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Sample records for adenovirus obp-301 telomelysin

  1. A novel molecular therapy using bioengineered adenovirus for human gastrointestinal cancer.

    PubMed

    Fujiwara, Toshiyoshi

    2011-06-01

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

  2. Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

    PubMed Central

    Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

    2016-01-01

    Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301. PMID:27673332

  3. Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging.

    PubMed

    Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

    Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301.

  4. [Oncolytic virotherapy for human solid tumors].

    PubMed

    Fujiwara, Toshiyoshi

    2009-05-01

    Replication-selective tumor-specific viruses present a novel approach for treatment of neoplastic disease. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity correlates closely with human telomerase reverse transcriptase(hTERT)expression. We constructed an attenuated adenovirus 5 vector(Telomelysin, OBP-301), in which the hTERT promoter element drives expression of E1 genes. Telomelysin replicated efficiently and induced marked cell killing in a panel of human cancer cell lines, whereas replication as well as cytotoxicity was highly attenuated in normal human cells lacking telomerase activity. We further modified the E3 region of OBP-301 to contain green fluorescent protein(GFP)gene for monitoring viral replication(TelomeScan, OBP-401). When TelomeScan was intratumorally injected into human tumors orthotopically implanted into the rectum in mice, para-aortic lymph node metastasis could be visualized at laparotomy with a three-chip color cooled charged-coupled device camera. This article reviews recent highlights in this rapidly evolving field of cancer therapeutic and diagnostic approaches using telomerase-specific oncolytic adenoviruses.

  5. Synergistic interaction of telomerase-specific oncolytic virotherapy and chemotherapeutic agents for human cancer.

    PubMed

    Fujiwara, Toshiyoshi; Kagawa, Shunsuke; Tazawa, Hiroshi

    2012-07-01

    Replication-selective tumor-specific viruses present a novel approach for treatment of neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis through the maintenance of telomeres, and its activity correlates closely with human telomerase reverse transcriptase (hTERT) expression. We constructed an attenuated adenovirus 5 vector, in which the hTERT promoter element drives expression of E1 genes, OBP-301 (Telomelysin). Since only tumor cells that express telomerase activity would activate this promoter, the hTERT proximal promoter allows for preferential expression of viral genes in tumor cells, leading to selective viral replication and oncolytic cell death. OBP-301 alone exhibited substantial antitumor effects both in animal models and in clinical trials; data regarding combination therapy with OBP-301 and chemotherapeutic agents are preliminary but encouraging. This article reviews synergistic interaction of virotherapy and chemotherapy, and illustrates the potential application for the treatment of human cancer.

  6. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

    Adenoviruses are associated with numerous disease outbreaks, particularly those involving d-cares, schools, children's camps, hospitals and other health care centers, and military settings. In addition, adenoviruses have been responsible for many recreational water outbreaks, including a great number of swimming pool outbreaks than any other waterborne virus (Gerba and Enriquez 1997). Two drinking water outbreaks have been documented for adenovirus (Divizia et al. 2004; Kukkula et al. 1997) but none for food. Of the 51 known adenovirus serotypes, one third are associated with human disease, while other infections are asymptomatic. Human disease associated with adenovirus infections include gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (Table 2). Children and the immunocompromised are more severely impacted by adenovirus infections. Subsequently, adenovirus is included in the EPA's Drinking Water Contaminant Candidate List (CCL), which is a list of unregulated contaminants found in public water systems that may pose a risk to public health (National Research Council 1999). Adenoviruses have been detected in various waters worldwide including wastewater, river water, oceans, and swimming pools (Hurst et al. 1988; Irving and Smith 1981; Pina et al. 1998). Adenoviruses typically outnumber the enteroviruses, when both are detected in surface waters. Chapron et al. (2000) found that 38% of 29 surface water samples were positive for infectious Ad40 and Ad41. Data are lacking regarding the occurrence of adenovirus in water in the US, particularly for groundwater and drinking water. Studies have shown, however, that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez et al. 1995), which may be due to their double-stranded DNA. Risk assessments have been conducted on waterborne adenovirus (Crabtree et al. 1997; van Heerden et al. 2005c). Using dose-response data for inhalation

  7. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  8. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  9. Adenovirus structure.

    PubMed

    Rux, John J; Burnett, Roger M

    2004-12-01

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

  10. Innate Immunity to Adenovirus

    PubMed Central

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka

    2014-01-01

    Abstract Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate–adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features. PMID:24512150

  11. Adenovirus (For Parents)

    MedlinePlus

    ... common in late winter, spring, and early summer conjunctivitis (pinkeye) and pharyngoconjunctival fever caused by adenovirus tend to ... cystitis usually resolves on its own. Eye infections: Pinkeye (conjunctivitis) is a mild inflammation of the conjunctiva ( ...

  12. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  13. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  14. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

    Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

  15. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and antisera used in serological tests to identify antibodies to adenovirus in serum. Additionally... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus...

  16. Canine adenovirus based rabies vaccines.

    PubMed

    Tordo, N; Foumier, A; Jallet, C; Szelechowski, M; Klonjkowski, B; Eloit, M

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control.

  17. Adenoviruses in the immunocompromised host.

    PubMed Central

    Hierholzer, J C

    1992-01-01

    Adenoviruses are among the many pathogens and opportunistic agents that cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe, and the serotypes involved are clustered according to the age of the patient and the nature of the immunosuppression. Over 300 adenovirus infections in immunocompromised patients, with an overall case fatality rate of 48%, are reviewed in this paper. Children with severe combined immunodeficiency syndrome and other primary immunodeficiencies are exposed to the serotypes of subgroups B and C that commonly infect young children, and thus their infections are due to types 1 to 7 and 31 of subgenus A. Children with bone marrow and liver transplants often have lung and liver adenovirus infections that are due to an expanded set of subgenus A, B, C, and E serotypes. Adults with kidney transplants have viruses of subgenus B, mostly types 11, 34, and 35, which cause cystitis. This review indicates that 11% of transplant recipients become infected with adenoviruses, with case fatality rates from 60% for bone marrow transplant patients to 18% for renal transplant patients. Patients with AIDS become infected with a diversity of serotypes of all subgenera because their adult age and life-style expose them to many adenoviruses, possibly resulting in antigenically intermediate strains that are not found elsewhere. Interestingly, isolates from the urine of AIDS patients are generally of subgenus B and comprise types 11, 21, 34, 35, and intermediate strains of these types, whereas isolates from stool are of subgenus D and comprise many rare, new, and intermediate strains that are untypeable for practical purposes. It has been estimated that adenoviruses cause active infection in 12% of AIDS patients and that 45% of

  18. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  19. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

    PubMed

    Lee, Choon-Taek; Park, Kyung-Ho; Yanagisawa, Kiyoshi; Adachi, Yasushi; Ohm, Joyce E; Nadaf, Sorena; Dikov, Mikhail M; Curiel, David T; Carbone, David P

    2004-09-15

    Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

  20. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    DOE PAGES

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; ...

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved tomore » have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.« less

  1. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    SciTech Connect

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; Borducchi, Erica N.; Iampietro, M. Justin; Bricault, Christine A.; Teigler, Jeffrey E.; Blackmore, Stephen; Parenteau, Lily; Wagh, Kshitij; Handley, Scott A.; Zhao, Guoyan; Virgin, Herbert W.; Korber, Bette; Barouch, Dan H.

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

  2. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

    Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adenoviruses one of (if not the) best models for studying viral evolution on a larger time scale. Phylogenetic calculation can infer the evolutionary distance among adenovirus strains on serotype, species, and genus levels, thus helping the establishment of a correct taxonomy on the one hand, and speeding up the process of typing new isolates on the other. Initially, four major lineages corresponding to four genera were recognized. Later, the demarcation criteria of lower taxon levels, such as species or types, could also be defined with phylogenetic calculations. A limited number of possible host switches have been hypothesized and convincingly supported. Application of the web-based BLAST and MultAlin programs and the freely available PHYLIP package, along with the TreeView program, enables everyone to make correct calculations. In addition to step-by-step instruction on how to perform phylogenetic analysis, critical points where typical mistakes or misinterpretation of the results might occur will be identified and hints for their avoidance will be provided.

  3. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  4. Adenovirus sensing by the immune system.

    PubMed

    Atasheva, Svetlana; Shayakhmetov, Dmitry M

    2016-12-01

    The host immune system developed multiple ways for recognition of viral pathogens. Upon disseminated adenovirus infection, the immune system senses adenovirus invasion from the moment it enters the bloodstream. The soluble blood factors, FX, antibodies, and complement, can bind and activate plethora of host-protective immune responses. Adenovirus binding to the cellular β3 integrin and endosomal membrane rupture trigger activation of IL-1α/IL-1R1 proinflammatory cascade leading to attraction of cytotoxic immune cells to the site of infection. Upon cell entry, adenovirus exposes its DNA genome in the cytoplasm and triggers DNA sensors signaling. Even when inside the nucleus, the specialized cellular machinery that recognizes the double-strand DNA breaks become activated and triggers viral DNA replication arrest. Thus, the host employs very diverse mechanisms to prevent viral dissemination.

