNIFICANCE: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the

  6. 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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  8. Evaluation of a rapid immunochromatographic assay for the detection of rotavirus, norovirus and adenovirus from children hospitalized with acute watery diarrhea.

    Science.gov (United States)

    Kas, Monalisa P; Maure, Tobias; Soli, Kevin W; Umezaki, Masahiro; Morita, Ayako; Bebes, Sauli; Jonduo, Marinjho H; Larkins, Jo-Ann; Luang-Suarkia, Dagwin; Siba, Peter M; Greenhill, Andrew R; Horwood, Paul F

    2013-01-01

    We evaluated the IP-Triple I immunochromatographic rapid test for the detection of rotavirus, norovirus and adenovirus using stool samples from children with diarrhoea. The detection of norovirus and adenovirus was poor compared to polymerase chain reaction assays. However, high sensitivity (92%) and specificity (99%) were obtained for the detection of rotavirus.

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

    Science.gov (United States)

    Freimuth, Paul I.

    2010-04-06

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

  10. 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.

  11. Repair of segmental bone defects with bone marrow and BMP-2 adenovirus in the rabbit radius

    Science.gov (United States)

    Cheng, Lijia; Lu, Xiaofeng; Shi, Yujun; Li, Li; Xue, Jing; Zhang, Li; Xia, Jie; Wang, Yujia; Zhang, Xingdong; Bu, Hong

    2012-12-01

    Bone tissue engineering (BTE) is approached via implantation of autogenous mesenchymal stem cells (MSCs), marrow cells, or platelet-rich plasma, etc. To the contrary, gene therapy combining with the bone marrow (BM) has not been often reported. This study was performed to investigate whether a modified BTE method, that is, the BM and a recombinant human bone morphogenetic protein-2 adenovirus (Ad.hBMP-2) gene administering in hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramics could accelerate the healing of segmental defects in the rabbit radius. In our study, ceramics were immersed in the adenovirus overnight, and half an hour before surgery, autologous BM aspirates were thoroughly mixed with the ceramics; at the same time, a 15-mm radius defect was introduced in the bilateral forelimbs of all animals, after that, this defect was filled with the following: (1) Ad.hBMP-2 + HA/β-TCP + autologous BM (group 1); (2) HA/β-TCP + Ad.hBMP-2 (group 2); (3) HA/β-TCP alone (group 3); (4) an empty defect as a control (group 4). Histological observation and μ-CT analyses were performed on the specimens at weeks 2, 4, 8, and 12, respectively. In group 1, new bone was observed at week 4 and BM appeared at week 12, in groups 2 and 3, new bone was observed at week 8 and it was more mature at week 12, in contrast, the defect was not bridged in group 4 at week 12. The new bone area percentage in group 1 was significantly higher than that in groups 2 and 3. Our study indicated that BM combined with hBMP-2 adenovirus and porous ceramics could significantly increase the amount of newly formed bone. And this modified BTE method thus might have potentials in future clinical application.

  12. Avian Adenoviruses Infections with Special Attention to Inclusion Body Hepatitis/ Hydropericardium Syndrome and Egg Drop Syndrome

    Directory of Open Access Journals (Sweden)

    Hafez Mohamed Hafez*

    2011-04-01

    Full Text Available The first avian adenovirus (AAV associated with clinical disease was isolated from an outbreak of respiratory disease in quail in 1950 (Olson, 1950. Since that time, AAVs have been found in all types and breeds of chickens and from a variety of other avian species. The infections may be asymptomatic or associated with several clinical and pathological conditions. Vertical transmission via the egg is the most common way of transmission. Also horizontal transmission through faeces, contaminated egg trays, crates and trucks play a role in the infection route. Studies have demonstrated the presence of antibodies in healthy poultry, and viruses have been isolated from normal birds. Avian adenoviruses in chickens are the etiological agents of 2 diseases known as inclusion body hepatitis (IBH and hydropericardium syndrome (HP. In some cases each condition is observed separately, however, recently the 2 conditions have frequently been observed as a single entity; therefore, the name hepatitis hydropericardium has been widely used to describe the pathologic condition. The syndrome is an acute disease of young chickens associated with anemia, haemorrhagic disorders, hydropericardium and high mortality. Egg-Drop-Syndrome (EDS is caused also by an adenovirus. The disease is characterised by a severe drop in egg production as well as the production of shell-less, thin-shelled, discoloured or misshapen eggs in apparently healthy birds. Ducks and geese are the natural host of the EDS virus. It was first described in chickens in the 1970s and spread to several countries world wide. The birds usually do not show any other signs of disease, and mortality is not expected. There is no specific treatment of the AAV infections. Active immunization by vaccination using an inactivated is wide spread.

  13. Respiratory syncytial virus, adenoviruses, and mixed acute lower respiratory infections in children in a developing country.

    Science.gov (United States)

    Rodríguez-Martínez, Carlos E; Rodríguez, Diego Andrés; Nino, Gustavo

    2015-05-01

    There is growing evidence suggesting greater severity and worse outcomes in children with mixed as compared to single respiratory virus infections. However, studies that assess the risk factors that may predispose a child to a mixture of respiratory syncytial virus (RSV) and adenoviral infections, are scarce. In a retrospective cohort study, the study investigated the epidemiology of RSV and adenovirus infections and predictors of mixed RSV-adenoviral infections in young children hospitalized with acute lower respiratory infection in Bogota, Colombia, South America, over a 2-year period 2009-2011. Of a total of 5,539 children admitted with a diagnosis of acute lower respiratory infection, 2,267 (40.9%) who were positive for RSV and/or adenovirus were selected. Out the total number of cases, 1,416 (62.5%) infections occurred during the 3-month period from March to May, the first rainy season of Bogota, Colombia. After controlling for gender, month when the nasopharyngeal sample was taken, and other pre-existing conditions, it was found that an age greater than 6 months (OR:1.74; CI 95%:1.05-2.89; P = 0.030) and malnutrition as a comorbidity (OR:9.92; CI 95%:1.01-100.9; P = 0.049) were independent predictors of mixed RSV-adenoviral infections in the sample of patients. In conclusion, RSV and adenovirus are significant causes of acute lower respiratory infection in infants and young children in Bogota, Colombia, especially during the first rainy season. The identified predictors of mixed RSV-adenoviral infections should be taken into account when planning intervention, in order to reduce the burden of acute lower respiratory infection in young children living in the country.

  14. San Martín, C., Correction: Latest Insights on Adenovirus Structure and Assembly. Viruses 2012, 4, 847-877.

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    Carmen San Martín

    2012-12-01

    Full Text Available It has come to my attention that my article "Latest Insights on Adenovirus Structure and Assembly" (Viruses 2012, 4, 847-877 [1] contains an inaccurate statement. On page 864, the caption for Figure 7 reads: "There are four potential cleavage sites in pTP but they have not been experimentally verified". However, three of these sites have been experimentally confirmed in vitro using recombinant AVP and pTP, as described in Webster A, Leith I.R., Hay R.T.: Activation of adenovirus-coded protease and processing of preterminal protein. J. Virol. 1994, 68, 7292-7300 [2].

  15. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

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    Montgomery Roy D

    2006-04-01

    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  16. Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China

    Directory of Open Access Journals (Sweden)

    Zhu Yuan-Mao

    2011-12-01

    Full Text Available Abstract Background Bovine adenovirus type 3 (BAV-3 belongs to the Mastadenovirus genus of the family Adenoviridae and is involved in respiratory and enteric infections of calves. The isolation of BAV-3 has not been reported prior to this study in China. In 2009, there were many cases in cattle showing similar clinical signs to BAV-3 infection and a virus strain, showing cytopathic effect in Madin-Darby bovine kidney cells, was isolated from a bovine nasal swab collected from feedlot cattle in Heilongjiang Province, China. The isolate was confirmed as a bovine adenovirus type 3 by PCR and immunofluorescence assay, and named as HLJ0955. So far only the complete genome sequence of prototype of BAV-3 WBR-1 strain has been reported. In order to further characterize the Chinese isolate HLJ0955, the complete genome sequence of HLJ0955 was determined. Results The size of the genome of the Chinese isolate HLJ0955 is 34,132 nucleotides in length with a G+C content of 53.6%. The coding sequences for gene regions of HLJ0955 isolate were similar to the prototype of BAV-3 WBR-1 strain, with 80.0-98.6% nucleotide and 87.5-98.8% amino acid identities. The genome of HLJ0955 strain contains 16 regions and four deletions in inverted terminal repeats, E1B region and E4 region, respectively. The complete genome and DNA binding protein gene based phylogenetic analysis with other adenoviruses were performed and the results showed that HLJ0955 isolate belonged to BAV-3 and clustered within the Mastadenovirus genus of the family Adenoviridae. Conclusions This is the first study to report the isolation and molecular characterization of BAV-3 from cattle in China. The phylogenetic analysis performed in this study supported the use of the DNA binding protein gene of adenovirus as an appropriate subgenomic target for the classification of different genuses of the family Adenoviridae on the molecular basis. Meanwhile, a large-scale pathogen and serological epidemiological

  17. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    Science.gov (United States)

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  18. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    Science.gov (United States)

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  19. Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transp...

  20. ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

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    Lacher Markus D

    2011-07-01

    Full Text Available Abstract Background We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9:2088-95 and TGF-β (Cancer Res. 2006 Feb 1;66(3:1648-57 signaling negatively regulate coxsackie virus and adenovirus receptor (CAR cell-surface expression and adenovirus uptake. In the case of TGF-β, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT, a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration. Results Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic and MDA-MB-231 (breast human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-β-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-β-induced mesenchymal phenotype of PANC-1 cells, TGF-β did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-β may inhibit CAR expression by regulating factor(s that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal

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

    OpenAIRE

    2012-01-01

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

  2. Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens

    OpenAIRE

    2013-01-01

    The present study was carried out for detection and molecular characterization of fowl adenoviruses (FAdVs) associated with hydropericardium syndrome or inclusion body hepatitis in commercial broiler chickens. The FAdVs were detected in liver samples from 33 commercial broiler chicken flocks by polymerase chain reaction (PCR) using hexon gene specific primers. The restriction enzyme analysis using StyI, BsiWI, MluI, AspI, BglI and ScaI enzymes of all the 33 FAdV-positive samples revealed FAdV...

  3. 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.

  4. The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro.

    Science.gov (United States)

    Grill, Jacques; Lamfers, Martine L M; van Beusechem, Victor W; Dirven, Clemens M; Pherai, D Shareen; Kater, Mathijs; Van der Valk, Paul; Vogels, Ronald; Vandertop, W Peter; Pinedo, Herbert M; Curiel, David T; Gerritsen, Winald R

    2002-11-01

    The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

  5. The role of Cajal bodies in the expression of late phase adenovirus proteins.

    Science.gov (United States)

    James, Nicola J; Howell, Gareth J; Walker, John H; Blair, G Eric

    2010-04-10

    Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

  6. 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.

  7. Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

    Science.gov (United States)

    Simão, D; Pinto, C; Fernandes, P; Peddie, C J; Piersanti, S; Collinson, L M; Salinas, S; Saggio, I; Schiavo, G; Kremer, E J; Brito, C; Alves, P M

    2016-01-01

    Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

  8. Cryo-EM structures of two bovine adenovirus type 3 intermediates

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    Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xiong, Wei [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Sun, Wei; Yang, Chongwen; Zhang, Kai; Wang, Ying [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Hongrong [College of Physics and Information Science, Hunan Normal University, Changsha, Hunan 410081 (China); Huang, Xiaojun; Ji, Gang; Sun, Fei [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Zheng, Congyi, E-mail: cctcc202@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Zhu, Ping, E-mail: zhup@ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-02-15

    Adenoviruses (Ads) infect hosts from all vertebrate species and have been investigated as vaccine vectors. We report here near-atomic structures of two bovine Ad type 3 (BAd3) intermediates obtained by cryo-electron microscopy. A comparison between the two intermediate structures reveals that the differences are localized in the fivefold vertex region, while their facet structures are identical. The overall facet structure of BAd3 exhibits a similar structure to human Ads; however, BAd3 protein IX has a unique conformation. Mass spectrometry and cryo-electron tomography analyses indicate that one intermediate structure represents the stage during DNA encapsidation, whilst the other intermediate structure represents a later stage. These results also suggest that cleavage of precursor protein VI occurs during, rather than after, the DNA encapsidation process. Overall, our results provide insights into the mechanism of Ad assembly, and allow the first structural comparison between human and nonhuman Ads at backbone level. - Highlights: • First structure of bovine adenovirus type 3. • Some channels are located at the vertex of intermediate during DNA encapsidation. • Protein IX exhibits a unique conformation of trimeric coiled–coiled structure. • Cleavage of precursor protein VI occurs during the DNA encapsidation process.

  9. Occurrence of adenovirus infection and clinical impact in paediatric stem cell transplant recipients

    Directory of Open Access Journals (Sweden)

    Gabriele Bianco

    2016-10-01

    Full Text Available In this study, the occurrence and clinical impact of adenovirus (AdV infection was investigated in paediatric hematopoietic stem cell transplantation (HSCT recipients. A number of 603 specimens (including whole blood, respiratory and other samples from 181 patients were tested by real-time polymerase chain reaction; clinical outcome was investigated. Overall, 118/603 (19.6% specimens from 21/181 (11.6% patients resulted positive to AdV (including 17.3, 29.9, 17.6, and 15.8% of total number of whole blood, respiratory, urine and other specimens, respectively. On whole blood specimens, viral loads ranged from <600 (limit of detection to >5×106 copies/mL, with a median value 2×104. Multiple specimens were positive in patients in which viral load on whole blood was high. Adenoviral positivity on whole blood was associated to poor prognosis, as death occurred in three of ten (30% patients with persistent positivity on whole blood specimens, also despite the administration of an antiviral agent (cidofovir. Adenovirus infection can account for systemic and/or organ-specific signs/symptoms in approximately 10% of paediatric HSCT recipients. At moment, there is no indication for routine monitor of AdV in these patients, although AdV aetiology of infectious transplant complications should be taken in account.

  10. The Role of NK Cell in T Cell Recruitment in Murine Liver Infected with Adenovirus

    Institute of Scientific and Technical Information of China (English)

    游上游; 艾洪武; 黄巍; 张楚瑜

    2003-01-01

    To study the role of natural killer (NK) cells in T cell recruitment in murine liver infected with virus, mice wereintravenously injected daily with anti-NK1.1+ antibody to deplete NK cells. Lymphocytes in the liver tissue of mice infectedwith type 5 adenovirus depleted in the E1 and E3 regions were assessed by fluorometric activated cell sorting (FACS). Ex-pression of chemokine IP-10 and its receptor CXCR3 mRNA in the liver, hepatic lymphocytes and spleen tissue were examined by reverse transcription polymerase chain reaction (RT-PCR). Serum almfine aminotransferase (ALT) was measured asan indicator of liver injury. It was found that infection of adenovims and anfi-Fas monoclonal antibody (mAb) into mice caused liver injury and high expression of interfemn-γ inducible protein-10 (IP-10) mRNA in the liver. Anfi-NK1.1+ mAb, which was intraperitoneally injected into the mice infected with adenovirus, suppresses T cell recruitment and expression of IP-10 mRNA in the hver. Slighter hver injury was also observed. After vires infection, expression of CXCR3 mRNAin spleen and hver tissue was observed at different time. The results suggested that T cell recruitment was initiated by NKcell dependent chemokine IP-10, which induced activated T cells priming in the spleen to the hver of the mouse. NK cells played a key role in T cell recruitment in the liver of mouse infected with adenovims.

  11. Investigating the role of Acanthamoeba polyphaga in protecting Human Adenovirus from water disinfection treatment.

    Science.gov (United States)

    Verani, Marco; Di Giuseppe, Graziano; Tammaro, Carmine; Carducci, Annalaura

    2016-06-01

    Human adenoviruses are responsible for a wide range of clinical infections and are present in aquatic environments, including river, seawater, drinking-water and sewage. Free-living amoebae (Acanthamoeba) in the same environments may internalize them and other microorganisms can act as a reservoir for the internalized viruses. In this study, we studied the interaction between Acanthamoeba polyphaga and Human Adenovirus type 5 (HAdV 5) to determine whether the amoeba played a role in protecting the internalized viruses from chemical disinfection. The efficacy of sodium hypochlorite disinfection against A. polyphaga and HAdV 5 either singly or in combination was assessed at three different concentrations. Individually, the amoeba were more resistant to chemical disinfection than HAdV 5 and remained alive after exposure to 5mg/l of sodium hypochlorite. In contrast, HAdV 5 lost infectivity following exposure to 2.5mg/l of sodium hypochlorite. When the amoeba and HAdV 5 were co-cultured, infectious virus was found in the cytoplasm of the amoeba at 5mg/l disinfectant concentration. These findings suggest that the A. polyphaga is providing protection for the HAdV 5.

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

    Science.gov (United States)

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

    2008-12-01

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

  13. 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.

  14. Derivation of a myeloid cell-binding adenovirus for gene therapy of inflammation.

    Directory of Open Access Journals (Sweden)

    Michael O Alberti

    Full Text Available The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5. Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR, with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP, with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease.

  15. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    Science.gov (United States)

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  16. Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHAO Dun-yan; SHEN Ai-guo; LIU Fan; ZHANG Feng; SUN Yu; WU Hong-fu; LU Chun-feng; SHI Hong-guang

    2007-01-01

    Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and β-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml ) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.

  17. Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ya Ning; LI Qiang

    2014-01-01

    Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

  18. 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.

  19. Adenovirus with p16 gene exerts antitumor effect on laryngeal carcinoma Hep2 cells.

    Science.gov (United States)

    Yang, Zhengang; Hu, Jingxia; Li, Dajun; Pan, Xinliang

    2016-08-01

    Laryngeal cancer is an uncommon form of cancer. The tumor suppressor P16, known to be mutated or deleted in various types of human tumor, including laryngeal carcinoma, is involved in the formation and development of laryngeal carcinoma. It has been previously reported that the inactivation or loss of P16 is associated with the acquisition of malignant characteristics. The current study hypothesized that restoring wild‑type P16 activity into P16‑null malignant Hep2 cells may exert an antitumor effect. A recombinant adenovirus carrying the P16 gene (Ad‑P16) was used to infect and express high levels of P16 protein in P16‑null Hep2 cells. Cell proliferation and invasion assays and polymerase chain reaction were performed to evaluate the effects of the P16 gene on cell proliferation and the antitumor effect on Hep2 cells. The results demonstrated that the Hep2 cells infected with Ad‑P16 exhibited significantly reduced cell proliferation, invasion and tumor volume compared with untreated or control adenovirus cells. Furthermore, the expression of laryngeal carcinoma‑associated genes, EGFR, survivin and cyclin D1, were measured in Ad‑P16‑infected cells and were significantly reduced compared with control groups. The results of the current study demonstrate that restoring wild‑type P16 activity into P16-null Hep2 cells exerts an antitumor effect.

  20. CAR-associated vesicular transport of an adenovirus in motor neuron axons.

    Science.gov (United States)

    Salinas, Sara; Bilsland, Lynsey G; Henaff, Daniel; Weston, Anne E; Keriel, Anne; Schiavo, Giampietro; Kremer, Eric J

    2009-05-01

    Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

  1. 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.

  2. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine.

    Science.gov (United States)

    Toro, Haroldo; Suarez, David L; Tang, De-chu C; van Ginkel, Frederik W; Breedlovea, Cassandra

    2011-03-01

    We evaluated protection conferred by mucosal vaccination with replication-competent adenovirus-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdTW68.H5ck). Commercial, layer-type chicken groups were either singly vaccinated ocularly at 5 days of age, singly vaccinated via spray at 5 days of age, or ocularly primed at 5 days and ocularly boosted at 15 days of age. Only chickens primed and boosted via the ocular route developed AI systemic antibodies with maximum hemagglutination inhibition mean titers of 3.9 log2 at 32 days of age. In contrast, single vaccination via the ocular or spray routes maintained an antibody status similar to unvaccinated controls. All chickens (16/16) subjected to ocular priming and boosting with AdTW68.H5ck survived challenge with highly pathogenic AI virus A/chicken/Queretaro/14588-19/95 (H5N2). Single ocular vaccination resulted in 63% (10/16) of birds surviving the challenge followed by a 44% (7/16) survival of single-sprayed vaccinated birds. Birds vaccinated twice via the ocular route also showed significantly lower (P < 0.05) AI virus RNA concentrations in oropharyngeal swabs compared to unvaccinated-challenged controls.

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

    Directory of Open Access Journals (Sweden)

    Briana Jill Williams

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

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

    Science.gov (United States)

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

    2012-01-01

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

  5. Suppression of gastric cancer growth by adenovirus-mediated transfer of the PTEN gene

    Institute of Scientific and Technical Information of China (English)

    Ying Hang; Yong-Chen Zheng; Yan Cao; Qing-Shan Li; Yu-Jie Sui

    2005-01-01

    AIM: To investigate the tumor-suppressive effect of the phosphatase and tensin homologue deleted from chromosome (PTEN) in human gastric cancer cells th atwere wild type for PTEN.METHODS: Adenoviruses expressing PTEN or luciferase as a control were introduced into gastric cancer cells.The effect of exogenous PTEN gene on the growth and apoptosis of gastric cancer cells that are wtPTEN were examined in vitro and in vivo.RESULTS: Adenovirus-mediated transfer of PTEN (AdPTEN) suppressed cell growth and induced apoptosis significantly in gastric cancer cells (MGC-803, SGC-7901)carrying wtPTEN in comparison with that in normal gastric epithelial cells (GES-1) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase and mitogen-activated protein kinase and cell-cycle arrest at the G2/M phase but not at the G1 phase. Furthermore,treatment of human gastric tumor xenografts (MGC-803,SGC-7901) with Ad-PTEN resulted in a significant (P<0.01)suppression of tumor growth.CONCLUSION: These results indicate a significant tumorsuppressive effect of Ad-PTEN against human gastric cancer cells. Thus, Ad-PTEN may be used as a potential therapeutic strategy for treatment of gastric cancers.

  6. Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months

    Science.gov (United States)

    Pelliccia, Maria; Andreozzi, Patrizia; Paulose, Jayson; D'Alicarnasso, Marco; Cagno, Valeria; Donalisio, Manuela; Civra, Andrea; Broeckel, Rebecca M.; Haese, Nicole; Jacob Silva, Paulo; Carney, Randy P.; Marjomäki, Varpu; Streblow, Daniel N.; Lembo, David; Stellacci, Francesco; Vitelli, Vincenzo; Krol, Silke

    2016-11-01

    Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight ~8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ~48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Julius W Kim

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

  9. Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes.

    Science.gov (United States)

    Tresilwised, Nittaya; Pithayanukul, Pimolpan; Holm, Per Sonne; Schillinger, Ulrike; Plank, Christian; Mykhaylyk, Olga

    2012-01-01

    Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.

  10. Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

    Institute of Scientific and Technical Information of China (English)

    朱瑾; 吴军; 陈希炜; 易绍萱; 罗高兴; 贺伟峰

    2003-01-01

    Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

  11. De Novo Synthesis of Marburg Virus Antigens from Adenovirus Vectors Induce Potent Humoral and Cellular Immune Responses

    Science.gov (United States)

    2005-12-09

    cells and purified by ultra-centrifugation in esium chloride gradients. Briefly, adenoviral lysates from hirty 150-mm plates were banded twice on CsCl...differences of individuals with cystic fibrosis con- sequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther

  12. Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

    Science.gov (United States)

    Kotha, Poornima L N; Sharma, Priyanka; Kolawole, Abimbola O; Yan, Ran; Alghamri, Mahmoud S; Brockman, Trisha L; Gomez-Cambronero, Julian; Excoffon, Katherine J D A

    2015-03-01

    Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

  13. Electrostatic Interactions between Complement Regulator CD46(SCR1-2 and Adenovirus Ad11/Ad21 Fiber Protein Knob

    Directory of Open Access Journals (Sweden)

    Carl Z. Chen

    2015-01-01

    Full Text Available Adenoviruses bind to a variety of human cells to cause infection. Both the B2 adenovirus 11 and B1 adenovirus 21 use protein knobs to bind to complement regulator CD46(SCR1-2 in order to gain entry into host cells. In each complex, the two proteins are highly negatively charged but bind to each other at an interface with oppositely charged surface patches. We computationally generated single-alanine mutants of charged residues in the complexes CD46(SCR1-2-Ad11k and CD46(SCR1-2-Ad21k. We used electrostatic clustering and Poisson-Boltzmann free energy calculations to propose a hypothesis on the role of electrostatics in association. Our results delineate specific interfacial electrostatic interactions that are critical for association in both CD46(SCR1-2-Ad11k and CD46(SCR1-2-Ad21k. These results will serve as a predictive tool in the selection of mutants with desired binding affinity in experimental mutagenesis studies. This study will also serve as a foundation for the design of inhibitors to treat adenovirus infections.

  14. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that f

  15. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    DEFF Research Database (Denmark)

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J

    2008-01-01

    Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity...

  16. Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters.

    Science.gov (United States)

    Shoji, Masaki; Yoshizaki, Shinji; Mizuguchi, Hiroyuki; Okuda, Kenji; Shimada, Masaru

    2012-01-01

    Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.

  17. Molecular characterisation of fowl adenovirus type 7 isolated from poultry associated with inclusion body hepatitis in Poland.

    Science.gov (United States)

    Niczyporuk, Jowita Samanta

    2017-02-03

    The fowl adenovirus field strain FAdV-JSN-5/10j (GenBank accession number KP879219) was isolated from the intestine of a 7-week-old chicken diagnosed with inclusion body hepatitis and simultaneously with Marek's disease, and for that reason, it was chosen for molecular study. It was identified as fowl adenovirus genotype 7 (species Fowl aviadenovirus E) based on nucleotide sequence analysis of the loop L1 region of the hexon gene. Nucleotide sequence alignment of this strain, FAdV-7 reference strains B-3A ATCC VR-832 (AF339922) and YR36 (AF508955), and eight additional FAdV-7 field strains confirmed its classification as FAdV-JS-5/10j and showed that these viruses are very similar to each other. Additionally, we described mutations and their influence on the amino acid sequence, nucleotide composition, and relative synonymous codon usage. Immunofluorescence of cell cultures infected with 10(4.5) TCID 50 per 0.1-ml dose of the FAdV-JSN-5/10j strain demonstrated the presence of a cytopathic effect. Infection of fowl with adenoviruses raises concerns for poultry production, and thus, the efficient detection of adenovirus infection is crucial. This is the first attempt to describe the molecular characteristics of FadV-7 strains isolated in Poland.

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

    Science.gov (United States)

    2013-01-01

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

  19. Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014

    Science.gov (United States)

    This study aimed to characterize a novel adenovirus (AdV) isolated from diseased Muscovy ducks in China. After the AdV was successfully propagated in duck embryo fibroblasts, the morphological and physicochemical properties of the virions were studied by electron microscopy and different tests. The ...

  20. 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.

  1. Adenovirus 36 Seropositivity is Strongly Associated With Race and Gender, But Not Obesity, Among U.S. Military Personnel

    Science.gov (United States)

    2010-01-01

    36).11 Further research showing such a relationship in animals included studies on rats,12 and marmosets and rhesus monkeys.13 Demonstrating the...Whigham LD, Abbott DH, Schultz-Darken NJ, Israel BA, Bradley SM et al. Human adenovirus Ad-36 promotes weight gain in male rhesus and marmoset

  2. Adenovirus surveillance, 1982-1993, Japan. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

    Science.gov (United States)

    Yamadera, S; Yamashita, K; Akatsuka, M; Kato, N; Hashido, M; Inouye, S; Yamazaki, S

    1995-08-01

    The Infectious Agents Surveillance Center, the National Institute of Health, Japan, received 17,265 reports from 1982 to 1993 on cases from whom adenovirus was isolated or detected; 85% from 57 public health institutes and the other 15% from two national hospitals and two commercial diagnostic laboratories. The followings were found. Three major diseases caused by adenovirus were upper respiratory tract infection, gastroenteritis, and conjunctivitis. Patients of upper respiratory tract infection numbered 6,837 (40% of all patients due to adenovirus), the identified serotypes being in order of frequency types 3, 2, 1, and 5. Those of gastroenteritis numbered 1,636 (9.5%). From 40% of the gastroenteritis patients, adenovirus was detected by electron microscopy or immunochemical methods without cultivation. From the remaining 60%, virus was isolated in tissue culture; the serotypes of the isolates resembled those causing upper respiratory tract infection. Patients of conjunctivitis numbered 3,437 (20%), the frequency being in order of types 3, 4, 8, 37, and 19. Conjunctivitis due to types 3 and 4 prevailed every summer; type 3 was isolated often from children with pharyngo-conjunctival fever and the other four types were mostly from adults with epidemic keratoconjunctivitis. Type 3 had a unique feature not seen in other types: it was most frequently isolated, causing upper respiratory tract infection, gastroenteritis, conjunctivitis, and pharyngo-conjunctival fever. Reports on isolation of type 7, which has been reported to cause severe pneumonia in many other countries, were as few as 28 (0.2%).

  3. Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

    Directory of Open Access Journals (Sweden)

    Poornima L N Kotha

    2015-03-01

    Full Text Available Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR, a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

  4. COINFECTION OF CALIFORNIA SEA LION ADENOVIRUS 1 AND A NOVEL POLYOMAVIRUS IN A HAWAIIAN MONK SEAL (NEOMONACHUS SCHAUINSLANDI).

    Science.gov (United States)

    Cortés-Hinojosa, Galaxia; Doescher, Bethany; Kinsel, Michael; Lednicky, John; Loeb, Julia; Waltzek, Thomas; Wellehan, James F X

    2016-06-01

    The Hawaiian monk seal (Neomonachus schauinslandi) is an endangered species. Here, we present a clinical case of a 26-yr-old male Hawaiian monk seal (HMS) kept in an aquarium with a history of intermittent anorexia and evidence of renal disease. Histologic examination revealed eosinophilic intranuclear inclusions in the liver. Conventional nested PCR protocols were used to test for viruses, and it tested positive for adenovirus and polyomavirus, and negative for herpesvirus. The adenovirus partial polymerase gene is 100% homologous to that of California sea lion adenovirus 1 (CSLAdV-1). CSLAdV-1 causes viral hepatitis in CSL, and has recently been reported in different species of otariids in an aquarium in Japan ( Otaria flavescens and Arctocephalus pusillus ) and a sequence from Spain has been submitted in NCBI as Otaria flavescens adenovirus-1. The polyomavirus in this animal is a novel virus, and is the first polyomavirus discovered in Hawaiian monk seals. This new virus is designated Hawaiian monk seal polyomavirus (HMSPyV-1), and is 83% homologous to California sea lion Polyomavirus-1 (CSLPyV-1). This is the first report of viral coinfection in a HMS and clinical significance in this case remains unclear but may be associated with advanced age.