  5. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

  6. Molecular Analysis of Adenovirus Isolates from Previously Vaccinated Young Adults

    DTIC Science & Technology

    2004-04-01

    NAVAL HEALTH RESEARCH CENTER MOLECULAR ANALYSIS OF ADENOVIRUS ISOLATES FROM PREVIOUSLY VACCINATED YOUNG ADULTS D. A. Blasiole...Molecular Analysis of Adenovirus Isolates From Previously Vaccinated Young Adults . 6. AUTHORS Daniel A Blasiole, David Metzgar, Luke T Daum, Margaret AK

  7. Adenovirus Serotype 14 Infection, New Brunswick, Canada, 2011

    PubMed Central

    Garceau, Richard; Thibault, Louise; Oussedik, Youcef; Bastien, Nathalie; Li, Yan

    2013-01-01

    We describe 3 culture-proven cases of adenovirus serotype 14 infection in New Brunswick, Canada, during the summer of 2011. Strains isolated from severely ill patients were closely related to strains of a genomic variant, adenovirus 14p1, circulating in the United States and Ireland. Physicians in Canada should be aware of this emerging adenovirus. PMID:23260201

  8. Elasticity and Binding of Adenovirus

    NASA Astrophysics Data System (ADS)

    Matthews, Garrett; Negishi, Atsuko; Seeger, Adam; McCarty, Doug; Taylor, Russell; Samulshi, Jude; Superfine, Richard

    1999-11-01

    Adenovirus was the first human virus found to cause the transformation of cells and is one of the more common vectors being used for the development of gene therapy. As such, much is known about the viral structure and genome; however, the events of the early infection cycle, such as binding of the virus to the cell membrane and the release of genetic material from the capsid, for this and other nonenveloped viruses, are not fully understood. With the atomic force microscope (AFM) we are able to image the virus in both air and liquids, allowing us to change the surrounding environment, varying such physiologically relevant parameters as osmolality or pH. We additionally have the ability to do manipulations on single virus particles in these environments using the nanoManipulator. The nanoManipulator is an advanced interface for AFM that allows real time three dimensional rendering of the topographical data, allows the sample surface to be non-destructively felt using a hand held stylus that responds to the information being sensed at the tip, and allows controlled modification of the surface. Using this tool we have translated single virions over various surfaces, allowing us to measure the adhesion between the capsid and these surfaces. Additionally, we are able to place the tip directly atop individual viruses and measure their elasticity under a compressive load being supplied by that tip. We can explore how this property changes as a function of the properties of the surrounding liquid.

  9. Components of Adenovirus Genome Packaging

    PubMed Central

    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

  10. Immunologic and Genetic Selection of Adenovirus Vaccine Strains: Synthesis and Characterization of Adenovirus Antigens.

    DTIC Science & Technology

    1984-08-01

    exhibited strikingly different chromatographic characteristics. 2. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble...antigens. The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine , an acridine dye...chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis

  11. Structure and Uncoating of Immature Adenovirus

    SciTech Connect

    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.

  12. Chimpanzee Adenovirus Vector Ebola Vaccine.

    PubMed

    Ledgerwood, Julie E; DeZure, Adam D; Stanley, Daphne A; Coates, Emily E; Novik, Laura; Enama, Mary E; Berkowitz, Nina M; Hu, Zonghui; Joshi, Gyan; Ploquin, Aurélie; Sitar, Sandra; Gordon, Ingelise J; Plummer, Sarah A; Holman, LaSonji A; Hendel, Cynthia S; Yamshchikov, Galina; Roman, Francois; Nicosia, Alfredo; Colloca, Stefano; Cortese, Riccardo; Bailer, Robert T; Schwartz, Richard M; Roederer, Mario; Mascola, John R; Koup, Richard A; Sullivan, Nancy J; Graham, Barney S

    2017-03-09

    Background The unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. Methods We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10(10) particle units or 2×10(11) particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. Results In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10(11) particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10(11) particle-unit dose than in the group that received the 2×10(10) particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×10(11) particle-unit dose than among those who received the 2×10(10) particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×10(11) particle-unit dose. Conclusions Reactogenicity and immune responses

  13. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    PubMed

    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.

  14. Molecular characterization of adenoviruses among finnish military conscripts.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Rönkkö, Esa; Virkki, Maria; Nikkari, Simo; Ziegler, Thedi

    2016-04-01

    Although adenoviruses were identified as important respiratory pathogens many years ago, little information is available concerning the prevalence of different adenovirus serotypes, which are circulating and causing epidemics in Finnish military training centers. Over a period of five years from 2008 to 2012, 3577 respiratory specimens were collected from military conscripts presenting with symptoms compatible with acute respiratory tract infection. Upon initial testing for certain respiratory viruses by real-time PCR, 837 of these specimens were identified as adenovirus-positive. For 672 of these specimens, the serotype of the adenovirus responsible was successfully determined by DNA sequencing. Serotypes 1, 2, 3, and 4 were detected in 1, 3, 181, and 487 samples, respectively. Adenovirus epidemics were observed during each year of this study. Based on these findings, adenovirus vaccination should be considered for military conscripts in the Finnish Defence Forces.

  15. Adenovirus dodecahedron, a new vector for human gene transfer.

    PubMed

    Fender, P; Ruigrok, R W; Gout, E; Buffet, S; Chroboczek, J

    1997-01-01

    Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

  16. Structure, function and dynamics in adenovirus maturation.

    PubMed

    Mangel, Walter F; San Martín, Carmen

    2014-11-21

    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 remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed.

  17. Structure, function and dynamics in adenovirus maturation

    DOE PAGES

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    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 remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  18. Structure and uncoating of immature adenovirus

    PubMed Central

    Pérez-Berná, Ana J.; Marabini, Roberto; Scheres, Sjors H. W.; Menéndez-Conejero, Rosa; Dmitriev, Igor P.; Curiel, David T.; Mangel, Walter F.; Flint, S. Jane; Martín, Carmen San

    2009-01-01

    Summary Maturation via proteolytical 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 proteolytical processing are not infectious. We present the 3D structure of immature adenovirus particles, as represented by the thermosensitive mutant Ad2 ts1 grown under non-permissive conditions, and compare it with the mature capsid. Our 3DEM maps at subnanometer resolution indicate that adenovirus maturation does not involve large scale conformational changes in the capsid. Difference maps reveal the location of unprocessed peptides pIIIa and pVI and help to 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. PMID:19563809

  19. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    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 remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  20. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    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 remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  1. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    SciTech Connect

    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).

  2. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  3. Enhanced inactivation of adenovirus under polychromatic UV lamps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The US EPA has stipulated that a UV fluence (dose) of 186 mJ cm-2 is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date publi...

  4. Capturing and concentrating adenovirus using magnetic anionic nanobeads

    PubMed Central

    Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

    2016-01-01

    We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

  5. The search for adenovirus 14 in children in Houston, Texas.

    PubMed

    Laham, Federico R; Jewell, Alan M; Schoonover, Shauna L; Demmler, Gail J; Piedra, Pedro A

    2008-07-01

    Adenovirus (Ad)14 has recently emerged in the United States causing outbreaks of severe respiratory disease. To determine if Ad14 circulated in Houston, Texas, during the same time as an outbreak in military recruits in nearby San Antonio, 215 pediatric adenovirus isolates were serotyped using microneutralization. None were Ad14; Ad1, Ad2, and Ad3 were the most common identified serotypes.

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

    PubMed

    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. Adenovirus dodecahedron allows large multimeric protein transduction in human cells.

    PubMed

    Fender, P; Schoehn, G; Foucaud-Gamen, J; Gout, E; Garcel, A; Drouet, E; Chroboczek, J

    2003-04-01

    Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.

  8. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

    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. PMID:26483965

  9. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

    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.

  10. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2004-05-18

    Disclosed is a mutant 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 significantly weakened binding affinity for CARD1 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. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

  11. Mechanisms of pathogenesis of emerging adenoviruses

    PubMed Central

    Cook, James; Radke, Jay

    2017-01-01

    Periodic outbreaks of human adenovirus infections can cause severe illness in people with no known predisposing conditions. The reasons for this increased viral pathogenicity are uncertain. Adenoviruses are constantly undergoing mutation during circulation in the human population, but related phenotypic changes of the viruses are rarely detected because of the infrequency of such outbreaks and the limited biological studies of the emergent strains. Mutations and genetic recombinations have been identified in these new strains. However, the linkage between these genetic changes and increased pathogenicity is poorly understood. It has been observed recently that differences in virus-induced immunopathogenesis can be associated with altered expression of non-mutant viral genes associated with changes in viral modulation of the host innate immune response. Initial small animal studies indicate that these changes in viral gene expression can be associated with enhanced immunopathogenesis in vivo. Available evidence suggests the hypothesis that there is a critical threshold of expression of certain viral genes that determines both the sustainability of viral transmission in the human population and the enhancement of immunopathogenesis. Studies of this possibility will require extension of the analysis of outbreak viral strains from a sequencing-based focus to biological studies of relationships between viral gene expression and pathogenic responses. Advances in this area will require increased coordination among public health organizations, diagnostic microbiology laboratories, and research laboratories to identify, catalog, and systematically study differences between prototype and emergent viral strains that explain the increased pathogenicity that can occur during clinical outbreaks. PMID:28184296

  12. Isolation and Epidemiology of Falcon Adenovirus

    PubMed Central

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

    2005-01-01

    An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2005). In this study, the virus was successfully isolated and propagated in peregrine falcon embryo fibroblasts, in which it caused visible and reproducible cytopathology. Testing for serum neutralizing antibodies found that infection with this virus was limited almost exclusively to falcons. Serology also found that wild and captive peregrine falcons had high seropositivity rates of 80% and 100%, respectively, although clinical disease was rarely reported in this species. These data implicate peregrine falcons as the natural host and primary reservoir for the virus. Other species of North American falcons, including aplomado falcons, had lower seropositivity rates of 43 to 57%. Falcon species of tropical and/or island origin were uniformly seronegative, although deaths among adults of these species have been described, suggesting they are highly susceptible. Chickens and quail were uniformly seronegative and not susceptible to infection, indicating that fowl were not the source of infection. Based on the information from this study, the primary control of falcon adenovirus infections should be based on segregation of carrier and susceptible falcon species. PMID:16000467

  13. Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus 1 Myocarditis

    PubMed Central

    McCarthy, Mary K.; Procario, Megan C.; Twisselmann, Nele; Wilkinson, J. Erby; Archambeau, Ashley J.; Michele, Daniel E.; Day, Sharlene M.

    2014-01-01

    ABSTRACT Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon gamma (IFN-γ) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was composed predominantly of CD3+ T lymphocytes. Depletion of IFN-γ during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN-γ is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction. IMPORTANCE Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN-γ modulation as a therapeutic strategy. PMID:25320326

  14. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    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.

  15. IMMUNOFLUORESCENT STUDIES OF THE POTENTIATION OF AN ADENOVIRUS-ASSOCIATED VIRUS BY ADENOVIRUS 7

    PubMed Central

    Blacklow, Neil R.; Hoggan, M. David; Rowe, Wallace P.

    1967-01-01

    A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA. PMID:4225814

  16. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  17. An Oncotropic Adenovirus Vector System for Breast Cancer Treatment

    DTIC Science & Technology

    2005-09-01

    AD Award Number: DAMD17-03-1-0629 TITLE: An Oncotropic Adenovirus Vector System for Breast Cancer Treatment PRINCIPAL INVESTIGATOR: Igor P. Dmitriev...Aug 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An Oncotropic Adenovirus Vector System for Breast Cancer Treatment 5b. GRANT NUMBER DAMD17-03-1...epithelial cells, the origin of most human cancers. However, realization of the full potential of Ad vectors for targeted cancer treatment is currently

  18. Adenovirus-receptor interaction with human lymphocytes.

    PubMed

    Mentel, R; Döpping, G; Wegner, U; Seidel, W; Liebermann, H; Döhner, L

    1997-03-01

    Lymphocytes play a key role in cell-mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC-labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life-threatening diseases can develop.

  19. [Gene engineering of the adenovirus vector].

    PubMed

    Kondo, Saki; Terashima, Miho; Fukuda, Hiromitsu; Saito, Izumu; Kanegae, Yumi

    2007-06-01

    The adenovirus vector is very attractive tool not only for the gene therapy but also for the basic sciences. However, because a construction method of this vector had been complex, only limited scientists had constructed and enjoyed the benefits. Recently, various methods were developed and the researchers came to be able to choose an efficient method, which is the COS-TPC method, or a concise procedure, which is the intact-genome transfection method (in vitro ligation method). Here we described not only these methods but also new method to construct the various Ads simultaneously using the recombinase-mediated cassette exchange (RMCE) by the site-specific recombinase. And also we want to refer the possibility to the worth of the vector, especially the vector of the expression-switch.

  20. Adenovirus Infections in Immunocompetent and Immunocompromised Patients

    PubMed Central

    2014-01-01

    SUMMARY Human adenoviruses (HAdVs) are an important cause of infections in both immunocompetent and immunocompromised individuals, and they continue to provide clinical challenges pertaining to diagnostics and treatment. The growing number of HAdV types identified by genomic analysis, as well as the improved understanding of the sites of viral persistence and reactivation, requires continuous adaptions of diagnostic approaches to facilitate timely detection and monitoring of HAdV infections. In view of the clinical relevance of life-threatening HAdV diseases in the immunocompromised setting, there is an urgent need for highly effective treatment modalities lacking major side effects. The present review summarizes the recent progress in the understanding and management of HAdV infections. PMID:24982316

  1. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

    Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors. PMID:19578438

  3. Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity

    PubMed Central

    Nemerow, Glen R.; Stewart, Phoebe L.

    2016-01-01

    Human adenoviruses are large (150 MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause acute respiratory, gastrointestinal and ocular infections. Despite these disease associations, adenovirus has aided basic and clinical research efforts through studies of its association with cells and as a target of host antiviral responses. This review highlights the knowledge of adenovirus disassembly and nuclear transport gleaned from structural, biophysical and functional analyses of adenovirus interactions with soluble and membrane-associated host molecules. PMID:28009821

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

    PubMed

    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.

  5. Pertussis-like syndrome associated with adenovirus presenting with hyperleukocytosis: Case report

    PubMed Central

    Sarbay, Hakan; Polat, Aziz; Mete, Emin; Balci, Yasemin Isik; Akin, Mehmet

    2016-01-01

    Adenovirus is an infectious viral agent that causes variety of clinical presentations such as respiratory disease, conjunctivitis, and gastroenteritis. Hepatitis, pancreatitis, myocarditis, encephalitis, and disseminated infection are primarily seen in immunocompromised patients. Rarely, adenovirus infection can present with pertussis-like syndrome. Described here is case of pertussis-like syndrome associated with adenovirus presenting with hyperleukocytosis. PMID:28058402

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

    PubMed Central

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

    2003-01-01

    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. PMID:14576328

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

    PubMed

    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.

  8. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses.

    PubMed

    Takagi-Kimura, M; Yamano, T; Tagawa, M; Kubo, S

    2014-03-01

    Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

  9. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  10. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle.

  11. Gene targeting with a replication-defective adenovirus vector.

    PubMed Central

    Fujita, A; Sakagami, K; Kanegae, Y; Saito, I; Kobayashi, I

    1995-01-01

    Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy. PMID:7666520

  12. Latest insights on adenovirus structure and assembly.

    PubMed

    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.

  13. Latest Insights on Adenovirus Structure and Assembly

    PubMed Central

    San Martín, Carmen

    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 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. PMID:22754652

  14. Adenovirus 36 and Obesity: An Overview

    PubMed Central

    Ponterio, Eleonora; Gnessi, Lucio

    2015-01-01

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

  15. Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extend the number of species within the genus Aviadenovirus.

    PubMed

    Marek, Ana; Kaján, Győző L; Kosiol, Carolin; Harrach, Balázs; Schlötterer, Christian; Hess, Michael

    2014-08-01

    Complete genomes of the first isolates of pigeon adenovirus 1 (PiAdV-1) and Muscovy duck adenovirus (duck adenovirus 2, DAdV-2) were sequenced. The PiAdV-1 genome is 45,480bp long, and has a gene organization most similar to turkey adenovirus 1. Near the left end of the genome, it lacks ORF0, ORF1A, ORF1B and ORF1C, and possesses ORF52, whereas six novel genes were found near the right end. The DAdV-2 genome is 43,734bp long, and has a gene organization similar to that of goose adenovirus 4 (GoAdV-4). It lacks ORF51, ORF1C and ORF54, and possesses ORF55A and five other novel genes. PiAdV-1 and DAdV-2 genomes contain two and one fiber genes, respectively. Genome organization, G+C content, molecular phylogeny and host type confirm the need to establish two novel species (Pigeon aviadenovirus A and Duck aviadenovirus B) within the genus Aviadenovirus. Phylogenetic data show that DAdV-2 is most closely related to GoAdV-4.

  16. Inhibition of adenovirus replication in vitro by trifluridine.

    PubMed

    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.

  17. Capsid-like Arrays in Crystals of Chimpanzee Adenovirus Hexon

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

    The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 Angstroms, b = 433.0 Angstroms, c = 159.3 Angstroms, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 Angstroms resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.

  18. Genetic Relatedness Studies with Adenovirus-associated Viruses

    PubMed Central

    Rose, James A.; Hoggan, M. David; Koczot, Frank; Shatkin, Aaron J.

    1968-01-01

    Adenovirus-associated viruses (AAV) contain double-stranded deoxyribonucleic acid (DNA). DNA from each of the four AAV serotypes was used as template for the in vitro synthesis of complementary 3H-ribonucleic acids(RNA). An estimation of genetic interrelatedness was made on the basis of hybridization reactions between synthetic AAV RNA and AAV DNA. Heterologous reactions were 27 to 37% of homologous reactions, suggesting that the AAV serotypes are related to about the same extent. AAV-1 synthetic RNA was also reacted with DNA from helper adenovirus types 2, 7, and SV15. Very low levels of RNA binding were observed, but it is not likely that these reactions represent AAV-adenovirus genetic relatedness. PMID:5739847

  19. Characterization of a novel adenovirus isolated from a skunk.

    PubMed

    Kozak, Robert A; Ackford, James G; Slaine, Patrick; Li, Aimin; Carman, Susy; Campbell, Doug; Welch, M Katherine; Kropinski, Andrew M; Nagy, Éva

    2015-11-01

    Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus.

  20. Effects of cold atmospheric plasmas on adenoviruses in solution

    NASA Astrophysics Data System (ADS)

    Zimmermann, J. L.; Dumler, K.; Shimizu, T.; Morfill, G. E.; Wolf, A.; Boxhammer, V.; Schlegel, J.; Gansbacher, B.; Anton, M.

    2011-12-01

    Experiments were performed with cold atmospheric plasma (CAP) to inactivate adenovirus, a non-enveloped double stranded DNA virus, in solution. The plasma source used was a surface micro-discharge technology operating in air. Various plasma diagnostic measurements and tests were performed in order to determine the efficacy of CAPs and to understand the inactivation mechanism(s). Different stages of the adenovirus ‘life cycle’ were investigated—infectivity and gene expression as well as viral replication and spread. Within 240 s of CAP treatment, inactivation of up to 6 decimal log levels can be achieved.

  1. Transport of human adenoviruses in porous media

    NASA Astrophysics Data System (ADS)

    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

  2. Adenovirus Respiratory Tract Infections in Peru

    PubMed Central

    Ampuero, Julia S.; Ocaña, Víctor; Gómez, Jorge; Gamero, María E.; Garcia, Josefina; Halsey, Eric S.; Laguna-Torres, V. Alberto

    2012-01-01

    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. PMID:23056519

  3. Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression.

    PubMed Central

    Bruder, J T; Kovesdi, I

    1997-01-01

    Previous studies have shown that airway administration of adenovirus or adenovirus vectors results in a dose-dependent inflammatory response which limits the duration of transgene expression. We explored the possibility that adenovirus infection triggers signal transduction pathways that induce the synthesis of cytokines and thus contribute to the early inflammatory response. Since stimulation of the Raf/mitogen-activated protein kinase (MAPK) pathway activates transcription factors that control the expression of inflammatory cytokines, we examined the activation of this pathway following adenovirus infection. Adenovirus infection induced the rapid activation of Raf-1 and a transient increase in the tyrosine phosphorylation and activation of p42mapk at early times postinfection. Activation of the Raf/MAPK pathway by adenovirus is likely triggered by the infection process, since it occurred rapidly and with various mutant adenoviruses and adenovirus vectors. Moreover, interleukin-8 (IL-8) mRNA accumulation was evident at 20 min postinfection and was induced even in the presence of cycloheximide. Both MAPK activation and IL-8 production were inhibited by forskolin, a potent inhibitor of Raf-1. These results suggest that adenovirus-induced Raf/MAPK activation contributes to IL-8 production. Adenovirus-induced activation of the Raf/MAPK signaling pathway and IL-8 production may play critical roles in the inflammation observed following in vivo administration of adenovirus vectors for gene therapy. PMID:8985363

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

    SciTech Connect

    Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E.; Leeuwen, M. van; Boheemen, S. van; Jong, A.A.W. de; 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. - 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.