  5. Efficacy of topical cobalt chelate CTC-96 against adenovirus in a cell culture model and against adenovirus keratoconjunctivitis in a rabbit model

    Directory of Open Access Journals (Sweden)

    Srivilasa Charlie

    2006-06-01

    Full Text Available Abstract Background Adenovirus (Ad, associated with significant morbidity, has no topical treatment. A leading CTC compound (CTC-96, a CoIII chelate, was found to have potent in vitro and in vivo antiviral efficacy against herpes viruses. In this study CTC-96 is being tested for possible anti-Adenovirus activity. Methods The biological anti-adenovirus activity of CTC-96 in concentrations from 5 to 250 ug/ml, was evaluated initially by viral inactivation (viral exposure to CTC-96 followed by dilution and inoculation of cells, virucidal (viral exposure to CTC-96 and inoculation of cells without dilution and antiviral (effect of CTC-96 on previously adsorbed virus plaque assays on HeLa (human cervical carcinoma, A549 (human lung carcinoma and SIRC (rabbit corneal cells. After verifying the antiviral activity, New Zealand White rabbits were infected with Ad-5 into: 1 the anterior cul-de-sac scarifying the conjunctiva (Group "C+"; 2 the anterior cul-de-sac scarifying the conjunctiva and cornea (Group "CC+"; 3 the stroma (Group "CI+". Controls were sham-infected ("C-", "CC-", "CI-". Other rabbits, after "CC", were treated for 21 days with: 1 placebo, 9x/day ("-"; 2 CTC-96, 50 ug/ml, 9x/day ("50/9"; CTC-96, 50 ug/ml, 6x/day ("50/6"; CTC-96, 25 ug/ml, 6x/day ("25/6". All animals were monitored via examination and plaque assays. Results In vitro viral inactivation, virucidal and antiviral assays all demonstrated CTC-96 to be effective against Adenvirus type 5 (ad-5. The in vivo model of Ad keratoconjunctivitis most similar to human disease and producing highest viral yield was "CC". All eyes (6/6 developed acute conjunctivitis. "CI" yielded more stromal involvement (1/6 and iritis (5/6, but lower clinical scores (area × severity. Infection via "C" was inconsistent (4/6. Fifty (50 ug/ml was effective against Ad-5 at 6x, 9x dosings while 25 ug/ml (6x was only marginally effective. Conclusion CTC-96 demonstrated virucidal activity against Ad5 in tissue

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

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    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

  7. 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

  8. Neumonectomía en atelectasia masiva post infección por adenovirus Pneumonectomy in massive atelectasis post adenovirus infection

    Directory of Open Access Journals (Sweden)

    Andrés Koppmann A

    2006-03-01

    Full Text Available Se describen las características clínicas, la imagenología torácica y función pulmonar de tres pacientes (edades actuales: 8-14 años. Todos ellos cursaron con una neumonía grave por adenovirus en el período de lactante y cuya evolución se agravó por una atelectasia pulmonar masiva refractaria al tratamiento que determinó la indicación de neumonectomía. En los tres casos, el pulmón resecado presentaba atelectasia, bronquiectasias y fibrosis. Dos niños mejoraron su condición clínica posterior a la neumonectomía, logrando suspender el oxígeno adicional. A pesar de presentar una limitación ventilatoria restrictiva y obstructiva severa y una tomografía computarizada de pulmón con signos de daño crónico, han disminuido las exacerbaciones obstructivas y hacen vida normal. El tercer paciente ha evolucionado con deterioro clínico progresivo, requiere oxígeno adicional a permanencia y presenta hipertensión pulmonar, determinado por el extenso daño del pulmón remanente. Siendo la neumonectomía un procedimiento extremo en pediatría, a estos pacientes se les indicó por fracaso del tratamiento médico y empeoramiento del estado general. Dos niños han mostrado hasta ahora una adaptación satisfactoria a esta condiciónWe described the clinical and radiological evolution and pulmonary function tests in three patients aging 8 to 14 years old at present time. All of them suffered severe pneumonia caused by adenovirus infection during the infant period. During the acute viral disease, massive lung atelectasis refractory to treatment, determined the indication of pneumonectomy. All of the removed lungs presented bronchiectasis, atelectasis and fibrosis. In two children the medical condition improved after surgery, allowing to discontinue oxygen therapy. Wotwith standing, both of them had severe restrictive and obstructive limitation and signs of chronic damage at CTscan obstructive exacerbations decreased and the patients have been

  9. Inactivation of Adenovirus Type 5, Rotavirus WA and Male Specific Coliphage (MS2 in Biosolids by Lime Stabilization

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    Aaron B. Margolin

    2007-03-01

    Full Text Available The use of lime to reduce or eliminate pathogen content is a cost-effective treatment currently employed in many Class B biosolids production plants in the United States. A bench scale model of lime stabilization was designed to evaluate the survival of adenovirus type 5, rotavirus Wa, and the male specific bacteriophage, MS2, in various matrices. Each virus was initially evaluated independently in a reverse osmosis treated water matrix limed with an aqueous solution of calcium hydroxide for 24-hr at 22 ± 5°C. In all R/O water trials, adenovirus type 5, rotavirus Wa and MS2 were below detectable levels (<100.5 TCID50/mL and <1 PFU/mL respectively following 0.1-hr of liming. Adenovirus type 5, rotavirus Wa, and MS2, were inoculated into composted, raw and previously limed matrices, representative of sludge and biosolids, to achieve a final concentration of approximately 104 PFU or TCID50/mL. Each matrix was limed for 24-hr at 22 ± 5°C and 4 ± 2°C. In all trials virus was below detectable levels following a 24-hr incubation. The time required for viral inactivation varied depending on the temperature and sample matrix. This research demonstrates reduction of adenovirus type 5, rotavirus Wa, and male-specific bacteriophage, in water, sludge and biosolids matrices following addition of an 8% calcium hydroxide slurry to achieve a pH of 12 for 2-hr reduced to 11.5 for 22-hr by addition of 0.1 N HCl. In these trials, MS2 was a conservative indicator of the efficacy of lime stabilization of adenovirus Type 5 and rotavirus Wa and therefore is proposed as a useful indicator organism.

  10. Persistence of Giardia, Cryptosporidium, Rotavirus, and Adenovirus in treated sewage in São Paulo state, Brazil.

    Science.gov (United States)

    Tonani, K A A; Padula, J A; Julião, F C; Fregonesi, B M; Alves, R I S; Sampaio, C F; Beda, C F; Hachich, E M; Segura-Muñoz, S I

    2013-12-01

    Abstract :  The persistence of Giardia, Cryptosporidium, Rotavirus, and Adenovirus in samples of raw and treated sewage collected monthly in 2010 at the Biological Wastewater Treatment Plant of Ribeirão Preto, SP, Brazil, was analyzed. The USEPA Method 1623 was used to detect and quantify Giardia and Cryptosporidium. An enzyme immunoassay was carried out to test Rotavirus and Adenovirus antigen optical density (Rotascreen® and Adenoscreen®). The results show a significant decrease in the concentrations of Giardia, Rotavirus and Adenovirus (P Giardia concentrations ranged from 120 to 2,200 cysts/L in raw sewage and from 0.45 to 3.5 cysts/L in treated sewage. Cryptosporidium concentration ranged from undetectable to 28.9 oocysts/L in raw sewage and undetectable to 1.05 oocysts/L in treated sewage. Rotavirus presented absorbance values that ranged from 1.17 ± 0.81 in raw sewage to 0.46 ± 0.32 in treated sewage. Adenovirus, in turn, presented absorbance values of 0.64 ± 0.20 in raw sewage and of 0.45 ± 0.04 in treated sewage. There was no significant seasonal tendency observed in the distribution of protozoa (oo)cysts and in the viral antigen density in the monthly sewage samples during 2010 (P > 0.05). Even though these pathogenic agents decreased after treatment, the remaining loads observed in treated sewage can reach the watercourses receiving it. Giardia, Cryptosporidium, Rotavirus, and Adenovirus are pathogens with very low infectious doses, representing a public health risk especially for vulnerable groups, such as children living near these watercourses and homeless people using this water for various purposes. Studies addressing the environmental persistence of opportunistic pathogens in watercourses are hugely important in the public health sphere, especially in developing countries, where economic, social, cultural, and environmental factors still persist that are favorable to population's exposure to diarrhea-causing agents.

  11. Adenovirus-mediated expression of SSAT inhibits colorectal cancer cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Hui SUN; Bin LIU; Ya-pei YANG; Chun-xiao XU; Yun-fei YAN; Wei WANG; Xian-xi LIU

    2008-01-01

    Aim: To construct a recombinant adenovirus that can express human spermidine/ spermine N1-acetyltransferase (SSAT) and detect its inhibitory effect on colorectal cancer cell growth in vitro. Methods: A 516 bp eDNA of SSAT was amplified and cloned into a pGL3-hTERT plasmid. The pGL3-hTERT-SSAT recombinant was digested, and the small fragment was cloned into the shuttle vector pAdTrack. The pAdTrack-hTERT-SSAT plasmids were recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the HEK293 packaging cells (transformed human embryonic kidney cells) after they were lin-earized by PacI. The process of adenovirus packaging and amplification was monitored by green fluorescent protein (GFP) expression. The SSAT protein levels were determined by Western blotting, and the intracellular polyamine con-tent was detected by reverse-phase high performance liquid chromatography. The MTS (3-(4, 5-dimethylthiaol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(-4-sulfophenyl)-2H-tetrazolium, inner salt) and colony-forming assays were used to analyze the gene transduction efficiency and effect on the growth of HT-29 and LoVo cells. A viable cell count was used to determine the cell growth with or without exogenous polyamines. Results: The GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting results demonstrated that Ad-hTERT-SSAT could increase the expres-sion of SSAT, and consequently, spermidine and spermine were reduced to low levels. The MTS and colony-forming assay results showed that HT-29 and LoVo cell growth were significantly inhibited, and the inhibitory effect could be partially reversed by exogenous spermidine and spermine. Conclusion: The successfully constructed recombinant adenovirus Ad-hTERT-SSAT could accelerate polyamine catabolism and inhibit the colorectal cell growth in vitro. It also has therapeutic potential in the treatment of colorectal cancer.

  12. Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

    Science.gov (United States)

    Jerkofsky, M; Rapp, F

    1975-02-01

    Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.

  13. Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients with respiratory symptoms

    NARCIS (Netherlands)

    van de Pol, Alma C.; van Loon, Anton M.; Wolfs, Tom F. W.; Jansen, Nicolaas J. G.; Nijhuis, Monique; Breteler, Els Klein; Schuurman, Rob; Rossen, John W. A.

    2007-01-01

    Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-t

  14. 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.

  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

    is followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells do not appear to have been driven towards exhaustive differentiation. Based on these findings, we suggest that adenovirus based prime-boost regimens (including Ad5 and Ad5-like vectors) represent...... 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...... leads to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and KLRG1 expression. These memory CD8 T cells are capable of proliferating in response to viral challenge, and protect against infection with live virus. Furthermore, viral challenge...

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

    Science.gov (United States)

    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 identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections. PMID:24907427

  17. DETECTION OF HUMAN ENTEROVIRUS AND ADENOVIRUS IN SHELLFISH COLLECTED IN MOROCCO MEDITERRANEAN COAST

    Directory of Open Access Journals (Sweden)

    Laila Benabbes

    2013-10-01

    Full Text Available The aim of this study was the screening for the presence of enteric human virus in shellfish (clam and cockle collected from two production area in Moroccan Mediterranean coast. Between October 2006 and April 2008, forty four samples were collected and tested for viral contamination using cell culture (HEp-2 and Vero cells and integrated cell culture PCR. Overall, 88.6 % of all analysed samples were contaminated by at least one of the studied viruses, Adenovirus was detected in 52.3 % of the samples and Enterovirus in 36.3%. The presence of viruses in shellfish production area can represent a potential health risk by causing gastroenteritis. The procedure used in this study may be a tool for monitoring shellfish viral contamination in Morocco.

  18. 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.

  19. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

    Directory of Open Access Journals (Sweden)

    E. Gout

    2010-01-01

    Full Text Available Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

  20. Adenovirus Structure as Revealed by X-Ray Crystallography, Electron Microscopy, and Difference Imaging

    Science.gov (United States)

    Stewart, Phoebe L.; Burnett, Roger M.

    1993-03-01

    The three-dimensional structure of human type 2 adenovirus was studied by combining X-ray crystallography and electron microscopy in a novel way. The 2.9 Å crystal structure of the major capsid protein, hexon, was positioned into a three-dimensional image reconstruction of the intact virus that was derived from cryo-electron micrographs. A three-dimensional difference map was generated by subtracting 240 copies of the crystallographic hexon from the density of the intact virus. This map revealed several minor structural proteins acting as “cement” to stabilize the assembly. The current state of structural knowledge concerning the location of the polypeptide components and the viral DNA is presented.

  1. The long repeat region is dispensable for fowl adenovirus replication in vitro.

    Science.gov (United States)

    Ojkic, D; Nagy, E

    2001-05-10

    Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome. The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown. An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses. The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2. Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro. The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR.

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

    Science.gov (United States)

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

    2014-08-01

    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 identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.

  3. Ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro.

    Science.gov (United States)

    Hartline, Caroll B; Gustin, Kortney M; Wan, William B; Ciesla, Stephanie L; Beadle, James R; Hostetler, Karl Y; Kern, Earl R

    2005-02-01

    The acyclic nucleoside phosphonate cidofovir (CDV) and its closely related analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA) have been reported to have activity against many adenovirus (AdV) serotypes. A new series of orally active ether lipid-ester prodrugs of CDV and of (S)-HPMPA that have slight differences in the structure of their lipid esters were evaluated, in tissue-culture cells, for activity against 5 AdV serotypes. The results indicated that, against several AdV serotypes, the most active compounds were 15-2500-fold more active than the unmodified parent compounds and should be evaluated further for their potential to treat AdV infections in humans.

  4. The effect of purine and pyrimidine analogues and virazole on adenovirus replication.

    Science.gov (United States)

    Scheffler, P; Haghchenas, D; Wigand, R

    1975-04-01

    The multiplication of adenovirus 19 in HeLa cells was inhibited by various purine and pyrimidine analogues and by virazole. The formation of infectious virus and of capsid proteins (haemagglutin, group-specific complement-fixing antigen) was inhibited to the same degree, while the viral cytopathic effect (CPE) was not inhibited. The reversibility of the inhibition after removal of the substances was more complete for purine than for pyrimidine analogues. The inhibition was counteracted by simulataneous addition of the corresponding nucleosides. Adenosine was more effected than guanosine against purine analogues; both were partially effective against virazole, but none of them against arabinofuranosyladenine. The time-dependence of inhibition, the ensuing eclipse period after removal of the inhibitors, and the successive application of two inhibitors led to the conclusion that most of them affect the viral multiplication mainly by inhibition of DNA synthesis. Azacytidine inhibits the synthesis of structural proteins as well.

  5. A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus.

    Directory of Open Access Journals (Sweden)

    Mattia Gazzola

    2009-12-01

    Full Text Available Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors.

  6. A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus.

    Science.gov (United States)

    Gazzola, Mattia; Burckhardt, Christoph J; Bayati, Basil; Engelke, Martin; Greber, Urs F; Koumoutsakos, Petros

    2009-12-01

    Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors.

  7. A novel supervised trajectory segmentation algorithm identifies distinct types of human adenovirus motion in host cells.

    Science.gov (United States)

    Helmuth, Jo A; Burckhardt, Christoph J; Koumoutsakos, Petros; Greber, Urs F; Sbalzarini, Ivo F

    2007-09-01

    Biological trajectories can be characterized by transient patterns that may provide insight into the interactions of the moving object with its immediate environment. The accurate and automated identification of trajectory motifs is important for the understanding of the underlying mechanisms. In this work, we develop a novel trajectory segmentation algorithm based on supervised support vector classification. The algorithm is validated on synthetic data and applied to the identification of trajectory fingerprints of fluorescently tagged human adenovirus particles in live cells. In virus trajectories on the cell surface, periods of confined motion, slow drift, and fast drift are efficiently detected. Additionally, directed motion is found for viruses in the cytoplasm. The algorithm enables the linking of microscopic observations to molecular phenomena that are critical in many biological processes, including infectious pathogen entry and signal transduction.

  8. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    Science.gov (United States)

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  9. Use of an Automated Image Processing Program to Quantify Recombinant Adenovirus Particles

    Science.gov (United States)

    Obenauer-Kutner, Linda J.; Halperin, Rebecca; Ihnat, Peter M.; Tully, Christopher P.; Bordens, Ronald W.; Grace, Michael J.

    2005-02-01

    Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro® Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

  10. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    Science.gov (United States)

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C

    1998-01-01

    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  11. Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model

    Science.gov (United States)

    Simão, Daniel; Pinto, Catarina; Fernandes, Paulo; Peddie, Christopher J.; Piersanti, Stefania; Collinson, Lucy M.; Salinas, Sara; Saggio, Isabella; Schiavo, Giampietro; Kremer, Eric J.; Brito, Catarina; Alves, Paula M.

    2017-01-01

    Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in pre-clinical tests. For clinical translation, in-depth pre-clinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, while human adenovirus type 5 (HAdV5) showed increased tropism towards glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity. PMID:26181626

  12. Concentration of Enteroviruses, Adenoviruses, and Noroviruses from Drinking Water by Use of Glass Wool Filters▿

    Science.gov (United States)

    Lambertini, Elisabetta; Spencer, Susan K.; Bertz, Phillip D.; Loge, Frank J.; Kieke, Burney A.; Borchardt, Mark A.