  5. Phylogenetic and pathogenic characterization of novel adenoviruses isolated from long-tailed ducks (Clangula hyemalis).

    PubMed

    Counihan, Katrina L; Skerratt, Lee F; Franson, J Christian; Hollmén, Tuula E

    2015-11-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

  6. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  7. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  8. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  9. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  10. Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus.

    PubMed

    Ortega-Esteban, Alvaro; Condezo, Gabriela N; Pérez-Berná, Ana J; Chillón, Miguel; Flint, S Jane; Reguera, David; San Martín, Carmen; de Pablo, Pedro J

    2015-11-24

    Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore.

  11. Bioaccumulation of animal adenoviruses in the pink shrimp.

    PubMed

    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.

  12. Serologic and hexon phylogenetic analysis of ruminant adenoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to determine the antigenic relationship among ruminant adenoviruses and determine their phylogenetic relationship based on the deduced hexon gene amino acid sequence. Results of reciprocal cross-neutralization tests demonstrated antigenic relationships in either on...

  13. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  14. Acute respiratory infection with mouse adenovirus type 1

    PubMed Central

    Weinberg, Jason B.; Stempfle, Gregory S.; Wilkinson, John E.; Younger, John G.; Spindler, Katherine R.

    2005-01-01

    Studies of the pathogenesis of adenovirus respiratory disease are limited by the strict species-specificity of the adenoviruses. Following intranasal inoculation of adult C57BL/6 mice with mouse adenovirus type 1 (MAV-1), we detected MAV-1 early region 3 (E3) and hexon gene expression in the lungs at 7 days post-infection (dpi). We detected MAV-1 E3 protein in the respiratory epithelium 7 dpi. We did not detect viral mRNA or protein at 14 dpi, but MAV-1 DNA was detected by PCR at 21 dpi. Chemokine transcript levels increased between 7 and 14 dpi in the lungs of infected mice. MAV-1 infection induced a patchy cellular infiltrate in lungs at 7 and 14 dpi. This is the first report demonstrating the presence of MAV-1 in the respiratory epithelium of infected mice and describing chemokine responses in the lung induced by MAV-1 respiratory infection. MAV-1 infection of mice has the potential to serve as a model for inflammatory changes seen in human adenovirus respiratory disease. PMID:16054189

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

    PubMed

    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.

  16. Bioaccumulation of animal adenoviruses in the pink shrimp

    PubMed Central

    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. PMID:26413052

  17. Site-specific nicking within the adenovirus inverted terminal repetition.

    PubMed Central

    Chow, K C; Pearson, G D

    1984-01-01

    Site-specific nicking occurs on the l-strand, but not on the r-strand, of the adenovirus left inverted terminal repeat. Nicks are presumably introduced into double- or single-stranded DNA by a cellular endonuclease in an ATP-independent reaction. The consensus nick site has the sequence: (sequence in text). Images PMID:6322107

  18. Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

    PubMed Central

    Schaller, J. P.; Yohn, D. S.

    1974-01-01

    Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 ± 0.02 and 1.34 ± 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (102 to 103 VP per infectivity unit) in contrast to virions at 1.30 g/ml density (104 to 105 VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 × 106) and for simian adenovirus 7 (4.00 × 106) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification. Images PMID:4211167

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

    PubMed

    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.

  20. Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

    PubMed Central

    Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

    2001-01-01

    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. PMID:11462032

  1. Routine monitoring of adenovirus and norovirus within the health care environment.

    PubMed

    Pankhurst, Louise; Cloutman-Green, Elaine; Canales, Melisa; D'Arcy, Nikki; Hartley, John C

    2014-11-01

    This study investigated the presence of adenovirus and norovirus on ward surfaces using real-time polymerase chain reaction (PCR) to assist in the development of evidence-based infection control policy. Screening was carried out weekly for 6 months in the common areas of 2 pediatric wards. Additionally, a one-off screening was undertaken for adenovirus and norovirus on a day unit and for adenovirus only in patient cubicles while occupied. Over the 6-month screening of common areas, 2.4% of samples were positive for adenovirus or norovirus. In rooms occupied with adenovirus-infected children, all cubicle screening sites and almost all swabs were contaminated with adenovirus. In the day unit, 13% of samples were positive. Cleaning and environmental interaction strategies must therefore be designed to control nosocomial transmission of viruses outside of outbreak scenarios.

  2. Effect of adenovirus infection on expression of human histone genes.

    PubMed Central

    Flint, S J; Plumb, M A; Yang, U C; Stein, G S; Stein, J L

    1984-01-01

    The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA

  3. Province-wide adenovirus type 3 outbreak with severe cases in New Brunswick

    PubMed Central

    Girouard, Gabriel; Garceau, Richard; Thibault, Louise; Bourque, Christine; Bastien, Nathalie; Li, Yan

    2011-01-01

    Adenovirus is a commonly isolated virus in clinical samples. Life-threatening infections, although rare, are described worldwide. An epidemic spread of an adenovirus type 3 strain occurred in the province of New Brunswick during the fall of 2008 to the winter of 2009; it resulted in three severely ill patients, with one fatality. Adenovirus should be considered as a cause of severe community-acquired viral pneumonia, especially when the influenza test is negative. PMID:22379488

  4. Large Epidemic of Respiratory Illness Due to Adenovirus Types 7 and 3 in Healthy Young Adults

    DTIC Science & Technology

    2008-02-15

    Epidemic of Respiratory fliness Due to Adenovirus Types 7 and 3 in Healthy Young Adults Margaret A. K. Ryan, Gregory C. Gray," Besa Smith, Jamie A...immunization, respiratory infections due to adenoviruses have reemerged to threaten the health of young adults in the military. Shortly after the loss...challenges for young adults in the military in the postvaccine era. The US military has long had concern about the impact adenovirus serotypes 4 and 7

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

    DTIC Science & Technology

    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

  6. Adenovirus urethritis and concurrent conjunctivitis: a case series and review of the literature.

    PubMed

    Liddle, Olivia Louise; Samuel, Mannampallil Itty; Sudhanva, Malur; Ellis, Joanna; Taylor, Chris

    2015-03-01

    We present eight cases and review the literature of concurrent urethritis and conjunctivitis where adenovirus was identified as the causative pathogen. The focus of this review concerns the identification of specific sexual practices, symptoms, signs and any serotypes that seem more commonly associated with such adenovirus infections. We discuss the seasonality of adenovirus infection and provide practical advice for clinicians to give to the patient.

  7. Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors.

    PubMed

    Seregin, Sergey S; Amalfitano, Andrea

    2009-12-01

    Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.

  8. Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy

    PubMed Central

    Farzad, Lisa; Cerullo, Vincenzo; Yagyu, Shigeki; Bertin, Terry; Hemminki, Akseli; Rooney, Cliona; Lee, Brendan; Suzuki, Masataka

    2014-01-01

    Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy. PMID:27119096

  9. Phylogenetic analyses of novel squamate adenovirus sequences in wild-caught Anolis lizards.

    PubMed

    Ascher, Jill M; Geneva, Anthony J; Ng, Julienne; Wyatt, Jeffrey D; Glor, Richard E

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity.

  10. Addition of Polyadenylate Sequences to Virus-Specific RNA during Adenovirus Replication

    PubMed Central

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

    1971-01-01

    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. PMID:5315962

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  14. Phylogenetic Analyses of Novel Squamate Adenovirus Sequences in Wild-Caught Anolis Lizards

    PubMed Central

    Ascher, Jill M.; Geneva, Anthony J.; Ng, Julienne; Wyatt, Jeffrey D.; Glor, Richard E.

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity. PMID:23593364

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

    PubMed

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

    2013-10-01

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

  16. Adenovirus vector delivery stimulates natural killer cell recognition

    PubMed Central

    Tomasec, Peter; Wang, Eddie C. Y.; Groh, Veronika; Spies, Thomas; McSharry, Brian P.; Aicheler, Rebecca J.; Stanton, Richard J.; Wilkinson, Gavin W. G.

    2007-01-01

    We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery. PMID:17374753

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

    PubMed

    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.

  18. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    NASA Astrophysics Data System (ADS)

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2016-01-01

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

  19. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  20. An Analysis of Adenovirus Genomes Using Whole Genome Software Tools

    PubMed Central

    Mahadevan, Padmanabhan

    2016-01-01

    The evolution of sequencing technology has lead to an enormous increase in the number of genomes that have been sequenced. This is especially true in the field of virus genomics. In order to extract meaningful biological information from these genomes, whole genome data mining software tools must be utilized. Hundreds of tools have been developed to analyze biological sequence data. However, only some of these tools are user-friendly to biologists. Several of these tools that have been successfully used to analyze adenovirus genomes are described here. These include Artemis, EMBOSS, pDRAW, zPicture, CoreGenes, GeneOrder, and PipMaker. These tools provide functionalities such as visualization, restriction enzyme analysis, alignment, and proteome comparisons that are extremely useful in the bioinformatics analysis of adenovirus genomes. PMID:28293072

  1. Therapy of Breast Cancers Using Conditionally Replicating Adenovirus

    DTIC Science & Technology

    2005-08-01

    virotherapy on breast cancer cells in vitro. We have developed a CRAd using the fit-I promoter element for specific EIA gene expression (CRAdflt), RGD...replicating adenoviruses (CRAd) and investigate effects of CRAd virotherapy on endothelial cells and breast cancer cells in vitro. Vascular targeting...determined the capacity of CRAdRGDflt-mda-7 virotherapy to induce breast cancer cell death. To verify the levels of MDA-7/IL-24 protein expression in vitro

  2. Oncolytic adenovirus-mediated therapy for prostate cancer

    PubMed Central

    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

  3. [Adenovirus vectors and their clinical application in gene therapy].