    2008-01-01

    Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min−1), and its low cost makes it advantageous for studies requiring large numbers of samples. PMID:18359827

  13. Use of cidofovir in pediatric patients with adenovirus infection [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Lakshmi Ganapathi

    2016-04-01

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

  14. CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter

    Directory of Open Access Journals (Sweden)

    Bhatia S

    2016-11-01

    Full Text Available Shilpa Bhatia,1 Samia M O’Bryan,1 Angel A Rivera,2 David T Curiel,3 J Michael Mathis1 1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 2Departments of Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL, 3Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO, USA Abstract: Ad vectors are promising delivery vehicles for cancer therapeutic interventions. However, their application is limited by promiscuous tissue tropism and hepatotoxicity. This limitation can be avoided by altering the native tropism of Ads so that they can be redirected to the target cells through alternate cellular receptors. The CXCR4 chemokine receptor belongs to a large superfamily of G-protein-coupled receptors and is known to be upregulated in a wide variety of cancers, including breast cancer and melanoma. These receptors have been associated with cancer cell survival, progression, and metastasis. In the current study, an Ad to cancer cells overexpressing CXCR4 by using a bispecific adapter, sCAR-CXCL12, was retargeted. The sCAR-CXCL12 adapter contained the soluble ectodomain form of the native Ad5 receptor (sCAR, which was fused to a mature human chemokine ligand, CXCL12, through a short peptide linker. A dramatic increase in the infectivity of cancer cells using a targeted Ad vector compared with an untargeted vector was observed. Furthermore, sCAR-CXCL12 attenuated Ad infection of liver ex vivo and in vivo and enhanced Ad vector infection of xenograft tumors implanted in immunodeficient SCID-bg mice. Thus, the sCAR-CXCL12 adapter could be used to retarget Ad vectors to chemokine receptor-positive tumors. Keywords: adapter, adenovirus serotype 5, cancer, hCAR, human coxsackievirus and adenovirus receptor, chemokine receptor, CXCL12, CXCR4, gene therapy, retargeting, viral vector

  15. Adenovirus replication as an in vitro probe for drug sensitivity in human tumors.

    Science.gov (United States)

    Parsons, P G; Maynard, K R; Little, J H; McLeod, G R

    1986-04-01

    The feasibility of using adenovirus 5 as an in vitro probe for chemosensitivity in short-term cultures of human tumors was evaluated using human melanoma cell lines and primary cultures of melanoma biopsies. A convenient immunoperoxidase method was developed for quantitating viral replication 2 days after infection. Two different approaches were explored: the host cell reactivation assay (HCR) using drug-treated virus; and the viral capacity assay using drug-treated cells. The HCR assay detected sensitivity to 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) in Mer- (methyl excision repair deficient) cell lines as decreased ability of the cells to replicate MTIC-treated virus. This test should be applicable to DNA-damaging agents and repair-deficient tumors. Adenovirus replicated readily in nonproliferating primary cultures of melanoma biopsies; application of the HCR assays to this material identified one Mer- sample of 11 tested. Herpes viruses were not suitable for use in HCR because herpes simplex virus type 1 failed to distinguish Mer- from Mer+ melanoma cells; and nonproductive infection of MTIC-sensitive lymphoid cells with Epstein-Barr virus yielded an MTIC-resistant cell line. The second assay (viral capacity) involved determination of the inhibition of replication of untreated virus in treated cells. This approach correctly predicted sensitivity to hydroxyurea and deoxyadenosine in melanoma cell lines when compared with clonogenic survival assay. Viral capacity was also inhibited by cytosine arabinoside, fluorouracil, vincristine, adriamycin, 6-mercaptopurine and ionising radiation, and may therefore be useful for detecting sensitivity to a wide range of antitumor agents.

  16. Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines.

    Science.gov (United States)

    Musk, P; Stowers, A; Parsons, P G

    1990-02-15

    Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.

  17. 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.

  18. GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; HAN De-min; WANG Wen-ge; WU Zu-ze; ZHANG Wei

    1999-01-01

    Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer cell line Hep-2 was used.Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

  19. Pathogenicity and cytokine gene expression pattern of a serotype 4 fowl adenovirus isolate.

    Directory of Open Access Journals (Sweden)

    Helena Grgić

    Full Text Available Hydropericardium-hepatitis syndrome (HHS, a recently emerged disease of chickens, is caused by some strains of fowl adenovirus serotype 4 (FAdV-4. In this study, a Canadian FAdV-4 isolate, designated as FAdV-4 ON1, was evaluated for pathogenicity after oral and intramuscular (im infection of specific pathogen free (SPF chickens. Pathogenicity was evaluated by observation of clinical signs and gross and histological lesions. The highest viral DNA copy numbers, irrespective of the inoculation route, were detected in the cecal tonsils. Virus titers in cloacal swabs collected over the entire study period were compared between the orally and im inoculated chickens, and the difference in titers between the two groups was significant (P<0.001, the oral group had a higher rank. The antibody response of infected chickens tested by an adenovirus-specific ELISA showed a statistically significant (P<0.001 difference between the orally and im inoculated chickens. The im inoculated chickens had higher values than birds inoculated orally (P<0.001. Serum samples from both groups collected at 14 days post-infection completely neutralized FAdV-4 ON1. In addition, the effects of FAdV-4 ON1 infection on transcription of a number of avian cytokines were studied in vivo. The expression of interferon (IFN-γ and interleukin (IL-10 in the liver was induced at early times after infection. This FAdV-4 ON1 potentially could be used as a live vaccine against HHS and developed as vaccine vector. The GenBank/EMBL/DDBJ accession number for the FAdV-4 ON1 sequence is GU188428.

  20. Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

    Science.gov (United States)

    Bradshaw, Angela C.; Coughlan, Lynda; Miller, Ashley M.; Alba, Raul; van Rooijen, Nico; Nicklin, Stuart A.; Baker, Andrew H.

    2012-01-01

    The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton–αvintegrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1 + sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery. PMID:22626939

  1. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Ayesha; Ellenson, Lora Hedrick, E-mail: lora.ellenson@med.cornell.edu

    2011-07-01

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten{sup +/-} mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten{sup +/-} mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ER{alpha} as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  2. Protection against Mucosal SHIV Challenge by Peptide and Helper-Dependent Adenovirus Vaccines

    Directory of Open Access Journals (Sweden)

    K. Jagannadha Sastry

    2009-11-01

    Full Text Available Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env peptides and first generation adenovirus serotype 5 (FG-Ad5 vaccines expressing the same peptides were immunized intramuscularly three times with helperdependent adenovirus (HD-Ad vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5-immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in the HD-Ad5 group. In this study, neutralizing antibody and T cell responses were compared between the groups before and after rectal challenge with CCR5-tropic SHIV-SF162P3. When serum samples were assayed for neutralizing antibodies, only weak activity was observed. T cell responses against env epitopes were higher in the serotype-switched group. When these animals were challenged rectally with SHIV-SF162P3, both the Ad5 and serotype-switch groups significantly reduced peak viral loads 2 to 10-fold 2 weeks after infection. Peak viral loads were significantly lower for the serotype-switched group as compared to the HD-Ad5-immunized group. Viral loads declined over 18 weeks after infection with some animals viremia reducing nearly 4 logs from the peak. These data demonstrate significant mucosal vaccine effects after immunization with only env antigens. These data also demonstrate HD-Ad vectors are a robust platform for vaccination.

  3. Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

    Institute of Scientific and Technical Information of China (English)

    Xin-Bo Xue; Kun Chen; Cong-Jun Wang; Jian-Wei Zheng; Yuan Yu; Zhi-Hai Peng; Zai-De Wu

    2008-01-01

    BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modiifed Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was conifrmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and lfow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P CONCLUSIONS: mda-7 selectively induces growth inhibi-tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

  4. Role of cellular heparan sulfate proteoglycans in infection of human adenovirus serotype 3 and 35.

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    Sebastian Tuve

    2008-10-01

    Full Text Available Species B human adenoviruses (Ads are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs. We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.

  5. 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.

  6. Chapter three--Syrian hamster as an animal model to study oncolytic adenoviruses and to evaluate the efficacy of antiviral compounds.

    Science.gov (United States)

    Wold, William S M; Toth, Karoly

    2012-01-01

    The Syrian (golden) hamster (Mesocricetus auratus) has served as a useful model for different aspects of biology for at least 50 years, and its use has been expanding recently. In earlier years, among other things, it was a model for cancer development. More recently, it has become a model for many different infectious diseases. It has also become an alternative model for the study of oncolytic adenovirus vectors for cancer gene therapy. Among several other human pathogens, the hamster is permissive for the replication of human species C adenoviruses, which are the parental virus for the majority of adenovirus vectors in use today. These vectors replicate in some of the established hamster tumor cell lines that can be used to generate tumors in vivo, that is, one can study oncolytic (replication competent) adenoviruses in a permissive, immunocompetent model. This has afforded the opportunity to study the effect of the host immune system on the vector-infected tumor and has allowed the use of a more relevant animal model to determine the safety and biodistribution of replication-competent adenoviruses. The hamster has also been used to evaluate antiviral compounds and vaccines against many viruses, including adenoviruses, flaviviruses, alphaviruses, arenaviruses, bunyaviruses, and paramyxoviruses.

  7. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui

    2011-01-01

    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

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

    Science.gov (United States)

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

    2017-01-01

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

  9. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    Science.gov (United States)

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  11. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  12. [Autoreactive TCD8+ lymphocytes in patients with chronic myeloid leukemia in association with HLA and adenovirus infection].

    Science.gov (United States)

    Rivera, Sergio E; Echeverría, Miriam; Salcedo, Pedro; Márquez, Georgina; Carrillo, Zuhey; Parra, Yennis; Cipriani, Ana María; Núñez, José R; Álvarez de Mon, Melchor; Farruco, Atilio

    2016-01-01

    Antecedentes: algunos adenovirus se han señalado como activadores clonales en leucemias. El alelo HLA-DRB1* 14 subtipos DRB1*14:21, 14:22, 14:45, 14:26, 14:33, 14:51, 14:35 se asociaron con leucemia mieloide crónica (LMC) en pacientes venezolanos. Objetivo: evaluar el mimetismo molecular entre el adenovirus y la estructura del antígeno HLA-DRB1*14 que exhiben el mismo cambio en la posición de aminoácido del epítopo DR53. Material y método: estudio experimental realizado en el IHO Banco de Sangre del Estado Zulia, Venezuela en muestras de sangre periférica de pacientes con LLA, LMC y controles sanos. Se realizaron cultivo mixto de linfocitos, serología, proliferación linfocitaria y citofluorometría. Resultados: los linfocitos DRB1*14 del paciente reaccionaron en 48 horas versus los linfocitos DRB1*14 estimuladores, que exhibieron aumento de los linfocitos T CD8+. Los pacientes con LMC tuvieron un perfil serológico diferente contra el adenovirus. Sólo pacientes con LMC reaccionaron frente al péptido secuencia LLERRRA con incremento de las células TCD8+. Conclusión: se estableció que la relación leucemia mieloide crónica, HLA-DRB1*14, células TCD8+ de memoria autorreactivas y TCD8+ en respuesta específica frente al adenovirus podría estar en el origen de la leucemia mieloide crónica de pacientes venezolanos.

  13. Adenovirus Activates Complement by Distinctly Different Mechanisms In Vitro and In Vivo: Indirect Complement Activation by Virions In Vivo▿

    OpenAIRE

    Tian, Jie; Xu, Zhili; Jeffrey S Smith; Hofherr, Sean E.; Barry, Michael A.; Byrnes, Andrew P.

    2009-01-01

    Understanding innate immunity is key to improving the safety of adenovirus (Ad) vectors for systemic gene therapy. Ad has been shown to activate complement in vitro, but activation of complement after Ad injection in vivo has not been directly measured. Using complement protein C3a as a marker of complement activation, we show that types 2 and 5 human Ads cause rapid complement activation after intravenous injection in mice. Unexpectedly, the mechanisms in vivo were different than those in vi...

  14. 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.

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

    OpenAIRE

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

    2004-01-01

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

  16. Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study

    OpenAIRE

    Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Chengjun BAN; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen

    2014-01-01

    Introduction Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. Methods We conducted a prospective, single-center observational study of pneumonia with A...

  17. Acceleration of adenovirus replication and increased virion production by treatment with the steroid hormone 17 beta-estradiol.

    Science.gov (United States)

    James, C B; Vanderpool, E A; Roane, P

    1992-01-01

    We report here that concentration of an estrogen known to promote enhanced transformation and to increase oncogenicity of rat embryo cells, accelerate the production and increase the yield of progeny virions in adenovirus type 12 (Ad 12)-infected HEp-2 cells. Further, measurement of the incorporation of radioactive RNA and DNA precursors indicated that macromolecular synthesis in the estrogen-treated, infected cells was accelerated. Possible explanations for this observation are discussed.

  18. 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.

  19. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    OpenAIRE

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing

    2013-01-01

    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The effica...

  20. Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.

    Science.gov (United States)

    Schagen, F H; Rademaker, H J; Rabelink, M J; van Ormondt, H; Fallaux, F J; van der Eb, A J; Hoeben, R C

    2000-09-01

    In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

  1. Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

    Institute of Scientific and Technical Information of China (English)

    SU Jia-qiang; CHI Bao-rong; LI Xiao; LIU Lei; LIU Li-ming; QI Yan-xin; WANG Zhuo-yue; JIN Ning-yi

    2012-01-01

    We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer(EC).The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses (Ad-GP,Ad-Apoptin,Ad-EGFP) in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro.In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assays,the growth of EC-109 cells was slightly inhibited by Ad-GP.Ad-Apoptin and Ad-EGFP.However,Ad-VP induced a significant cytotoxic effect.Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro,detected by 4′,6-diamidino-2-phenylindole(DAPI) or acridine orange and ethidium bromide staining.The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential(△ψm),the release of cytochrome c and the activation of caspase-3,6 and 7 in Ad-VP infiected EC-109 cells.In contrast,all these assays show almost no effects of the recombinant adenoviruses on L02 cells.These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells.Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

  2. Survival of adenovirus types 2 and 41 in surface and ground waters measured by a plaque assay.

    Science.gov (United States)

    Rigotto, C; Hanley, K; Rochelle, P A; De Leon, R; Barardi, C R M; Yates, M V

    2011-05-01

    To manage artificial recharge systems, it is necessary to understand the inactivation process of microorganisms within aquifers so that requirements regarding storage times and treatment strategies for ground and surface waters can be developed and modeled to improve water management practices. This study was designed to investigate the survival of representative adenoviruses in surface- and groundwaters using a cell culture plaque assay with human lung carcinoma cells (A549) to enumerate surviving viruses. Adenovirus types 2 (Ad2) and 41 (Ad41) were seeded into 50 mL of three sterilized surface waters and groundwaters, and incubated at 10 and 19 °C for up to 301 days. Concentrations of Ad2 and Ad41 were relatively stable in all waters at 10 °C for at least 160 days and in some instances up to 301 days. At 19 °C, virus concentrations were reduced by 99.99% (4 log) after 301 days in surface water. There was approximately 90% (1 log) reduction of both viruses at 19 °C after 160 days of incubation in groundwater samples. There was no overall difference in survival kinetics in surface waters compared to groundwaters. The relatively high stability and long-term survival of adenoviruses in environmental waters at elevated temperatures should be considered in risk assessment models and drinking water management strategies.

  3. Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinheng [College of Animal Science, South China Agricultural University & Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642 (China); Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642 (China); South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642 (China); Zhong, Yangjin; Zhou, Zhenhai; Liu, Yang [College of Animal Science, South China Agricultural University & Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642 (China); Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642 (China); Zhang, Huanmin [USDA, Agriculture Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI 48823 (United States); Chen, Feng [College of Animal Science, South China Agricultural University & Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642 (China); Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642 (China); Chen, Weiguo [College of Animal Science, South China Agricultural University & Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642 (China); Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642 (China); South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642 (China); Xie, Qingmei, E-mail: qmx@scau.edu.cn [College of Animal Science, South China Agricultural University & Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642 (China); Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642 (China); South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642 (China)

    2016-06-15

    This study aimed to characterize a novel adenovirus (AdV) isolated from diseased Muscovy ducks in China. After the AdV was successfully propagated in duck embryo fibroblasts, the morphological and physicochemical properties of the virions were studied by electron microscopy and different tests. The results of the analyses were in conformity with AdV properties. The full genome sequence was determined and analyzed. The new isolate (named CH-GD-12-2014) shared over 91% sequence identity with duck AdV-2 representing the species Duck aviadenovirus B. The most important distinguishing feature between the two DAdV strains was the presence of a second fiber gene in the Chinese isolate. Phylogeny reconstruction confirmed the affiliation of the virus with goose and duck AdVs in the genus Aviadenovirus. Experimental infection resulted in embryo death, and intramuscular inoculation provoked morbidity and mortality among ducks and chickens. - Highlights: • A duck adenovirus type 3 was isolated and the complete genome of DAdV-3 was obtained. • Physicochemical properties and electron microscopy were researched. • Pathogenicity of duck adenovirus type 3 was researched.

  4. 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.

  5. 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.

  6. Adenovirus-based strategies overcome temozolomide resistance by silencing the O6-methylguanine-DNA methyltransferase promoter.

    Science.gov (United States)

    Alonso, Marta M; Gomez-Manzano, Candelaria; Bekele, B Nebiyou; Yung, W K Alfred; Fueyo, Juan

    2007-12-15

    Currently, the most efficacious treatment for malignant gliomas is temozolomide; however, gliomas expressing the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) are resistant to this drug. Strong clinical evidence shows that gliomas with methylation and subsequent silencing of the MGMT promoter are sensitive to temozolomide. Based on the fact that adenoviral proteins directly target and inactivate key DNA repair genes, we hypothesized that the oncolytic adenovirus Delta-24-RGD could be successfully combined with temozolomide to overcome the reported MGMT-mediated resistance. Our studies showed that the combination of Delta-24-RGD and temozolomide induces a profound therapeutic synergy in glioma cells. We observed that Delta-24-RGD treatment overrides the temozolomide-mediated G(2)-M arrest. Furthermore, Delta-24-RGD infection was followed by down-modulation of the RNA levels of MGMT. Chromatin immunoprecipitation assays showed that Delta-24-RGD prevented the recruitment of p300 to the MGMT promoter. Importantly, using mutant adenoviruses and wild-type and dominant-negative forms of the p300 protein, we showed that Delta-24-RGD interaction with p300 was required to induce silencing of the MGMT gene. Of further clinical relevance, the combination of Delta-24-RGD and temozolomide significantly improved the survival of glioma-bearing mice. Collectively, our data provide a strong mechanistic rationale for the combination of oncolytic adenoviruses and temozolomide, and should propel the clinical testing of this therapy approach in patients with malignant gliomas.

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

    Science.gov (United States)

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

    1998-03-01

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

  8. Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

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    Takeshi Nishimoto

    Full Text Available The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

  9. Effects of capsid-modified oncolytic adenoviruses and their combinations with gemcitabine or silica gel on pancreatic cancer.

    Science.gov (United States)

    Kangasniemi, Lotta; Parviainen, Suvi; Pisto, Tommi; Koskinen, Mika; Jokinen, Mika; Kiviluoto, Tuula; Cerullo, Vincenzo; Jalonen, Harry; Koski, Anniina; Kangasniemi, Anna; Kanerva, Anna; Pesonen, Sari; Hemminki, Akseli

    2012-07-01

    Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.

  10. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy.

    Science.gov (United States)

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing

    2014-01-15

    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.

  11. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection

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    Zhao, Hongxing, E-mail: Hongxing.Zhao@igp.uu.se [The Beijer Laboratory, Dept. of Immunology, Genetics and Pathology, Uppsala University, S-751 85 Uppsala (Sweden); Chen, Maoshan [Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086 (Australia); Lind, Sara Bergström [Department of Chemistry-BMC, Analytical Chemistry, Science for Life Laboratory, Uppsala University, Box 599, SE-751 24 Uppsala (Sweden); Pettersson, Ulf [The Beijer Laboratory, Dept. of Immunology, Genetics and Pathology, Uppsala University, S-751 85 Uppsala (Sweden)

    2016-05-15

    The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phase and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction. - Highlights: • The expression of 398 lncRNAs showed a distinct temporal pattern during Ad2 infection. • 80% of the deregulated lncRNAs were up-regulated during the late phase of infection. • The deregulated lncRNAs potentiallyinteract with 33 cellular RNA binding proteins. • These RBPs are involved in RNA splicing, nuclear export and translation. • Adenovirus exploits the cellular lncRNA network to optimize its replication.

  12. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  14. Lung parenchymal change after the resolution of adenovirus pneumonia : chest radiographs and high-resolution CT findings

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    Yoon, Jung Hee; Kim, Joung Sook; Kim, Chang Kuen; Kang, Seung Pyung; Lee, Soo Hyun; Hur Gham [Inje Univ. Sanggye Paik Hospital, Seoul (Korea, Republic of)

    1998-07-01

    To evaluate lung parenchymal change as seen on chest radiographs and high-resolution CT (HRCT) after the resolution of adenovirus pneumonia (a common cause of lower respiratory infection in infants and children),and the usefulness of HRCT during follow-up. Material and Methods : Four to 13(mean, 8) months after recovery, ten patients infected with adenovirus pneumonia underwent HRCT and chest radiographs. Eight were boys and two were girls, and their mean age was 26(range, 14-45) months. Adenovirus pneumonia had been confirmed by viral isolation in culture or serologic test. CT scanning was performed during quiet breathing ; collimation was 2 mm and the interval from apex to diaphragm was 5-10 mm. Lung settings were 1600 HU (window width) and -700 HU(level). CT findings were assessed and compared with chest radiographs by two chest radiologists, who reached a consensus. The patients were clinically followed up for one year. Result : On chest radiographs, hyperlucent lung was seen in 8 of 10 patients (80%) ; in one other there was partial collapse, and in one, findings were normal. The most common HRCT finding was a mosaic pattern of lung attenuation with decreased pulmonary vascularity in the area of lower attenuation ; this was seen in 8 of 10 patients (80%). Other findings were partial collapse, bronchiectasis, and bronchial wall thickening, each seen in two patients, and reticulonodular density, seen in one. In two patients HRCT findings were normal ; in one of these, chest findings were normal but a mosaic pattern of lung attenuation was found in all lobes. During follow-up, three patients wheezed continuously. Conclusion : In cases of adenovirus pneumonia, HRCT demonstrated more specific parenchymal change than did chest radiographs ; a mosaic pattern of lung attenuation was seen, with decreased pulmonary vascularity in areas of lower attenuation ; bronchiectasis,bronchial wall thickening, and reticulo-odular density were also noted. These findings were

  15. Severe Community-Acquired Pneumonia Caused by Human Adenovirus in Immunocompetent Adults: A Multicenter Case Series.

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    Dingyu Tan

    Full Text Available Severe community-acquired pneumonia (CAP caused by human adenovirus (HAdV, especially HAdV type 55 (HAdV-55 in immunocompetent adults has raised increasing concerns. Clinical knowledge of severe CAP and acute respiratory distress syndrome induced by HAdV-55 is still limited, though the pathogen has been fully characterized by whole-genome sequencing.We conducted a multicentre retrospective review of all consecutive patients with severe CAP caused by HAdV in immunocompetent adults admitted to the Emergency Department Intensive Care Unit of two hospitals in Northern China between February 2012 and April 2014. Clinical, laboratory, radiological characteristics, treatments and outcomes of these patients were collected and analyzed.A total of 15 consecutive severe CAP patients with laboratory-confirmed adenovirus infections were included. The median age was 30 years and all cases were identified during the winter and spring seasons. HAdV-55 was the most frequently (11/15 detected HAdV type. Persistent high fever, cough and rapid progression of dyspnea were typically reported in these patients. Significantly increased pneumonia severity index (PSI, respiratory rate, and lower PaO2/FiO2, hypersensitive CRP were reported in non-survivors compared to survivors (P = 0.013, 0.022, 0.019 and 0.026, respectively. The rapid development of bilateral consolidations within 10 days after illness onset were the most common radiographic finding, usually accompanied by adjacent ground glass opacities and pleural effusions. Total mortality was 26.7% in this study. Corticosteroids were prescribed to 14 patients in this report, but the utilization rate between survivors and non-survivors was not significant.HAdV and the HAdV-55 sub-type play an important role among viral pneumonia pathogens in hospitalized immunocompetent adults in Northern China. HAdV should be tested in severe CAP patients with negative bacterial cultures and a lack of response to antibiotic

  16. Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

    Institute of Scientific and Technical Information of China (English)

    Li Li; Jia-Ling Huang; Qi-Cai Liu; Pei-Hong Wu; Ran-Yi Liu; Yi-Xin Zeng; Wen-Lin Huang

    2004-01-01

    AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402xenografted tumors.METHODS: Immunohistochemistry analysis with an anti endostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of 1×109 pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-turmoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELTSA).RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/ LacZ control group (t=2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin.Plasma endostatin levels peaked at 87.52±8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23±2.54 ng/mL). By d 7,plasma levels dropped to nearly half the peak level(40.34±4.80 ng/mL).CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous

  17. Key role of splenic myeloid DCs in the IFN-alphabeta response to adenoviruses in vivo.

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    György Fejer

    2008-11-01

    Full Text Available The early systemic production of interferon (IFN-alphabeta is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-alphabeta response to human adenoviruses (Ads in mice. By comparing the responses of normal, myeloid (mDC- and plasmacytoid (pDC-depleted mice and by measuring IFN-alphabeta mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-alphabeta production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-alphabeta response does not require Toll-like receptors (TLR, known cytosolic sensors of RNA (RIG-I/MDA-5 and DNA (DAI recognition and interferon regulatory factor (IRF-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-alphabeta and IL-6 in vivo by distinct pathways and confirm that IFN-alphabeta positively regulates the IL-6 response. Finally, by measuring TNF-alpha responses to LPS in Ad-infected wild type and IFN-alphabetaR(-/- mice, we show that IFN-alphabeta is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-alphabeta response, which is responsible for the bulk of IFN-alphabeta production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-alphabeta induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the

  18. The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species.

    Science.gov (United States)

    Dicks, Matthew D J; Guzman, Efrain; Spencer, Alexandra J; Gilbert, Sarah C; Charleston, Bryan; Hill, Adrian V S; Cottingham, Matthew G

    2015-02-25

    Adenovirus vaccine vectors generated from new viral serotypes are routinely screened in pre-clinical laboratory animal models to identify the most immunogenic and efficacious candidates for further evaluation in clinical human and veterinary settings. Here, we show that studies in a laboratory species do not necessarily predict the hierarchy of vector performance in other mammals. In mice, after intramuscular immunization, HAdV-5 (Human adenovirus C) based vectors elicited cellular and humoral adaptive responses of higher magnitudes compared to the chimpanzee adenovirus vectors ChAdOx1 and AdC68 from species Human adenovirus E. After HAdV-5 vaccination, transgene specific IFN-γ(+) CD8(+) T cell responses reached peak magnitude later than after ChAdOx1 and AdC68 vaccination, and exhibited a slower contraction to a memory phenotype. In cattle, cellular and humoral immune responses were at least equivalent, if not higher, in magnitude after ChAdOx1 vaccination compared to HAdV-5. Though we have not tested protective efficacy in a disease model, these findings have important implications for the selection of candidate vectors for further evaluation. We propose that vaccines based on ChAdOx1 or other Human adenovirus E serotypes could be at least as immunogenic as current licensed bovine vaccines based on HAdV-5.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  20. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    Science.gov (United States)

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  1. Characteristics of Adenovirus Pneumonia in Korean Military Personnel, 2012-2016.

    Science.gov (United States)

    Yoon, Hee; Jhun, Byung Woo; Kim, Hojoong; Yoo, Hongseok; Park, Sung Bum

    2017-02-01

    Adenovirus (AdV) can cause severe pneumonia in non-immunocompromised host, but limited data exist on the distinctive characteristics of AdV pneumonia in non-immunocompromised patients. We evaluated distinctive clinico-laboratory and radiological characteristics and outcomes of AdV pneumonia (n = 179), compared with non-AdV pneumonia (n = 188) in Korean military personnel between 2012 and 2016. AdV pneumonia patients had a higher rate of consolidation with ground-glass opacity (101/152) in lobar distribution (89/152) on computed tomography (CT) (P pneumonia patients. Bacterial co-infection was identified in 28.5% of AdV pneumonia. Most of the AdV isolates typed (69/72, 95.8%) were AdV-55. Patients with a pneumonia severity index ≥ class III were more commonly observed in AdV pneumonia patients compared with non-AdV pneumonia patients (11.2% vs. 2.1%, P pneumonia patients compared with the non-AdV pneumonia patients (3.8 vs. 2.6 days, P pneumonia patients, one of whom died due to AdV-55. Our data showed that AdV pneumonia in non-immunocompromised patients had distinct characteristics and most of the isolates typed in our study were AdV-55. It is suggested that AdV-55 is an important pathogen of pneumonia in Korean military personnel.

  2. Polyethylene glycol-grafted polyethylenimine used to enhance adenovirus gene delivery.

    Science.gov (United States)

    Singarapu, Kumar; Pal, Ivy; Ramsey, Joshua D

    2013-07-01

    An improved adenoviral-based gene delivery vector was developed by complexing adenovirus (Ad) with a biocompatible, grafted copolymer PEG-g-PEI composed of polyethylene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tropism, tendency to elicit an immune response, and susceptibility to inactivating antibodies makes the virus less than ideal. The goal of the current study was to determine whether Ad could be complexed with a PEG-g-PEI copolymer that would enable the virus to transduce cells lacking the Ad receptor, while avoiding the issues commonly associated with PEI. A copolymer library was synthesized using 2 kDa PEG and either linear or branched PEI (25 kDa) with a PEG to PEI grafting ratio of 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negative NIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropriately sized than PEI/Ad complexes.

  3. Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

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    Joselma Siqueira-Silva

    2009-08-01

    Full Text Available The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

  4. Outcomes of early administration of cidofovir in non-immunocompromised patients with severe adenovirus pneumonia.

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    Se Jin Kim

    Full Text Available The benefits of treatment with antiviral therapy for severe adenovirus (AdV pneumonia are not well established. We described the clinical characteristics and treatment outcomes of early cidofovir treatment of severe AdV pneumonia in non-immunocompromised patients. We retrospectively reviewed the medical records of all patients diagnosed with severe AdV pneumonia between 2012 and 2014. A total of seven non-immunocompromised patients with severe AdV pneumonia were identified, and all isolates typed (n = 6 were human AdV-B55. All patients had progressive respiratory failure with lobar consolidation with or without patchy ground glass opacity. Three patients required vasopressors and mechanical ventilation. All patients had abnormal laboratory findings including: leukopenia, thrombocytopenia, or elevated liver enzymes. After admission, all patients received antiviral therapy with cidofovir, and the median time from admission to cidofovir administration was 48 h and median the time from onset of symptoms to cidofovir administration was 7.1 days. After cidofovir administration, complete symptomatic improvement occurred after a median of 12 days and radiographic resolution occurred after a median of 21 days. Consequently, all patients completely improved without complications. Our data suggest that early administration of cidofovir in the course of treatment for respiratory failure as a result of AdV pneumonia in non-immunocompromised patients could be a treatment strategy worth considering, especially in cases of HAdV-55 infection.

  5. Adenovirus and “Culture-Negative Sepsis” in a Preterm Neonate

    Science.gov (United States)

    Moallem, Mohannad; Song, Eunkyung; Jaggi, Preeti; Conces, Miriam R.; Kajon, Adriana E.; Sánchez, Pablo J.

    2016-01-01

    Background Respiratory viral infections remain an underrecognized cause of morbidity and mortality among preterm infants in the neonatal intensive care unit (NICU). Case Report An eight day old, 650 gram birth weight, 23 weeks' gestational age female developed “culture-negative” sepsis manifested by respiratory deterioration, hypoxia, leukocytosis, and thrombocytopenia. She was diagnosed with pneumonia and hepatitis due to adenovirus HAdV-D (H29F9) by polymerase chain reaction (PCR) testing, but died at the age of 18 days despite treatment with cidofovir and immune globulin intravenous. Conclusion As the ability to diagnosis respiratory viral infections in the NICU has improved greatly with the use of PCR testing, the impact and contribution of these viruses to neonatal disease is now being recognized and the notion of “culture-negative” sepsis needs reassessment. The diagnosis of these infections in high risk infants is important not only for etiologic and epidemiologic reasons but ultimately for informing antimicrobial stewardship efforts. PMID:27924246

  6. Outcomes of early administration of cidofovir in non-immunocompromised patients with severe adenovirus pneumonia.

    Science.gov (United States)

    Kim, Se Jin; Kim, Kang; Park, Sung Bum; Hong, Duck Jin; Jhun, Byung Woo

    2015-01-01

    The benefits of treatment with antiviral therapy for severe adenovirus (AdV) pneumonia are not well established. We described the clinical characteristics and treatment outcomes of early cidofovir treatment of severe AdV pneumonia in non-immunocompromised patients. We retrospectively reviewed the medical records of all patients diagnosed with severe AdV pneumonia between 2012 and 2014. A total of seven non-immunocompromised patients with severe AdV pneumonia were identified, and all isolates typed (n = 6) were human AdV-B55. All patients had progressive respiratory failure with lobar consolidation with or without patchy ground glass opacity. Three patients required vasopressors and mechanical ventilation. All patients had abnormal laboratory findings including: leukopenia, thrombocytopenia, or elevated liver enzymes. After admission, all patients received antiviral therapy with cidofovir, and the median time from admission to cidofovir administration was 48 h and median the time from onset of symptoms to cidofovir administration was 7.1 days. After cidofovir administration, complete symptomatic improvement occurred after a median of 12 days and radiographic resolution occurred after a median of 21 days. Consequently, all patients completely improved without complications. Our data suggest that early administration of cidofovir in the course of treatment for respiratory failure as a result of AdV pneumonia in non-immunocompromised patients could be a treatment strategy worth considering, especially in cases of HAdV-55 infection.