    PubMed

    Adám, E; Nász, I

    2001-09-23

    The potential therapeutic application of the gene transfer technology with adenovirus vectors seems to be enormous. Adenovirus vectors offer several advantages over other vectors, but several important limitations of adenovirus mediated gene transfer are also known. Great number of studies in inherited diseases and in different cancer therapy clinical trials have provided information of critical importance for design of efficient clinical protocols. Clinical trials have been extended to the treatment of many other diseases, too. There are about thirty currently active gene therapy protocols for the treatment only of HIV-1 infection in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are in risk of infection, to develop preventive or therapeutic vaccines for patients with AIDS and other infectious diseases. Gene therapy represents one of the most important developments in oncology, however, before this can be realised as standard treatment the technical problems of gene delivery and higher safety must be overcome. The early--first and second generation--adenovirus vectors are now likely to be phased out for most diseases, and further experiments seem to be necessary. It might be change to the third generation or other, more modern vector application in clinical trials, as the helper dependent vectors. Almost all transcriptional unit is removed from the DNA of these vectors ("gutless vectors"), therefore they cannot reproduce, give higher gene expression and far less inflammatory. Despite the latest achievement reported in vector design it is not possible to predict yet to what extent and when gene therapy will be effective.

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

    PubMed

    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.

  5. Effect of CD4 gene expression on adenovirus replication.

    PubMed Central

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

    1994-01-01

    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. Images PMID:7933112

  6. Effect of CD4 gene expression on adenovirus replication.

    PubMed

    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.

  7. Ion etching of human adenovirus 2: structure of the core

    SciTech Connect

    Newcomb, W.W.; Boring, J.W.; Brown, J.C.

    1984-07-01

    The surface of human adenovirus 2 was etched by irradiating intact virions with low-energy (1-keV) Ar/sup +/ ions in a Technics Hummer V sputter coater. Viral structures exposed by the etching process were shadowed and then examined in the electron microscope. Periods of etching that were sufficient to reduce the viral diameter by 20 to 30 nm revealed distinct substructural elements in the virion core. Cores were found to consist of a cluster of 12 large, uniformly sized spheres which abutted one another in the intact virion. The spheres, for which we suggest the name adenosomes, had a diameter of 23.0 +/- 2.3 nm, and they were related to each other by two-, three-, and fivefold axes of rotational symmetry. The results support the view, originally suggested by Brown et al. that the adenovirus 2 core is composed of 12 large spheres packed tightly together in such a way that each is directed toward the vertex of an icosahedron. Such a structure, constructed of 23.0-nm-diameter spheres, would have an outside diameter (vertex-to-vertex distance) of 67.0 nm and a face-to-face distance of 58.2 nm. It could be accommodated inside the icosahedral adenovirus capsid if each large sphere were located beneath a capsid vertex.

  8. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

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

    2015-01-01

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

  9. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

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

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8+ T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides ‘self-adjuvanting’ activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  10. Structural organization and polypeptide composition of the avian adenovirus core.

    PubMed Central

    Li, P; Bellett, A J; Parish, C R

    1984-01-01

    CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500. The core was similar to that of human adenoviruses, with some evidence of compact subcore domains. Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern. Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel. The organization of the core is thus quite unlike that of chromatin. Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core. We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside. Images PMID:6092686

  11. Template requirements for the initiation of adenovirus DNA replication.

    PubMed Central

    Challberg, M D; Rawlins, D R

    1984-01-01

    The first step in the replication of the adenovirus genome is the covalent attachment of the 5'-terminal nucleotide, dCMP, to the virus-encoded terminal protein precursor (pTP). This reaction can be observed in vitro and has been previously shown to be dependent upon either viral DNA or linearized plasmid DNA containing viral terminal sequences. Plasmids containing deletions or point mutations within the viral terminal sequence were constructed by site-directed mutagenesis. In the case of linear double-stranded templates, pTP-dCMP formation required sequences located within the first 18 base pairs of the viral genome. This sequence contains a segment of 10 base pairs that is conserved in all human adenovirus serotypes. Point mutations within the conserved segment greatly reduced the efficiency of initiation, while a point mutation at a nonconserved position within the first 18 base pairs had little effect. Single-stranded DNAs can also support pTP-dCMP formation in vitro. In contrast to the results obtained with duplex templates, experiments with a variety of single-stranded templates, including phage M13-adenovirus recombinants, denatured plasmids, and synthetic oligodeoxynucleotides, failed to reveal any requirements for specific nucleotide sequences. With single-stranded templates containing no dG residues, the specific deoxynucleoside triphosphate requirements of the initiation reaction were altered. Images PMID:6320160

  12. Adenovirus infection elevates levels of cellular topoisomerase I.

    PubMed Central

    Chow, K C; Pearson, G D

    1985-01-01

    We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme. Images PMID:2986107

  13. Prevalence of human adenoviruses in raw and treated water.

    PubMed

    van Heerden, J; Ehlers, M M; van Zyl, W B; Grabow, W O K

    2004-01-01

    Human adenoviruses (HAds), of which there are 51 antigenic types, are associated aetiologically with gastrointestinal, respiratory, urinary tract and eye infections. The clinical importance of HAds and the potential health risks constituted by HAds in water environments are widely recognised. This study was conducted to assess the use of an optimised integrated cell culture molecular-based technique to determine the prevalence of HAds in raw and treated drinking-water supplies in South Africa. Selected supplies were monitored weekly for the presence of adenoviruses over a one-year period (July 2001 to June 2002). Drinking-water supplies were derived from acceptable quality surface water sources using treatment processes that conformed to international standards for the production of safe drinking water. Adenoviruses were detected by amplification in cell cultures, followed by amplifying the extracted nucleic acids using molecular techniques (nested PCR). HAds were detected in 29.8% (59/198) of the treated drinking water, 16% (8/50) of dam water and 44% (22/50) of river-water samples tested. The results of this study confirmed the presence of HAds in some raw and treated drinking water supplies in South Africa.

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

    PubMed Central

    Bru, Thierry; Salinas, Sara; Kremer, Eric J.

    2010-01-01

    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. PMID:21994722

  15. History of the restoration of adenovirus type 4 and type 7 vaccine, live oral (Adenovirus Vaccine) in the context of the Department of Defense acquisition system.

    PubMed

    Hoke, Charles H; Snyder, Clifford E

    2013-03-15

    Respiratory pathogens cause morbidity and mortality in US military basic trainees. Following the influenza pandemic of 1918, and stimulated by WWII, the need to protect military personnel against epidemic respiratory disease was evident. Over several decades, the US military elucidated etiologies of acute respiratory diseases and invented and deployed vaccines to prevent disease caused by influenza, meningococcus, and adenoviruses. In 1994, the Adenovirus Vaccine manufacturer stopped its production. By 1999, supplies were exhausted and adenovirus-associated disease, especially serotype 4-associated febrile respiratory illness, returned to basic training installations. Advisory bodies persuaded Department of Defense leaders to initiate restoration of Adenovirus Vaccine. In 2011, after 10 years of effort by government and contractor personnel and at a cost of about $100 million, the Adenovirus Vaccine was restored to use at all military basic training installations. Disease and adenovirus serotype 4 isolation rates have fallen dramatically since vaccinations resumed in October 2011 and remain very low. Mindful of the adage that "The more successful a vaccine is, the more quickly the need for it will be forgotten.", sustainment of the supply of the Adenovirus Vaccine may be a challenge, and careful management will be required for such sustainment.

  16. Tropism modification of adenovirus vectors by peptide ligand insertion into various positions of the adenovirus serotype 41 short-fiber knob domain.

    PubMed

    Hesse, Andrea; Kosmides, Daniela; Kontermann, Roland E; Nettelbeck, Dirk M

    2007-03-01

    Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

  17. The Evaluation of Polyhexamethylene Biguanide (PHMB) as a Disinfectant for Adenovirus

    PubMed Central

    Romanowski, Eric G.; Yates, Kathleen A.; O’Connor, Katherine E.; Mah, Francis S.; Shanks, Robert M. Q.; Kowalski, Regis P.

    2013-01-01

    Purpose Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs. The current study determined whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs. Methods The direct disinfecting activity of PHMB was determined in triplicate assays by incubating nine human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with 50 and 0 PPM (µg/ml) of PHMB for 24 hours at room temperature, to simulate swimming pool temperatures, or 40°C, to simulate hot tub temperatures. Plaque assays determined adenovirus titers after incubation. Titers were Log10 converted and mean ± standard deviation Log10 reductions from controls were calculated. Virucidal (greater than 99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested. Results At room temperature, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for all adenovirus types tested. At 40°C, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for two adenovirus types and greater than 1 Log10, but less than 3 Log10, for seven of nine adenovirus types. Conclusions 50 PPM of PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Clinical Relevance Recreational water maintained and sanitized with PHMB has the potential to serve as a vector for the transmission of ocular adenovirus infections. PMID:23450376

  18. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2010 CFR

    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...

  19. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 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...

  20. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  1. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  5. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 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...

  6. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  7. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 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...

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

    PubMed Central

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

    1996-01-01

    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. PMID:8627709

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene (AdTW68.H5ck). Commercial layer-type chicken groups were singly vaccinated ocularly at 5 days of age, or singly v...

  11. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding a H7 hemagglutinin gene from a low pathogenic North American isolate (AdChNY94.H7). Chickens vaccinate...

  12. Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

    SciTech Connect

    Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam; Abu-Asab, Mones S.; Wildner, Oliver; Miles, Brian K.; Yim, Kevin C.; Ramanan, Vijay; Prince, Gregory A.; Morris, John C.

    2007-12-05

    Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

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

    PubMed

    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.

  14. Comparison of throat swab and nasopharyngeal aspirate specimens for rapid detection of adenovirus.

    PubMed

    Hara, Michimaru; Takao, Shinichi; Shimazu, Yukie

    2015-06-01

    Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.

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

    PubMed

    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.

  16. Location of the Origin of DNA Replication in Adenovirus Type 2

    PubMed Central

    Horwitz, Marshall S.

    1974-01-01

    Utilizing the isolated left and right halves of both adenovirus type 2 and the nondefective adenovirus simian virus 40 hybrid (Ad2+ND1), studies were undertaken to find the site on the DNA molecules at which replication begins. The data are consistent with several models which include an initiation event at both ends and bidirectional growth. PMID:4363250

  17. Subgenomic viral DNA species synthesized in simian cells by human and simian adenoviruses.

    PubMed Central

    Daniell, E

    1981-01-01

    DNA synthesized after infection of simian tissue culture cells (BSC-1 or CV-1) with human adenovirus type 2 or 5 or with simian adenovirus 7 was characterized. It was demonstrated that as much as 40% of the virus-specific DNA in nuclei of infected monkey cells consists of subgenomic pieces. No subgenomic viral DNA species were detected in the nuclei of human (HeLa) cells infected with these adenovirus types. Restriction analysis showed that these short viral DNA molecules contain normal amounts of the sequences from the ends of the viral genome, whereas internal regions are underrepresented. The production of subgenomic DNAs is not correlated with semipermissive infection. Although adenovirus types 2 and 5 are restricted in monkey cells, these cells are fully permissive for simian adenovirus 7. HR404, an adenovirus type 5 mutant which is not restricted in monkey cells, produced the same percentage of subgenomic DNAs as did its wild type (restricted) parent, and coinfection of monkey cells with adenovirus type 5 DNAs. The array of predominant size classes among the heterogeneously sized short DNAs is serotype specific. Extensive plaque purification and comparison of wild-type adenovirus type 5 with several viral mutants indicated that the distribution of aberrant sizes of DNA is characteristic of the virus and not a result of random replicative errors and then enrichment of particular species. Images PMID:6261009

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

    EPA Pesticide Factsheets

    This dataset includes the recoveries of spiked adenovirus through various stages of experimental optimization procedures. This dataset is associated with the following publication:McMinn , B., A. Korajkic, and A. Grimm. Optimization and evaluation of a method to detect adenoviruses in river water. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 231(1): 8-13, (2016).

  19. Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

    PubMed

    Vragniau, Charles; Hübner, Jens-Martin; Beidler, Peter; Gil, Sucheol; Saydaminova, Kamola; Lu, Zhuo-Zhuang; Yumul, Roma; Wang, Hongjie; Richter, Maximilian; Sova, Pavel; Drescher, Charles; Fender, Pascal; Lieber, André

    2017-03-15

    Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our

  20. Human adenoviruses in water: occurrence and health implications: a critical review.

    PubMed

    Jiang, Sunny C

    2006-12-01

    Adenoviruses are important human pathogens that are responsible for both enteric illnesses and respiratory and eye infections. Recently, these viruses have been found to be prevalent in rivers, coastal waters, swimming pool waters, and drinking water supplies worldwide. United Sates Environmental Protection Agency (USEPA) listed adenovirus as one of nine microorganisms on the Contamination Candidate List for drinking water because their survival characteristic during water treatment is not yet fully understood. Adenoviruses have been found to be significantly more stable than fecal indicator bacteria and other enteric viruses during UV treatment. Adenovirus infection may be caused by consumption of contaminated water or inhalation of aerosolized droplets during water recreation. The goal of this review is to summarize the state of technology for adenovirus detection in natural and drinking waters and the human health risk imposed by this emerging pathogen. The occurrence of these viruses in natural and treated waters is summarized from worldwide reports.

  1. Rotavirus and adenovirus prevalence at Tepecik education and research hospital (Turkey).

    PubMed

    Ece, Gulfem; Samlioglu, Pinar; Ulker, Temel; Kose, Sukran; Ersan, Gursel

    2012-06-01

    Diarrhoea affects many people globally. Rotaviruses and enteric adenovirus types 40 and 41 are the most common viruses causing childhood gastroenteritis. The aim of this study was to determine the prevalence of rotavirus and adenovirus from the faecal samples obtained at the Infectious Diseases and Clinical Microbiology Laboratory of Tepecik Education and Research Hospital. The faecal samples were screened for rotavirus, and adenovirus by commercially available immunochromatographic EIA kit (Rotavirus/Adenovirus Combo Rapid Test Device) (San Diego, CA, USA). A total of 1112 stool samples were collected from May 23rd 2008 to May 25th 2010. Of these faecal samples, 201(18.07%) were positive for rotavirus and 14 (1.2 %) for adenovirus antigen. In our study the most common agent detected was rotavirus. Viral antigen analysis in stool specimens is important for diagnosis. Detection of the viral aetiology in gastroenteritis cases will prevent unnecessary antibiotic consumption.

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

    PubMed

    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.

  3. Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force.

    PubMed

    Tresilwised, Nittaya; Pithayanukul, Pimolpan; Mykhaylyk, Olga; Holm, Per Sonne; Holzmüller, Regina; Anton, Martina; Thalhammer, Stefan; Adigüzel, Denis; Döblinger, Markus; Plank, Christian

    2010-08-02

    Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the

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

    PubMed

    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.

  5. Packaging capacity and stability of human adenovirus type 5 vectors.

    PubMed Central

    Bett, A J; Prevec, L; Graham, F L

    1993-01-01

    Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population. Images PMID:8371349

  6. Canine adenovirus type 1 in a fennec fox (Vulpes zerda).

    PubMed

    Choi, Jeong-Won; Lee, Hyun-Kyoung; Kim, Seong-Hee; Kim, Yeon-Hee; Lee, Kyoung-Ki; Lee, Myoung-Heon; Oem, Jae-Ku

    2014-12-01

    A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing.

  7. The Length of the Terminal Repetition in Adenovirus-2 DNA

    PubMed Central

    Roberts, Richard J.; Arrand, John R.; Keller, Walter

    1974-01-01

    Adenovirus-2 DNA was end-labeled by partial digestion with Escherichia coli exonuclease III and resynthesis with the DNA polymerase from avian myeloblastosis virus and α-32P-labeled deoxyribonucleoside triphosphates. This end-labeled DNA was cleaved with several specific endonucleases and the terminal fragments were characterized by gel electrophoresis and pyrimidine tract analysis. Two endonucleases gave identical fragments from both ends, presumably from cleavage within the inverted terminal repetition, while all other endonucleases gave dissimilar fragments from the two ends. From the sizes of these fragments it is estimated that the inverted terminal repetition is between 100 and 140 nucleotide pairs long. Images PMID:4139703

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

    PubMed

    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.

  9. Phylogenetic and pathogenic characterization of novel adenoviruses from long-tailed ducks (Clangula hyemalis)

    USGS Publications Warehouse

    Counihan, Katrina; Skerratt, Lee; Franson, J. Christian; Hollmen, Tuula E.

    2015-01-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

  10. E1A RNA transcripts amplify adenovirus-mediated tumor reduction.

    PubMed

    Dion, L D; Goldsmith, K T; Strong, T V; Bilbao, G; Curiel, D T; Garver, R I

    1996-11-01

    Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.

  11. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    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

  12. Fluctuating expression of microRNAs in adenovirus infected cells.

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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.

  15. Investigation of the mechanical and interfacial properties of adenovirus

    NASA Astrophysics Data System (ADS)

    Matthews, William Garrett

    The ability to investigate materials at the single molecule and macromolecule level became a reality with the introduction of the atomic force microscope (AFM) by Bennig et al. in 1986. Presented in this dissertation is a modification to the AFM that facilitates imaging of delicate samples under liquids. The instrument was subsequently used to investigate a variety of material and interfacial properties of adenovirus. Firstly, the elasticity of adenovirus particles in air and in water was measured. The virus was found to be some fifty fold more compliant in water than in air, with the measured elastic modulus changing from 15 MPa to 770 MPa. Individual viruses also were translated across a variety of surfaces, and the force required to do these manipulations was recorded. A variety of behaviors were observed, including evidence for rolling of the particles. This measurement constitutes one of the first direct observations of rolling behavior on this length scale. Finally, viruses were observed to shed their capsid proteins when deposited on positively charged magnesium intercalated mica. The resulting core structure was investigated, and the uncoating process was captured in a series of images. These images represent one of the first molecular level processes to be observed at the single molecule level.

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

    PubMed

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

    2016-02-05

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

  17. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

    Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.; Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J.; Mercier, George T.; Barry, Michael A.

    2015-08-15

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

  18. Directed adenovirus evolution using engineered mutator viral polymerases

    PubMed Central

    Uil, Taco G.; Vellinga, Jort; de Vrij, Jeroen; van den Hengel, Sanne K.; Rabelink, Martijn J. W. E.; Cramer, Steve J.; Eekels, Julia J. M.; Ariyurek, Yavuz; van Galen, Michiel; Hoeben, Rob C.

    2011-01-01

    Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing. PMID:21138963

  19. Recombinant adenovirus vectors for gene therapy and clinical trials.

    PubMed

    Nász, I; Adám, E

    2001-01-01

    In the last decade adenovirus (AdV) vectors have emerged as promising technology in gene therapy. They have been used for genetic modification of a variety of somatic cells in vitro and in vivo. They have been widely used as gene delivery vectors in experiments both with curative and preventive purposes. AdV vectors have been used in the experimental and in some extent in the clinical gene therapy of a variety of cancers. The combination of recombinant AdV technology with chemotherapy (pro drug system) seems to be promising, too. AdV vectors offer several advantages over other vectors. Replication defective vectors can be produced in very high titers (10(11) pfu/ml) thus allowing a substantially greater efficiency of direct gene transfer; they have the capacity to infect both replicating and nonreplicating (quiescent) cells from a variety of tissues and species. Several important limitations of adenovirus mediated gene transfer are also known, such as the relatively short-term (transient) expression of foreign genes, induction of the host humoral and cellular immune response to viral proteins and viral infected cells, which may substantially inhibit the effect of repeated treatment with AdV vectors, the limited cloning capacity and the lack of target cell specificity. However, the well-understood structure, molecular biology and host cell interactions of AdV-s offer some potential solutions to these limitations.