  7. Antigen Gene Transfer to Human Plasmacytoid Dendritic Cells Using Recombinant Adenovirus and Vaccinia Virus Vectors

    Directory of Open Access Journals (Sweden)

    Hetty J. Bontkes

    2005-01-01

    Full Text Available Recombinant adenoviruses (RAd and recombinant vaccinia viruses (RVV expressing tumour-associated antigens (TAA are used as anti-tumour vaccines. It is important that these vaccines deliver the TAA to dendritic cells (DC for the induction of a strong immune response. Infection of myeloid DC (MDC with RAd alone is relatively inefficient but CD40 retargeting significantly increases transduction efficiency and DC maturation. Infection with RVV is efficient without DC maturation. Plasmacytoid dendritic cells (PDC play a role in the innate immune response to viral infections through the secretion of IFNα but may also play a role in specific T-cell induction. The aim of our study was to investigate whether PDC are better targets for RAd and RVV based vaccines. RAd alone hardly infected PDC (2% while CD40 retargeting did not improve transduction efficiency, but it did increase PDC maturation (25% CD83 positive cells. Accordingly, specific CTL activation by RAd infected PDC was limited (the number of IFNγ producing CTL was reduced by 75% compared to stimulation with peptide loaded PDC. RVV infected PDC specifically stimulated CTL but PDC were not activated. These Results indicate that PDC are not ideal targets for RAd and RVV based vaccines. However, PDC induced specific CTL activation after pulsing with recombinant protein, indicating that PDC can also cross-present antigens released from surrounding infected cells.

  8. Potential of mesenchymal stem cells by adenovirus-mediated erythropoietin gene therapy approaches for bone defect.

    Science.gov (United States)

    Li, Chen; Ding, Jian; Jiang, Liming; Shi, Ce; Ni, Shilei; Jin, Han; Li, Daowei; Sun, Hongchen

    2014-11-01

    Regeneration of large bone defects is a common clinical problem. Recent studies have shown that mesenchymal stem cells (MSCs) have emerged as a promising alternative to traditional surgical techniques. However, it is still a key question how to enhance the osteogenic potential of MSCs for possible clinical trials. The aim of the present study was to investigate the effect of adenovirus-mediated erythropoietin (Ad-EPO) transfer on BMSCs, we performed extensive in vitro/in vivo assays in this study. Flow cytometry analysis and the result of MTT showed that EPO could promote BMSCs proliferation. QPCR data demonstrated that EPO increased expressions of Runx2, Sp7, and Col1 in osteoblast at various time points and also increased alkaline phosphatase activity and the calcium deposition. These results indicate that EPO can increase the differentiation of osteoblast. Importantly, in vivo assays clearly demonstrate that EPO can efficiently induce new bone formation in the bone defect model. Our results strongly suggest that EPO can affect osteoblast differentiation and play important roles in bone regeneration leading to an increase in bone formation.

  9. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    Science.gov (United States)

    Wickham, T J; Tzeng, E; Shears, L L; Roelvink, P W; Li, Y; Lee, G M; Brough, D E; Lizonova, A; Kovesdi, I

    1997-11-01

    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.

  10. Activation of the human beta interferon gene by the adenovirus type 12 E1B gene

    Energy Technology Data Exchange (ETDEWEB)

    Shiroki, K.; Toth, M.

    1988-01-01

    The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogeneously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.

  11. 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

  12. Immunosuppression enhances oncolytic adenovirus replication and antitumor efficacy in the Syrian hamster model.

    Science.gov (United States)

    Thomas, Maria A; Spencer, Jacqueline F; Toth, Karoly; Sagartz, John E; Phillips, Nancy J; Wold, William S M

    2008-10-01

    We recently described an immunocompetent Syrian hamster model for oncolytic adenoviruses (Ads) that permits virus replication in tumor cells as well as some normal tissues. This model allows exploration of interactions between the virus, tumor, normal organs, and host immune system that could not be examined in the immunodeficient or nonpermissive animal models previously used in the oncolytic Ad field. Here we asked whether the immune response to oncolytic Ad enhances or limits antitumor efficacy. We first determined that cyclophosphamide (CP) is a potent immunosuppressive agent in the Syrian hamster and that CP alone had no effect on tumor growth. Importantly, we found that the antitumor efficacy of oncolytic Ads was significantly enhanced in immunosuppressed animals. In animals that received virus therapy plus immunosuppression, significant differences were observed in tumor histology, and in many cases little viable tumor remained. Notably, we also determined that immunosuppression allowed intratumoral virus levels to remain elevated for prolonged periods. Although favorable tumor responses can be achieved in immunocompetent animals, the rate of virus clearance from the tumor may lead to varied antitumor efficacy. Immunosuppression, therefore, allows sustained Ad replication and oncolysis, which leads to substantially improved suppression of tumor growth.

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

    Science.gov (United States)

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

    2003-09-01

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

  14. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

    Science.gov (United States)

    Pombo, A; Ferreira, J; Bridge, E; Carmo-Fonseca, M

    1994-11-01

    We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

  15. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  16. Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis.

    Science.gov (United States)

    Maroof, Asher; Brown, Najmeeyah; Smith, Barbara; Hodgkinson, Michael R; Maxwell, Alice; Losch, Florian O; Fritz, Ulrike; Walden, Peter; Lacey, Charles N J; Smith, Deborah F; Aebischer, Toni; Kaye, Paul M

    2012-03-01

    Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.

  17. ROLE OF COXSACKIEVIRUS AND ADENOVIRUS RECEPTOR ( CAR)IN CARDIOTOXICITY INFECTED BY COXSACKIEVIRUS B3

    Institute of Scientific and Technical Information of China (English)

    ZHAO Wu; ZHOU Ai-qing; Fu Li-jun; LIANG Ying; TANG Ning

    2005-01-01

    Objective To explore the role of coxsackievirus and adenovirus receptor(CAR)in cardiotoxicity infected by coxsackievirus B3. Methods A toxic cellular model was established in vitro by adding myocarditic coxsackievirus B3(CVB3m)into the culture of neonatal mouse cardiomyocytes.48 h later, the cardiomyocytes were divided into control, CVB3m, and CAR antibody+CVB3m groups. CVB3m-mediated myocytopathic effect of above three groups was observed after further culturing for 48h. At the same time,the cardiomyocytes'viability of above three groups was assessed by MTT assay. Results The degree of cytopathic effect(CPE)of CAR antibody+CVB3m group was significantly lower than CVB3m group (P<0.01)and there was a significant increase in cell viability in CAR antibody+CVB3m group compared with CVB3m group(P<0.01). No significant difference was found between CAR antibody+CVB3m group and control group. Conclusion CAR antibody possesses a protective effect on CVB3m infected cardiomyoctyes, which indicates that CAR may play an important role in mediating cardiotoxicity infected by CVB3m.

  18. The depuration dynamics of oysters (Crassostrea gigas artificially contaminated with hepatitis A virus and human adenovirus

    Directory of Open Access Journals (Sweden)

    Adriana de Abreu Corrêa

    2012-02-01

    Full Text Available Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV or human adenovirus type 5 (HAdV5. After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

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

    Science.gov (United States)

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

    2015-11-27

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

  20. The Microbial Hypothesis: Contributions of Adenovirus Infection and Metabolic Endotoxaemia to the Pathogenesis of Obesity

    Directory of Open Access Journals (Sweden)

    Amos Tambo

    2016-01-01

    Full Text Available The global obesity epidemic, dubbed “globesity” by the World Health Organisation, is a pressing public health issue. The aetiology of obesity is multifactorial incorporating both genetic and environmental factors. Recently, epidemiological studies have observed an association between microbes and obesity. Obesity-promoting microbiome and resultant gut barrier disintegration have been implicated as key factors facilitating metabolic endotoxaemia. This is an influx of bacterial endotoxins into the systemic circulation, believed to underpin obesity pathogenesis. Adipocyte dysfunction and subsequent adipokine secretion characterised by low grade inflammation, were conventionally attributed to persistent hyperlipidaemia. They were thought of as pivotal in perpetuating obesity. It is now debated whether infection and endotoxaemia are also implicated in initiating and perpetuating low grade inflammation. The fact that obesity has a prevalence of over 600 million and serves as a risk factor for chronic diseases including cardiovascular disease and type 2 diabetes mellitus is testament to the importance of exploring the role of microbes in obesity pathobiology. It is on this basis that Massachusetts General Hospital is sponsoring the Faecal Microbiota Transplant for Obesity and Metabolism clinical trial, to study the impact of microbiome composition on weight. The association of microbes with obesity, namely, adenovirus infection and metabolic endotoxaemia, is reviewed.

  1. 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.

  2. Treatment of chronical myocardial ischemia by adenovirus-mediated hypatocyte growth factor gene transfer in minipigs

    Institute of Scientific and Technical Information of China (English)

    YUAN Biao; ZHANG YouRong; ZHAO Zhong; WU DanLi; YUAN LiZhen; WU Bin; WANG LiSheng; HUANG Jun

    2008-01-01

    Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno-virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab-lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran-domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

  3. Random sequential adsorption of human adenovirus 2 onto polyvinylidene fluoride surface influenced by extracellular polymeric substances.

    Science.gov (United States)

    Lu, Ruiqing; Li, Qi; Nguyen, Thanh H

    2016-03-15

    Virus removal by membrane bioreactors depends on virus-membrane and virus-foulant interactions. The adsorption of human adenovirus 2 (HAdV-2) on polyvinylidene fluoride (PVDF) membrane and a major membrane foulant, extracellular polymeric substances (EPS), were measured in a quartz crystal microbalance. In 3-100mM CaCl2 solutions, irreversible adsorption of HAdV-2 was observed on both pristine and EPS-fouled PVDF surfaces. The HAdV-2 adsorption kinetics was successfully fitted with the random sequential adsorption (RSA) model. The applicability of the RSA model for HAdV-2 adsorption is confirmed by comparing the two fitting parameters, adsorption rate constant k(a) and area occupied by each adsorbed HAdV-2 particle a, with experimentally measured parameters. A linear correlation between the fitting parameter k(a) and the measured attachment efficiency was found, suggesting that the RSA model correctly describes the interaction forces dominating the HAdV-2 adsorption. By comparing the fitting parameter d(ads) with the hydrodynamic diameter of HAdV-2, we conclude that virus-virus and virus-surface interactions determine the area occupied by each adsorbed HAdV-2 particle, and thus influence the adsorption capacity. These results provide insights into virus retention and will benefit improving virus removal in membrane filtration.

  4. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    Energy Technology Data Exchange (ETDEWEB)

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R. [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Yousef, Ahmed F. [Department of Chemical and Environmental Engineering, Masdar Institute, Abu Dhabi (United Arab Emirates); Zhang, Zhiying [College of Animal Science and Technologies, Northwest A and F University, Yangling, Shaanxi 712100 (China); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Department of Oncology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada)

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  5. Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools

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    RODRIGO STAGGEMEIER

    2016-01-01

    Full Text Available ABSTRACT Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16 were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR. HAdVs were detected in 62.5% (10/16 of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

  6. Angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium

    Institute of Scientific and Technical Information of China (English)

    DONG Shu-qiang; ZHANG Bao-ren; MEI Ju; XU Zhi-yun; ZOU Liang-jian; HUANG Sheng-dong

    2002-01-01

    Objective: To study the angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium. Methods: Domestic pigs underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, Ad. VEGF-B, Ad. LacZ or PBS were administrated directly into the myocardium at 10 sites in the circumflex distribution (109 PFU or 100 μl) according to groups. Echocardiography and ex vivo coronary angiography were performed. The injection sites around myocardium were harvested and subjected to histological analysis and immunochemical staining. Results: Echocardiography assessment 4 weeks after vector administration demonstrated significant improvement of regional wall systolic function. Collateral vesseldevelopment assessed by angiography was also significantly greater in Ad. VEGF-B animals than that in control animals. Vascular density analysis revealed a mean of 43±5 neovessels per high-power field in Ad.VEGF-B group versus 19±4 and 17±6 in Ad.LacZ and PBS group. Conclusion:Direct intramyocardial administration of Ad.VEGF-B can induce focal angiogenesis and result in improvement in regional myocardial function, which may be useful in patients with ischemic heart disease who are not eligible for conventional therapies.

  7. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG

    2006-01-01

    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  8. Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.

    Science.gov (United States)

    Schulz, Ryan; Zhang, Yian-Biao; Liu, Chang-Jun; Freimuth, Paul

    2007-12-01

    Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

  9. Inhibition of HIV-1 replication in alveolar macrophages by adenovirus gene transfer vectors.

    Science.gov (United States)

    Rice, Joshua; Connor, Ruth; Worgall, Stefan; Moore, John P; Leopold, Philip L; Kaner, Robert J; Crystal, Ronald G

    2002-08-01

    To assess the hypothesis that infection of alveolar macrophages (AM) with adenovirus (Ad) gene transfer vectors might prevent subsequent human immunodeficiency virus (HIV)-1 replication in AM, AM isolated from normal volunteers were infected with increasing doses of first generation (E1(-)) Ad vectors, followed 72 h later by infection with HIV-1(JRFL), an R5/M-tropic strain that preferentially uses the CCR5 coreceptor. As a measure of HIV-1 replication, p24 Ag was quantified by enzyme-linked imunosorbent assay in supernatants on Days 4 to 14 after HIV-1infection. Pretreatment of the AM with an Ad vector resulted in a dose- and time-dependent suppression of subsequent HIV-1 replication. The Ad vector inhibition of HIV-1 replication was independent of the transgene in the Ad vector expression cassette and E4 genes in the Ad backbone. Moreover, it did not appear to be secondary to a soluble factor released by the AM, nor was it overridden by the concomitant transfer of the CCR5 or CXCR4 receptors to the AM before HIV-1 infection. These observations have implications regarding pulmonary host responses associated with HIV-1 infection, as well as possibly uncovering new therapeutic strategies against HIV-1 infection.

  10. 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.

  11. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    Directory of Open Access Journals (Sweden)

    Sumarheni

    2016-01-01

    Full Text Available Penton-dodecahedron (Pt-Dd derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.

  12. A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil

    Science.gov (United States)

    Portes, Silvana Augusta Rodrigues; Volotão, Eduardo de Mello; Rocha, Monica Simões; Rebelo, Maria Cristina; Xavier, Maria da Penha Trindade Pinheiro; de Assis, Rosane Maria; Rose, Tatiana Lundgren; Miagostovich, Marize Pereira; Leite, José Paulo Gagliardi; Carvalho-Costa, Filipe Anibal

    2016-01-01

    A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed. PMID:27223654

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

    Science.gov (United States)

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

    2001-12-01

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

  14. Adenovirus detection in shellfish and urban sewage in Morocco (Casablanca region) by the polymerase chain reaction.

    Science.gov (United States)

    Karamoko, Y; Ibenyassine, K; Aitmhand, R; Idaomar, M; Ennaji, M M

    2005-06-01

    Human enteric viruses are largely excreted in faeces. These resistance of these viruses in the environment makes their faecal-oral transmission easier. Filter feeder organisms such as the mussels are bio-accumulators of viruses contaminating their aquatic environment. Thus, undercooked shellfish consumption involves sanitary risks. Thirty samples of mussels (Mytilus sp.), were tested, half were from an aquaculture origin, the others were from an area more exposed to faecal pollution. Fifteen sewage samples from this last area were also examined. Viruses were extracted from the digestive tissue by direct elution method in a glycine/NaCl pH 9.5 buffer followed by PEG 8000 precipitation. The PEG pellets were used for DNA extraction by proteinase K and phenol/chloroform. The molecular characterization, by PCR using specific adenovirus primers revealed that shellfish growing on Mohammedia (a town in the Casablanca outskirts) littoral are contaminated whereas those chosen from aquaculture and bought in the central market were not contaminated.