  20. Inactivation kinetics of adenovirus serotype 2 with monochloramine.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-03-01

    The effect of pH (6-10), temperature (10-30 degrees C), disinfectant concentration (1-11mg/l as Cl(2)), and ammonia nitrogen-to-chlorine molar ratio (1.3-52) on the inactivation kinetics of adenovirus serotype 2 with monochloramine was investigated by performing batch-reactor experiments with synthetic 0.01M buffer (phosphate or borate) solutions. The inactivation kinetics was independent of monochloramine concentration and ammonia nitrogen-to-chlorine molar ratio but had strong pH dependence, with the rate of inactivation decreasing with increasing pH. The kinetics at pH 6 and 8 were consistent with pseudo-first-order kinetics, while curves at pH 10 were characterized by a lag phase followed by a pseudo-first-order phase. The rate of inactivation increased with increasing temperature-activation energies of 56.5kJ/mole (pH 8) and 72.6kJ/mole (pH 10). The results obtained in this study revealed that monochloramine disinfection might not generally provide adequate control of adenoviruses in drinking water at high pH and low temperature.

  1. Gene Transcript Abundance Profiles Distinguish Kawasaki Disease from Adenovirus Infection

    PubMed Central

    Popper, Stephen J.; Watson, Virginia E.; Shimizu, Chisato; Kanegaye, John T.; Burns, Jane C.; Relman, David A.

    2010-01-01

    Background Acute Kawasaki disease (KD) is difficult to distinguish from other illnesses that involve acute rash or fever, in part because the etiologic agent(s) and pathophysiology remain poorly characterized. As a result, diagnosis and critical therapies may be delayed. Methods We used DNA microarrays to identify possible diagnostic features of KD. We compared gene expression patterns in the blood of 23 children with acute KD and 18 age-matched febrile children with 3 illnesses that resemble KD. Results Genes associated with platelet and neutrophil activation were expressed at higher levels in patients with KD than in patients with acute adenovirus infections or systemic adverse drug reactions, but levels in patients with KD were not higher than those in patients with scarlet fever. Genes associated with B cell activation were also expressed at higher levels in patients with KD than in control subjects. A striking absence of interferon-stimulated gene expression in patients with KD was confirmed in an independent cohort of patients with KD. Using a set of 38 gene transcripts, we successfully predicted the diagnosis for 21 of 23 patients with KD and 7 of 8 patients with adenovirus infection. Conclusions These findings provide insight into the molecular features that distinguish KD from other febrile illnesses and support the feasibility of developing novel diagnostic reagents for KD based on the host response. PMID:19583510

  2. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    Hyndman, T; Shilton, C M

    2011-06-01

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

  6. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  7. Molecular Detection of Adenoviruses, Rhabdoviruses, and Paramyxoviruses in Bats from Kenya

    PubMed Central

    Conrardy, Christina; Tao, Ying; Kuzmin, Ivan V.; Niezgoda, Michael; Agwanda, Bernard; Breiman, Robert F.; Anderson, Larry J.; Rupprecht, Charles E.; Tong, Suxiang

    2014-01-01

    We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus. PMID:24865685

  8. Fatal pulmonary edema in white-tailed deer (Odocoileus virginianus) associated with adenovirus infection.

    PubMed

    Sorden, S D; Woods, L W; Lehmkuhl, H D

    2000-07-01

    Sporadic sudden deaths in adult white-tailed deer occurred from November 1997 through August 1998 on an Iowa game farm. Three of the 4 deer necropsied had severe pulmonary edema, widespread mild lymphocytic vasculitis, and amphophilic intranuclear inclusion bodies in scattered endothelial cells in blood vessels in the lung and abdominal viscera. Immunohistochemistry with bovine adenovirus 5 antisera and transmission electron microscopy demonstrated adenoviral antigen and nucleocapsids, respectively, within endothelial cells. Adenovirus was isolated in cell culture from 1 of the affected deer. The isolate was neutralized by California black-tailed deer adenovirus antiserum. These findings indicate that adenovirus should be considered in the differential diagnosis of both black-tailed and white-tailed deer with pulmonary edema and/or hemorrhagic enteropathy.

  9. Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

    PubMed

    Walters, Robert W; Freimuth, Paul; Moninger, Thomas O; Ganske, Ingrid; Zabner, Joseph; Welsh, Michael J

    2002-09-20

    Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

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

    PubMed Central

    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

  11. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells

    SciTech Connect

    Zorn, G.A.; Anderson, C.W.

    1981-02-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide.

  12. Inhibition of Adenovirus In Vitro DNA Replication by Vesicular Stomatitis Virus Leader RNA

    DTIC Science & Technology

    1986-08-18

    19 Inhibition of Macromolecular Synthesis •••••••••••••••••••••••••••• 22 Adenovirus Structure and Life Cycle...be possible to determine the affects of VSV and VSV leader RNA on eukaryotic DNA synthesis. ; . 1 .· 29 Adenovirus Structure and Life Cycle

  13. Ganciclovir Inhibits Human Adenovirus Replication and Pathogenicity in Permissive Immunosuppressed Syrian Hamsters

    PubMed Central

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

    2014-01-01

    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. PMID:25224011

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

    PubMed

    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.

  15. Unambiguous typing of canine adenovirus isolates by deoxyribonucleic acid restriction-endonuclease analysis.

    PubMed Central

    Assaf, R; Marsolais, G; Yelle, J; Hamelin, C

    1983-01-01

    Viral deoxyribonucleic acid extracted from a limited number of cells infected with canine adenovirus type 1 or type 2 was cleaved with several restriction endonucleases. Agarose gel electrophoresis of the limit digests showed stable differences between the canine adenovirus type 1 and type 2 cleavage patterns. Rapid and accurate typing of large numbers of clinical isolates may thus be done by deoxyribonucleic acid restriction-endonuclease analysis. Images Fig. 1. Fig. 2. PMID:6321002

  16. Adenovirus type 2 nuclear RNA accumulating during productive infection.

    PubMed Central

    Bachenheimer, S L

    1977-01-01

    The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus-type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Later nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and less than 32S after denaturation and a second having a narrow size distribution around 35S which is quite stable to Me2SO. Analysis of late RNA by hybridization to Sma fragments of Ad2 DNA suggests that the 35S RNA species is derived from a limited portion of the left half of the viral genome. PMID:864839

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

    PubMed Central

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

    2015-01-01

    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. PMID:26280277

  18. Coxsackievirus and adenovirus receptor is essential for cardiomyocyte development.

    PubMed

    Asher, Damon R; Cerny, Anna M; Weiler, Sarah R; Horner, James W; Keeler, Marilyn L; Neptune, Mychell A; Jones, Stephen N; Bronson, Roderick T; Depinho, Ronald A; Finberg, Robert W

    2005-06-01

    The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that is known to be a site of viral attachment and entry, but its physiologic functions are undefined. CAR expression is maximal in neonates and wanes rapidly after birth in organs such as heart, muscle, and brain, suggesting that CAR plays a role in the development of these tissues. Here, we show that CAR deficiency resulted in an embryonic lethal condition associated with cardiac defects. Specifically, commencing approximately 10.5 days postconception (dpc), CAR-/- cardiomyocytes exhibited regional apoptosis evidenced by both histopathologic features of cell death and positive staining for the apoptotic marker cleaved caspase 3. CAR-/- fetuses invariably suffered from degeneration of the myocardial wall and thoracic hemorrhaging, leading to death by 11.5 dpc. These findings are consistent with the view that CAR provides positive survival signals to cardiomyocytes that are essential for normal heart development.

  19. Oncolytic adenoviruses: A thorny path to glioma cure

    PubMed Central

    Ulasov, I.V.; Borovjagin, A.V.; Schroeder, B.A.; Baryshnikov, A.Y.

    2014-01-01

    Glioblastoma Multiforme (GBM) is a rapidly progressing brain tumor. Despite the relatively low percentage of cancer patients with glioma diagnoses, recent statistics indicate that the number of glioma patients may have increased over the past decade. Current therapeutic options for glioma patients include tumor resection, chemotherapy, and concomitant radiation therapy with an average survival of approximately 16 months. The rapid progression of gliomas has spurred the development of novel treatment options, such as cancer gene therapy and oncolytic virotherapy. Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach. Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment. PMID:25685829

  20. Adenovirus Membrane Penetration: Tickling the Tail of a Sleeping Dragon

    PubMed Central

    Wiethoff, Christopher M.; Nemerow, Glen R.

    2015-01-01

    As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (1), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus(HAdV). PMID:25798531

  1. Partial characterization of new adenoviruses found in lizards.

    PubMed

    Ball, Inna; Behncke, Helge; Schmidt, Volker; Geflügel, F T A; Papp, Tibor; Stöhr, Anke C; Marschang, Rachel E

    2014-06-01

    In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species.

  2. Mucosal vaccination by adenoviruses displaying reovirus sigma 1.

    PubMed

    Weaver, Eric A; Camacho, Zenaido T; Hillestad, Matthew L; Crosby, Catherine M; Turner, Mallory A; Guenzel, Adam J; Fadel, Hind J; Mercier, George T; Barry, Michael A

    2015-08-01

    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.

  3. Adenovirus receptors and their implications in gene delivery

    PubMed Central

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

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  4. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

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

  5. Role of preterminal protein processing in adenovirus replication.

    PubMed Central

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

    1997-01-01

    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. PMID:9261355

  6. Role of preterminal protein processing in adenovirus replication.

    PubMed

    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.

  7. Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

    PubMed Central

    Roy, Soumitra; Calcedo, Roberto; Medina-Jaszek, Angelica; Keough, Martin; Peng, Hui; Wilson, James M.

    2011-01-01

    Objective Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested. Conclusion Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora. PMID:21980361

  8. Use of cidofovir in pediatric patients with adenovirus infection

    PubMed Central

    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

  9. “Hit-and-Run” Transformation by Adenovirus Oncogenes

    PubMed Central

    Nevels, Michael; Täuber, Birgitt; Spruss, Thilo; Wolf, Hans; Dobner, Thomas

    2001-01-01

    According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy. PMID:11238835

  10. Adenovirus Vectors Targeting Distinct Cell Types in the Retina

    PubMed Central

    Sweigard, J. Harry; Cashman, Siobhan M.

    2010-01-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5ΔRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5ΔRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5ΔRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5ΔRGD vectors. PMID:19892875

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

    SciTech Connect

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    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.