  15. Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells.

    Science.gov (United States)

    Hosoya, Noriaki; Miura, Toshiyuki; Kawana-Tachikawa, Ai; Koibuchi, Tomohiko; Shioda, Tatsuo; Odawara, Takashi; Nakamura, Tetsuya; Kitamura, Yoshihiro; Kano, Munehide; Kato, Atsushi; Hasegawa, Mamoru; Nagai, Yoshiyuki; Iwamoto, Aikichi

    2008-03-01

    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients.

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

    Science.gov (United States)

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

    2002-03-01

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

  17. Adenovirus-36 Seropositivity and Its Relation with Obesity and Metabolic Profile in Children

    Directory of Open Access Journals (Sweden)

    Isela Parra-Rojas

    2013-01-01

    Full Text Available The human adenovirus 36 (Ad-36 is causally and correlatively associated in animals and humans, respectively, with increased adiposity and altered metabolic profile. In previous studies, the relationship between Ad-36 seropositivity with obesity was established in adults and children. We evaluated the association of positive antibodies to Ad-36 with obesity and metabolic profile in Mexican children. Seventy-five children with normal-weight and 82 with obesity were studied in this research. All children had a clinic assessment which included weight, height, body circumferences, and skinfold thickness. Laboratory analyzes included triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, and glucose and insulin levels. An enzyme-linked immunosorbent assay (ELISA was used to determine the antibodies to Ad-36 in the serum samples. The overall Ad-36 seroprevalence was 73.9%. Ad-36 seropositivity had a higher prevalence in obese children than in normal weight group (58.6 versus 41.4%, P=0.007. Ad-36 seropositivity was associated with obesity (OR=2.66, P=0.01 and high-density lipoprotein <40 mg/dL (OR=2.85, P=0.03. The Ad-36 seropositive group had greater risk of 4 metabolic abnormalities compared with those children without none alteration. In summary, Ad-36 seropositivity was associated with obesity and low HDL-c levels in the sample of children studied.

  18. Adenovirus-36 Seropositivity and Its Relation with Obesity and Metabolic Profile in Children

    Science.gov (United States)

    Del Moral-Hernández, Oscar; Salgado-Bernabé, Aralia B.; Guzmán-Guzmán, Iris P.; Salgado-Goytia, Lorenzo; Muñoz-Valle, José F.

    2013-01-01

    The human adenovirus 36 (Ad-36) is causally and correlatively associated in animals and humans, respectively, with increased adiposity and altered metabolic profile. In previous studies, the relationship between Ad-36 seropositivity with obesity was established in adults and children. We evaluated the association of positive antibodies to Ad-36 with obesity and metabolic profile in Mexican children. Seventy-five children with normal-weight and 82 with obesity were studied in this research. All children had a clinic assessment which included weight, height, body circumferences, and skinfold thickness. Laboratory analyzes included triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, and glucose and insulin levels. An enzyme-linked immunosorbent assay (ELISA) was used to determine the antibodies to Ad-36 in the serum samples. The overall Ad-36 seroprevalence was 73.9%. Ad-36 seropositivity had a higher prevalence in obese children than in normal weight group (58.6 versus 41.4%, P = 0.007). Ad-36 seropositivity was associated with obesity (OR = 2.66, P = 0.01) and high-density lipoprotein <40 mg/dL (OR = 2.85, P = 0.03). The Ad-36 seropositive group had greater risk of 4 metabolic abnormalities compared with those children without none alteration. In summary, Ad-36 seropositivity was associated with obesity and low HDL-c levels in the sample of children studied. PMID:24324491

  19. The relationship between adenovirus-36 seropositivity, obesity and metabolic profile in Turkish children and adults.

    Science.gov (United States)

    Karamese, M; Altoparlak, U; Turgut, A; Aydogdu, S; Karamese, S Aksak

    2015-12-01

    Obesity potentially arising from viral infection is known as 'infectobesity'. The latest reports suggest that adenovirus-36 (Adv36) is related to obesity in adults and children. Our aim was not only to determine the Adv36 seropositivity in both obese and non-obese children and adults, but also to investigate correlations between antibody positivity and serum lipid profiles. Both Adv36 positivity and tumour-necrosis-factor-alpha, leptin and interleukin-6 levels were detected in blood samples collected from 146 children and 130 adults by ELISA. Fasting plasma triglycerides, total cholesterol and low-density lipoprotein levels were also measured. Adv36 positivity was determined to be 27·1% and 6% in obese and non-obese children and 17·5% and 4% in obese and non-obese adults, respectively. There was no difference with regard to total cholesterol, low-density lipoprotein, triglyceride, tumour-necrosis-factor-alpha and interleukin-6 levels (P > 0·05). However, there was a significant difference between groups in terms of leptin levels (P obese children and adults. Our results showed that Adv36 may be an obesity agent for both adults and children, parallel with current literature data. However, the available data on a possible relationship between Adv36 infection and obesity both in children and adults do not completely solve the problem.

  20. 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.

    Science.gov (United States)

    Rojas, José M; Moreno, Héctor; Valcárcel, Félix; Peña, Lourdes; Sevilla, Noemí; Martín, Verónica

    2014-01-01

    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.

  1. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    during the late phase of infection. Pharmacologic inhibition of ERK phosphorylation reduced virus recovery by >100-fold. Blocking MEK/ERK signaling affected virus DNA replication and mRNA levels only weakly but strongly reduced the amount of viral proteins, independently of the kinases MNK1 and PKR....... Hence, adenovirus induces the oncogenic Raf/MEK/ERK signaling pathway to enhance viral progeny by sustaining the levels of viral proteins. Concerning therapy, our results suggest that the use of Raf/MEK/ERK inhibitors will interfere with the propagation of oncolytic adenoviruses.......The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...

  2. 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...

  3. ADENOVIRUS-MEDIATED EXPRESSION OF PEX, A NONCATALYTIC FRAGMENT OF MATRIX METALLOPROTEINASE-2, AND IT'S INHIBITION ON ANGIOGENESIS AND TUMOR GROWTH

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To develop an adenovirus system to deliver biologically active peptides or proteins such as angiogenesis inhibitors in vivo for the treatment of cancer. Methods: DNA recombination techniques were employed to construct adenovirus shuttle vector, in which angiogenesis inhibitor was put downstream of rat growth hormone signal peptide, and the C-terminal was the myc-epitope 10-amino-acid peptide for the following up of the protein. Adenovirus was made using the bacteria recombination method. We tested this system using an angiogenesis inhibitor chick MMP-2 C-terminal hemopexin-like fragment (PEX) in Sarcoma 180 (S-180) bearing Kunming mice. The anti-angiogenic effect was performed by chick chorioallantoic membrane assay. Results: PEX was readily secreted outside human stomach carcinoma BGC823 cells as demonstrated by immunofluorescent staining and western blot infected by adenovirus with rat growth hormone signal peptide (E-T-rGH-PEX). However, without signal peptide (E-T-PEX), PEX was expressed and localized in the cytoplasm of the infected cells, and formed large aggregates, which suggested that PEX was insoluble. The adenovirus E-T-rGH-PEX could inhibit angiogenesis, while E-T-rGH-PEX not. The adenoviruses of E-T-rGH-PEX inhibited the growth of S-180 tumor significantly compared with the empty virus control group E-T (P=0.026) and without signal peptide group E-T-PEX (P=0.006) respectively, while E-T-PEX had little effect. Conclusion: These results suggest that this adenoviral system is likely to be used in the gene therapy of cancer to deliver angiogenesis inhibitors.

  4. Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Bing ZHANG; Xian-xi LIU; Yan ZHANG; Chun-ying JIANG; Qing-shan TENG; Hai-yan HU; Wei WANG; Lei GONG

    2006-01-01

    Aim: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AdoMetDC) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. Methods: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by Pad. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. Results: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. Conclusion: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.

  5. Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3

    Science.gov (United States)

    Singh, Abhimanyu K.; Berbís, M. Álvaro; Ballmann, Mónika Z.; Kilcoyne, Michelle; Menéndez, Margarita; Nguyen, Thanh H.; Joshi, Lokesh; Cañada, F. Javier; Jiménez-Barbero, Jesús; Benkő, Mária; Harrach, Balázs; van Raaij, Mark J.

    2015-01-01

    The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component. PMID:26418008

  6. 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.

  7. Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

    Institute of Scientific and Technical Information of China (English)

    XIN LEI LIU; SHUNAG QING YU; XIA FENG; XIAO LI WANG; HONG MEI LIU; XIAO MEI ZHANG; HONG XIA LI; LING ZHOU; YI ZENG

    2006-01-01

    The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod. gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod. gag, rAd5-mod. gag or DNA and were boosted after 3 weeks.To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIVspecific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

  8. In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

    Science.gov (United States)

    Alyaqoub, Fadel S; Aldhamen, Yasser A; Koestler, Benjamin J; Bruger, Eric L; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Waters, Christopher M; Amalfitano, Andrea

    2016-02-15

    There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-β and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

  9. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

    Science.gov (United States)

    Griffin, Bryan D; Nagy, Eva

    2011-06-01

    Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-07-15

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

  11. 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.

  12. 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.

  13. Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

    Institute of Scientific and Technical Information of China (English)

    刘启功; 陆再英; 岳远坤; 林立; 张卫东; 颜进

    2004-01-01

    This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer.The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

  14. Adenovirus-associated health risks for recreational activities in a multi-use coastal watershed based on site-specific quantitative microbial risk assessment.

    Science.gov (United States)

    Kundu, Arti; McBride, Graham; Wuertz, Stefan

    2013-10-15

    We used site-specific quantitative microbial risk assessment (QMRA) to assess the probability of adenovirus illness for three groups of swimmers: adults with primary contact, children with primary contact, and secondary contact regardless of age. Human enteroviruses and adenoviruses were monitored by qPCR in a multi-use watershed and Adenovirus type 40/41 was detected in 11% of 73 samples, ranging from 147 to 4117 genomes per liter. Enterovirus was detected only once (32 genomes per liter). Seven of eight virus detections occurred when E. coli concentrations were below the single sample maximum water quality criterion for contact recreation, and five of eight virus detections occurred when fecal coliforms were below the corresponding criterion. We employed dose-harmonization to convert viral genome measurements to TCID50 values needed for dose-response curves. The three scenarios considered different amounts of water ingestion and Monte Carlo simulation was used to account for the variability associated with the doses. The mean illness risk in children based on adenovirus measurements obtained over 11 months was estimated to be 3.5%, which is below the 3.6% risk considered tolerable by the current United States EPA recreational criteria for gastrointestinal illnesses (GI). The mean risks of GI illness for adults and secondary contact were 1.9% and 1.0%, respectively. These risks changed appreciably when different distributions were fitted to the data as determined by Monte Carlo simulations. In general, risk was at a maximum for the log-logistic distribution and lowest for the hockey stick distribution in all three selected scenarios. Also, under default assumptions, the risk was lowered considerably when assuming that only a small proportion of Adenovirus 40/41 (3%) was as infectious as Adenovirus type 4, compared to the assumption that all genomes were Adenovirus 4. In conclusion, site-specific QMRA on water-borne adenoviruses in this watershed provided a similar

  15. E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

    OpenAIRE

    2008-01-01

    The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1980-07-15

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

  17. Acute post-infectious cerebellar ataxia due to co-infection of human herpesvirus-6 and adenovirus mimicking myositis.

    Science.gov (United States)

    Naselli, Aldo; Pala, Giovanna; Cresta, Federico; Finetti, Martina; Biancheri, Roberta; Renna, Salvatore

    2014-11-26

    Acute cerebellar ataxia (ACA) is a relatively common neurological disease in children. Most common types of ACA are acute post-infectious (APCA) and acute disseminated encephalomyelitis (ADEM). Less common but important causes include opsoclonus-myoclonus syndrome (OMS) and acute cerebellitis. Cerebellar neoplasms and acute hydrocephalus are additional causes of paediatric ataxia. APCA is the most common cause of ACA in children, comprising about 30-50% of total cases. This is a report about an immunocompetent 4-yrs-old male affected by APCA, due to co-infection by human herpesvirus-6 (HHV-6) and adenovirus, with symptoms mimicking myositis.

  18. SYNERGISTIC EFFICACY OF ADENOVIRUS-MEDIATED BCL-XS GENE TRANSFER AND TOPOTECAN IN OVARIAN CANCER CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To observe the synergistic efficacy between Adenovirus-mediated bcl-Xs(Adv-bcl-Xs) gene transfer and chemotherapy on ovarian cancer cell growth. Methods: NuTu-19 cells were infected by different titers of Adv-bcl-Xs and treated with topotecan in the meantime. Cell proliferation was measured 3 days later by MTT. Graphical representations and statistical analyses for their interaction in tumor cells were done. Results: The statistical result and Graphical representations of the statistical modeling showed synergy effect on cell growth inhibition (P<0.01). Conclusion: There were synergistic efficacies between Adv-bcl-Xs gene therapy and Topotecan in ovarian cancer cell growth.

  19. Analysen zur Funktion des E1B-55K-Proteins von Adenovirus Typ 5 im lytischen Replikationszyklus

    OpenAIRE

    Kindsmüller, Kathrin

    2007-01-01

    Das E1B-55K-Protein von Adenovirus Typ 5 ist ein multifunktionelles Phosphoprotein, das eine zentrale Rolle im produktiven Replikationszyklus von Ad5 einnimmt. Die lytischen Aktivitäten des E1B-Proteins werden zumindest teilweise im Komplex mit dem viralen Protein E4orf6 vermittelt. Soweit bekannt fördert der E1B-55K/E4orf6-Komplex (E1B/E4-Komplex) den nukleozytoplasmatischen Transport viraler mRNAs und fördert somit die Synthese viraler Kapsidproteine bzw. die Produktion von Nachkommenviren....

  20. Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation

    OpenAIRE

    Kindsmüller, Kathrin; Groitl, Peter; Härtl, Barbara; Blanchette, Paola; Hauber, Joachim; Dobner, Thomas

    2007-01-01

    We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral re...

  1. 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

  2. Inactivation of adenovirus, reovirus and bacteriophages in fecal sludge by pH and ammonia.

    Science.gov (United States)

    Magri, Maria Elisa; Fidjeland, Jørgen; Jönsson, Håkan; Albihn, Ann; Vinnerås, Björn

    2015-07-01

    The aim of this study was to evaluate the inactivation of adenovirus, reovirus and bacteriophages (MS2, ΦX174, 28B) in a fecal sludge. We conducted two experiments. In the first, we tested different compositions of the fecal sludge by mixing different amounts of water, feces and urine, totaling nine combinations which were kept at temperatures between 10 and 28°C. In the second study, urea was added to the mixtures, which were kept at temperatures from 5 to 33°C. The inactivation was based on a combination of temperature, pH and uncharged ammonia concentration. The increase in pH and ammonia was provided mainly by urine content (Experiment 1) and by urine and added urea (Experiment 2). The inactivation of bacteriophages was slower than the AdV and ReV. At 23°C and 28°, reasonable treatment times were obtained when pH was higher than 8.9 and NH3 concentrations were higher than 35 and 55 mM respectively. With those conditions, the maximum time for a 3 log reduction in viruses, according to this study, would be 35 days (23°C) and 21 days (28°C). However, in most applications where helminth eggs are present, the treatment time and NH3 for sanitization will be the scaling criteria, as they are more persistent. Concerning the sanitization of effluents from latrines, vacuum toilets or dry toilets in developing countries with tropical and sub-tropical climates, the use of intrinsic ammonia combined with high pH can be effective in producing a safe and highly valuable liquid that can be used as a fertilizer. In the case of the fecal sludge with very intrinsic ammonia concentration (<20 mM), sanitization could still be achieved by the addition of urea.

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

    Science.gov (United States)

    Green, M R; Roeder, R G

    1980-11-01

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

  4. Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models.

    Science.gov (United States)

    Staba, M J; Wickham, T J; Kovesdi, I; Hallahan, D E

    2000-01-01

    Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.

  5. Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs.

    Science.gov (United States)

    Logunov, Denis Y; Zubkova, Olga V; Karyagina-Zhulina, Anna S; Shuvalova, Eugenia A; Karpov, Andrei P; Shmarov, Maxim M; Tutykhina, Irina L; Alyapkina, Yulia S; Grezina, Natalia M; Zinovieva, Natalia A; Ernst, Lev K; Gintsburg, Alexsandr L; Naroditsky, Boris S

    2007-09-01

    Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.

  6. 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.

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

    Science.gov (United States)

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

    1981-04-01

    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) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

  8. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    Science.gov (United States)

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Dennis Hoffmann; Wibke Bayer; Oliver Wildner

    2007-01-01

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

  12. Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

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    Eichholz, Karsten; Bru, Thierry; Tran, Thi Thu Phuong; Fernandes, Paulo; Mennechet, Franck J. D.; Manel, Nicolas; Alves, Paula; Perreau, Matthieu

    2016-01-01

    Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs. PMID:27636895

  13. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.

    Science.gov (United States)

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2014-06-20

    Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

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

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    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.

  17. Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus

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    Lysa Nepomuceno Luiz

    2010-08-01

    Full Text Available Human adenoviruses (HAdV are a major cause of acute respiratory diseases (ARD, gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA and 3% (14/468 tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV, as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1% HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2 was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100% identical to a part of the feline adenovirus hexon gene sequence.

  18. Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chang-Qing Su; Xing-Hua Wang; Jie Chen; Yong-Jing Liu; Wei-Guo Wang; Lin-Fang Li; Meng-Chao Wu; Qi-Jun Qian

    2006-01-01

    AIM: To construct a tumor-selective replicationcompetent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase(hTERT).METHODS: The antitumor efficacy of SG300 in epatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, andin vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice.RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI.Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015.Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells.CONCLUSION: hTERT promoter-regulated replicative adenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.

  19. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  20. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    Science.gov (United States)

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  1. The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

    Science.gov (United States)

    Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

    2005-12-20

    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

  2. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  3. Serological detection of infection with canine distemper virus, canine parvovirus and canine adenovirus in communal dogs from Zimbabwe.

    Science.gov (United States)

    McRee, Anna; Wilkes, Rebecca P; Dawson, Jessica; Parry, Roger; Foggin, Chris; Adams, Hayley; Odoi, Agricola; Kennedy, Melissa A

    2014-09-05

    Domestic dogs are common amongst communities in sub-Saharan Africa and may serve as important reservoirs for infectious agents that may cause diseases in wildlife. Two agents of concern are canine parvovirus (CPV) and canine distemper virus (CDV), which may infect and cause disease in large carnivore species such as African wild dogs and African lions, respectively. The impact of domestic dogs and their diseases on wildlife conservation is increasing in Zimbabwe, necessitating thorough assessment and implementation of control measures. In this study, domestic dogs in north-western Zimbabwe were evaluated for antibodies to CDV, CPV, and canine adenovirus (CAV). These dogs were communal and had no vaccination history. Two hundred and twenty-five blood samples were collected and tested using a commercial enzyme-linked immunosorbent assay (ELISA) for antibodies to CPV, CDV, and CAV. Of these dogs, 75 (34%) had detectable antibodies to CDV, whilst 191 (84%) had antibodies to CPV. Antibodies to canine adenovirus were present in 28 (13%) dogs. Canine parvovirus had high prevalence in all six geographic areas tested. These results indicate that CPV is circulating widely amongst domestic dogs in the region. In addition, CDV is present at high levels. Both pathogens can infect wildlife species. Efforts for conservation of large carnivores in Zimbabwe must address the role of domestic dogs in disease transmission.

  4. GP73-regulated oncolytic adenoviruses possess potent killing effect on human liver cancer stem-like cells

    Science.gov (United States)

    Zhang, Rong; Ma, Buyun; Liu, Tao; Yang, Yu; Xie, Wenjie; Liu, Xianglei; Huang, Fang; Liu, Tao; Zhou, Xiumei; Liu, Xinyuan; Wang, Yigang

    2016-01-01

    Cancer stem cells (CSCs), also known as tumor-initiating cells, are highly metastatic, chemo-resistant and tumorigenic, and are critical for cancer development, maintenance and recurrence. Oncolytic adenovirus could targetedly kill CSCs and has been acted as a promising anticancer agent. Currently, a novel GP73-regulated oncolytic adenovirus GD55 was constructed to specifically treat liver cancer and exhibited obvious cytotoxicity effect. However, there remains to be confirmed that whether GD55 could effectively eliminate liver CSCs. We first utilized the suspension culture to enrich the liver CSCs-like cells, which acquires the properties of liver CSCs in self-renewal, differentiation, quiescence, chemo-resistance and tumorigenicity. The results indicated that GD55 elicited more significant cytotoxicity and stronger oncolytic effect in liver CSC-like cells compared to common oncolytic virus ZD55. Additionally, GD55 possessed the greater efficacy in suppressing the growth of implanted tumors derived from liver CSC-like cells than ZD55. Furthermore, GD55 induced remarkable apoptosis of liver CSC-like cells in vitro and in vivo, and inhibited the propogation of cells and angiogenesis in xenograft tumor tissues. Thus, GD55 may virtually represent an attractive therapeutic agent for targeting liver CSCs to achieve better clinical outcomes for HCC patients. PMID:27121064

  5. Serological detection of infection with canine distemper virus, canine parvovirus and canine adenovirus in communal dogs from Zimbabwe

    Directory of Open Access Journals (Sweden)

    Anna McRee

    2014-02-01

    Full Text Available Domestic dogs are common amongst communities in sub-Saharan Africa and may serve as important reservoirs for infectious agents that may cause diseases in wildlife. Two agents of concern are canine parvovirus (CPV and canine distemper virus (CDV, which may infect and cause disease in large carnivore species such as African wild dogs and African lions, respectively. The impact of domestic dogs and their diseases on wildlife conservation is increasing in Zimbabwe, necessitating thorough assessment and implementation of control measures. In this study, domestic dogs in north-western Zimbabwe were evaluated for antibodies to CDV, CPV, and canine adenovirus (CAV. These dogs were communal and had no vaccination history. Two hundred and twenty-five blood samples were collected and tested using a commercial enzyme-linked immunosorbent assay (ELISA for antibodies to CPV, CDV, and CAV. Of these dogs, 75 (34% had detectable antibodies to CDV, whilst 191 (84% had antibodies to CPV. Antibodies to canine adenovirus were present in 28 (13% dogs. Canine parvovirus had high prevalence in all six geographic areas tested. These results indicate that CPV is circulating widely amongst domestic dogs in the region. In addition, CDV is present at high levels. Both pathogens can infect wildlife species. Efforts for conservation of large carnivores in Zimbabwe must address the role of domestic dogs in disease transmission.

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

    Science.gov (United States)

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

    2003-01-01

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

  7. Fever, Haematuria, and Acute Graft Dysfunction in Renal Transplant Recipients Secondary to Adenovirus Infection: Two Case Reports

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    J. Ramírez

    2013-01-01

    Full Text Available We report two cases of adenoviral infection in kidney transplant recipients that presented with different clinical characteristics under similar demographic and posttransplant conditions. The first case presented with fever, gross haematuria, and acute graft dysfunction 15 days following renal transplantation. A graft biopsy, analyzed with immunohistochemistry, yielded negative results. However, the diagnosis was confirmed with blood and urine real-time PCR for adenovirus 3 days after the initial clinical manifestations. The immunosuppression dose was reduced, and ribavirin treatment was started, for which the patient quickly developed toxicity. Antiviral treatment allowed for transient response; however, a relapse occurred. The viral real-time PCR became negative upon immunosuppression reduction and administration of IVIG; graft function normalized. In the second case, the patient presented with fever and dysuria 1 month after transplantation. The initial imaging studies revealed graft enlargement and areas of hypoperfusion. In this case, the diagnosis was also confirmed with blood and urine real-time PCR for adenovirus 3 days after the initial clinical manifestations. Adenoviral nephritis was confirmed through a graft biopsy analyzed with light microscopy, immunohistochemistry, and PCR in frozen tissue. The immunosuppression dose was reduced, and IVIG was administered obtaining excellent clinical results along with a negative real-time PCR.

  8. Comparison of enterovirus and adenovirus concentration and enumeration methods in seawater from Southern California, USA and Baja Malibu, Mexico.

    Science.gov (United States)

    Sassoubre, Lauren M; Love, David C; Silverman, Andrea I; Nelson, Kara L; Boehm, Alexandria B

    2012-09-01

    Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

  9. Immunochromatographic test for detection of adenovirus from respiratory samples: is it a real solution for pediatric emergency department?

    Science.gov (United States)

    Romero-Gómez, María Pilar; López López, Rosario; González Montes, Remedios; Ots Ruiz, Cristina; Hierro Cuesta, Sara; Martín Crespo, María Antonia; García García, Santos

    2014-01-01

    Rapid diagnosis of adenoviral respiratory infections is required in order to decide optimal treatment strategies. The adenovirus antigen immunochromatography Adeno Respiratory Card Letitest (Leti diagnostics, Barcelona, Spain), was evaluated versus the shell-vial culture and multiplex PCR (Clart Pneumovir Version 3.0, Genomica, Madrid, Spain), in nasopharyngeal washes and oropharyngeal swabs specimens from subjects with respiratory tract infections. Between April 2011 and November 2012, 224 patients were included. The IC Adeno Respiratory Card Letitest was positive for 77.9% (74 of 95) of patients diagnosed at bedside. Using multiplex-PCR as the reference standard, the overall sensitivity was 77.9% and the specificity was 73.6%. Taking shell-viral culture as the reference method, the Adeno Respiratory Card Letitest (Leti diagnostics, Barcelona, Spain) sensitivity and specificity values were 80.0% and 60.9%, respectively. Using RT-PCR (Clart Pneumovir Version 3.0, Genomica, Madrid, Spain) as the reference standard, the viral culture sensitivity was 53.2% and the specificity was 100%. The Adeno Respiratory Card Letitest (Leti diagnostics, Barcelona, Spain) assay could be a simple and rapid method for antigenic detection of adenovirus in pediatric respiratory samples although it would be necessary to improve the specificity and sensitivity of the test.

  10. EFFECT OF ADENOVIRUS-MEDIATED p53 GENE TRANSFER ON APOPTOSIS AND RADIOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张珊文; 肖绍文; 吕有勇

    2003-01-01

    Objective: To evaluate the effect of adenovirus- mediated p53 gene (Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines. Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 protein expression was detected by immunohistochemistry assay and western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infected with Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flow cytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection at Adp53 at 100 MOI which caused high transfer rate of wild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53 status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

  11. High-Throughput Sequencing of MicroRNAs in Adenovirus Type 3 Infected Human Laryngeal Epithelial Cells

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    Yuhua Qi

    2010-01-01

    Full Text Available Adenovirus infection can cause various illnesses depending on the infecting serotype, such as gastroenteritis, conjunctivitis, cystitis, and rash illness, but the infection mechanism is still unknown. MicroRNAs (miRNA have been reported to play essential roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. We analyzed the miRNA expression profiles from adenovirus type 3 (AD3 infected Human laryngeal epithelial (Hep2 cells using a SOLiD deep sequencing. 492 precursor miRNAs were identified in the AD3 infected Hep2 cells, and 540 precursor miRNAs were identified in the control. A total of 44 miRNAs demonstrated high expression and 36 miRNAs showed lower expression in the AD3 infected cells than control. The biogenesis of miRNAs has been analyzed, and some of the SOLiD results were confirmed by Quantitative PCR analysis. The present studies may provide a useful clue for the biological function research into AD3 infection.

  12. Features of a clinical course of the acute respiratory diseases caused by adenoviruses of epidemic significant serotypes

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    N. I. L`vov

    2014-01-01

    Full Text Available The objective: To investigate etiological structure of adenoviral diseases in young people from organized groups and the clinical features of acute respiratory disease caused by different serotypes of adenovirus were identified.Materials and methods: A total 382 patients with adenovirusinfections were investigated. Virus isolation from nasopharyngeal swabs was carried out in cell cultures Vero, HeLa, Hep-2. Typing of adenoviruses was performed by virus neutralization test with polyclonal rabbit subtype specific sera. The average (M, standard deviation (SD and frequency of occurrence (% of clinical signs (respiratory and non-respiratory syndromes of pneumonia, protracted and recurrent course was calculated. Significance of the differences (p<0,05 of average frequency of cases compared to independent group of patients was evaluated by Student t-test and criterion φ2 (phi – square Fischer, respectively.Results: As result of investigation 199 adenovirus strains (52,1 % were isolated. 183 strains were serotyped: 64 (32,2 % – 3 serotype, 42 (21,1 % – 4 serotype, 38 (19,1 % – 7 serotype, 15 (7,5 % – 5 serotype, 11 (5,5 % – 21 serotype, 8 (4,0 % – 1 serotype, 3 (1,5 % – 2 serotype, 2 (1,0 % – 6 serotype. In assessing the features of the clinical course of adenoviral infection caused by the most actual serotypes (3, 4, 7 of adenovirus revealed that duration of diseases caused by serotype 7 was significantly longer and remained febrile fever (4,3±2,74 days, p<0,05, rhinitis (9,4±6,01 days, p<0,05, pharyngitis (7,9±2,87 days, p<0,05, laryngitis (7,3±2,87 days, p<0,05 and bronchitis (11,8±8,03 days, p<0,05, tonsillitis (63,0%, φ2=12,6, p<0,05, lymphadenopathy (63,0%, φ2=4,1, p<0,05, and pneumonia (34,2%, φ2=3,84, p<0,05 were registered significantly more frequently.Conclusion: The study showed that the adenoviruses of 3, 4 and 7 serotype have the greatest epidemiological significance. Clinical features

  13. Genome Sequences of Human Adenovirus 14 Isolates from Mild Respiratory Cases and a Fatal Pneumonia, Isolated during 2006-2007 Epidemics in North America

    Science.gov (United States)

    2010-01-01

    Hsiang Lin5, David Metzgar6 Abstract Background: Human adenovirus 14 (HAdV-14) is a recognized causative agent of epidemic febrile respiratory illness (FRI...Shih- Chuan 1st Road, Kaohsiung,80708, Taiwan. 6Department of Respiratory Diseases Research, Naval Health Research Center (NHRC), 140 Sylvester Rd San

  14. Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems

    Science.gov (United States)

    Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate of human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a pr...

  15. 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

  16. Delivery route, MyD88 signaling and cross-priming events determine the anti-tumor efficacy of an adenovirus based melanoma vaccine.

    NARCIS (Netherlands)

    Hangalapura, B.N.; Oosterhoff, D.; Gupta, T.; Groot, J. de; Wijnands, P.G.J.T.B.; Beusechem, V.W. van; Haan, J.; Tuting, T.; Eertwegh, A.J. van den; Curiel, D.T.; Scheper, R.J.; Gruijl, T.D. de

    2011-01-01

    Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intran

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

    Science.gov (United States)

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

    2008-12-19

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

  18. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

    Science.gov (United States)

    Human adenovirus is relatively resistant to UV radiation and has been used as a conservative testing microbe for evaluations of UV disinfection systems as components of water treatment processes. In this study, we attempted to validate the applicability of integrated cell culture...

  19. Latent class analysis of diagnostic tests for adenovirus, Bordetella pertussis and influenza virus infections in German adults with longer lasting coughs.

    Science.gov (United States)

    Sobotzki, C; Riffelmann, M; Kennerknecht, N; Hülsse, C; Littmann, M; White, A; Von Kries, R; Wirsing VON König, C H

    2016-03-01

    Laboratory tests in adult outpatients with longer lasting coughs to identify a potential causal pathogen are rarely performed, and there is no gold standard for these diagnostic tests. While the diagnostic validity of serological tests for pertussis is well established their potential contribution for diagnosing adenovirus and influenza virus A and B infections is unclear. A sentinel study into the population-based incidence of longer lasting coughs in adults was done in Rostock (former East Germany) and Krefeld (former West Germany). A total of 971 outpatients who consulted general practitioners or internists were included. Inclusion criteria were coughing for ⩾1 week and no chronic respiratory diseases. We evaluated the performance of polymerase chain reaction (PCR) as well as IgG and IgA serology, applying a latent class model for diagnosing infections with adenovirus, B. pertussis, and influenza virus A and B. The adult outpatients first sought medical attention when they had been coughing for a median of 3 weeks. In this situation, direct detection of infectious agents by PCR had a low sensitivity. Modelling showed that additional serological tests equally improved sensitivity and specificity for diagnosis for adenovirus, B. pertussis and influenza virus A and B infections. The combination of serology and PCR may improve the overall performance of diagnostic tests for B. pertussis and also for adenovirus, and influenza virus A and B infections.

  20. 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...