  12. Isolation of a novel adenovirus from California sea lions Zalophus californianus.

    PubMed

    Goldstein, T; Colegrove, K M; Hanson, M; Gulland, F M D

    2011-05-09

    Viral hepatitis associated with adenoviral infection has been reported in California sea lions Zalophus californianus admitted to rehabilitation centers along the California coast since the 1970s. Canine adenovirus 1 (CAdV-1) causes viral hepatitis in dogs and infects a number of wildlife species. Attempts to isolate the virus from previous sea lion hepatitis cases were unsuccessful, but as the hepatitis had morphologic features resembling canine infectious hepatitis, and since the virus has a wide host range, it was thought that perhaps the etiologic agent was CAdV-1. Here, we identify a novel adenovirus in 2 stranded California sea lions and associate the infection with viral hepatitis and endothelial cell infection. Phylogenetic analysis confirmed the classification of the sea lion adenovirus in the Mastadenovirus genus with the most similarity to tree shrew adenovirus 1 (TSAdV-1, 77%). However, as the sea lion adenovirus appeared to be equally distant from the other Mastadenovirus species based on phylogenetic analysis, results indicate that it represents an independent lineage and species. Although sequences from this novel virus, otarine adenovirus 1 (OtAdV-1), show some similarity to CAdV-1 and 2, it is clearly distinct and likely the cause of the viral hepatitis in the stranded California sea lions.

  13. Replication of adenovirus type 4 DNA by a purified fraction from infected cells.

    PubMed Central

    Temperley, S M; Hay, R T

    1991-01-01

    An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication. Images PMID:1829516

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

    PubMed Central

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

    2010-01-01

    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; Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre-existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad-immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre-existing immunity. PMID:19711370

  15. Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

    PubMed

    Miura, Y; Yoshida, K; Nishimoto, T; Hatanaka, K; Ohnami, S; Asaka, M; Douglas, J T; Curiel, D T; Yoshida, T; Aoki, K

    2007-10-01

    Targeting of gene transfer at the level of cell entry 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 proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

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

    PubMed Central

    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

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

    PubMed Central

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

    2016-01-01

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

  18. Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea.

    PubMed

    Lee, Seung-Hoon; Kim, Sang-Jong

    2002-01-01

    We investigated the viral contamination of tap water at 11 urban sites in Korea between 1997 and 1998 over a period of 11 months. A total of 23 tap water samples were examined for infectious enteroviruses and adenoviruses by a cell culture technique followed by polymerase chain reaction (PCR) amplification. To identify the recovered viruses, sequence analysis of PCR products was performed. Infectious enteroviruses and adenoviruses were detected in 11 (47.8%) and 9 (39.1%) of the samples, respectively. Both enteroviruses and adenoviruses were detected in five samples (21.7%). The level of viral contamination was quite high, ranging from 2 x 10(-3) to 2.9 x 10(-2) Most Probable Number of Infectious Unit L(-1), far above the recommended virus level in drinking water set by the US EPA. Poliovirus type I derived from vaccine was frequently detected and the remainder comprised coxsackievirus B type or echovirus type 6, which were causative agents of aseptic meningitis in Korea in 1997 and 1998, respectively. Several types of adenovirus were detected in tap water samples and some water samples were found to contain adenoviruses which were closely related to enteric adenovirus types 40 and 41.

  19. Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells

    SciTech Connect

    Richardson, W.D.; Anderson, C.W.

    1984-08-01

    Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 M/sub r/ virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus. 26 references, 2 figures, 1 table.

  20. Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre

    SciTech Connect

    El Bakkouri, Majida; Seiradake, Elena; Cusack, Stephen; Ruigrok, Rob W.H. Schoehn, Guy

    2008-08-15

    There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.

  1. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

  2. [Current state of oncolytic virotherapy in Japan].

    PubMed

    Nakamori, Mikihito; Yamaue, Hiroki

    2013-05-01

    Oncolytic virotherapy is an emerging treatment strategy that uses replication-competent viruses to destroy cancers. Recent advances include preclinical proof of feasibility for a single-shot virotherapy cure, identification of drugs that accelerate intratumoral virus propagation, and strategies to maximize the immunotherapeutic action of oncolytic viruses. The primary clinical milestone has been completion of accrual in a phase 3 trial of intratumoral herpes simplex virus therapy using OncoVEX for metastatic melanoma. In Japan, clinical treatments such as oncolytic adenoviruses(OBP-301)for esophageal cancer and oncolytic herpes simplex viruses(G47b)for brain cancer have accelerated considerably. We hope that a steady stream of new oncolytic viruses will enter the clinical arena in our country.

  3. Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

    PubMed Central

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

    2012-01-01

    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. PMID:22811532

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

    PubMed

    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.

  5. Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression.

    PubMed

    Lee, Cho-Hee; Kasala, Dayananda; Na, Youjin; Lee, Min Sang; Kim, Sung Wan; Jeong, Ji Hoon; Yun, Chae-Ok

    2014-07-01

    Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    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.

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

    PubMed

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

    2015-01-01

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

  9. An adenovirus linked to mortality and disease in long-tailed ducks (Clangula hyemalis) in Alaska

    USGS Publications Warehouse

    Hollmén, Tuula E.; Franson, J.C.; Flint, P.L.; Grand, J.B.; Lanctot, Richard B.; Docherty, D.E.; Wilson, H.M.

    2003-01-01

    An adenovirus was isolated from intestinal samples of two long-tailed ducks (Clangula hyemalis) collected during a die-off in the Beaufort Sea off the north coast of Alaska in 2000. The virus was not neutralized by reference antiserum against known group I, II, or III avian adenoviruses and may represent a new serotype. The prevalence of the virus was determined in live-trapped long-tailed ducks at the mortality site and at a reference site 100 km away where no mortality was observed. Prevalence of adenovirus antibodies in serum samples at the mortality site was 86% compared to 10% at the reference site. Furthermore, 50% of cloacal swabs collected at the mortality site and only 7% of swabs from the reference site were positive for adenoviruses. In 2001, no mortality was observed at either of the study areas, and virus prevalence in both serum and cloacal samples was low, providing further evidence that the adenovirus was linked to the mortality event in 2000. The virus was used to infect long-tailed ducks under experimental conditions and resulted in lesions previously described for avian adenovirus infections and similar to those observed in long-tailed duck carcasses from the Beaufort Sea. The status of long-tailed ducks has recently become a concern in Alaska due to precipitous declines in breeding populations there since the mid-1970s. Our findings suggest that the newly isolated adenovirus is a disease agent and source of mortality in long-tailed ducks, and thus could be a contributing factor in population declines.

  10. An adenovirus linked to mortality and disease in long-tailed ducks (Clangula hyemalis) in Alaska.

    PubMed

    Hollmén, Tuula E; Franson, J Christian; Flint, Paul L; Grand, James B; Lanctot, Richard B; Docherty, Douglas E; Wilson, Heather M

    2003-01-01

    An adenovirus was isolated from intestinal samples of two long-tailed ducks (Clangula hyemalis) collected during a die-off in the Beaufort Sea off the north coast of Alaska in 2000. The virus was not neutralized by reference antiserum against known group I, II, or III avian adenoviruses and may represent a new serotype. The prevalence of the virus was determined in live-trapped long-tailed ducks at the mortality site and at a reference site 100 km away where no mortality was observed. Prevalence of adenovirus antibodies in serum samples at the mortality site was 86% compared to 10% at the reference site. Furthermore, 50% of cloacal swabs collected at the mortality site and only 7% of swabs from the reference site were positive for adenoviruses. In 2001, no mortality was observed at either of the study areas, and virus prevalence in both serum and cloacal samples was low, providing further evidence that the adenovirus was linked to the mortality event in 2000. The virus was used to infect long-tailed ducks under experimental conditions and resulted in lesions previously described for avian adenovirus infections and similar to those observed in long-tailed duck carcasses from the Beaufort Sea. The status of long-tailed ducks has recently become a concern in Alaska due to precipitous declines in breeding populations there since the mid-1970s. Our findings suggest that the newly isolated adenovirus is a disease agent and source of mortality in long-tailed ducks, and thus could be a contributing factor in population declines.

  11. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine.

    PubMed

    Pandey, Aseem; Singh, Neetu; Vemula, Sai V; Couëtil, Laurent; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2012-01-01

    The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

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

    PubMed Central

    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

  13. The Dual Nature of Nek9 in Adenovirus Replication

    PubMed Central

    Jung, Richard; Radko, Sandi

    2015-01-01

    ABSTRACT 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 silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks

  14. Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

    PubMed

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

    2015-06-01

    Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor

  15. Adenovirus disease in six small bowel, kidney and heart transplant recipients; pathology and clinical outcome.

    PubMed

    Mehta, Vikas; Chou, Pauline C; Picken, Maria M

    2015-11-01

    Adenoviruses are emerging as important viral pathogens in hematopoietic stem cell and solid organ transplant recipients, impacting morbidity, graft survival, and even mortality. The risk seems to be highest in allogeneic hematopoietic stem cell transplant recipients as well as heart, lung, and small bowel transplant recipients. Most of the adenovirus diseases develop in the first 6 months after transplantation, particularly in pediatric patients. Among abdominal organ recipients, small bowel grafts are most frequently affected, presumably due to the presence of a virus reservoir in the mucosa-associated lymphoid tissue. Management of these infections may be difficult and includes the reduction of immunosuppression, whenever possible, combined with antiviral therapy, if necessary. Therefore, an awareness of the pathology associated with such infections is important in order to allow early detection and specific treatment. We reviewed six transplant recipients (small bowel, kidney, and heart) with adenovirus graft involvement from two institutions. We sought to compare the diagnostic morphology and the clinical and laboratory findings. The histopathologic features of an adenovirus infection of the renal graft and one native kidney in a heart transplant recipient included a vaguely granulomatous mixed inflammatory infiltrate associated with rare cells showing a cytopathic effect (smudgy nuclei). A lymphocytic infiltrate, simulating T cell rejection, with admixture of eosinophils was also seen. In the small bowel grafts, there was a focal mixed inflammatory infiltrate with associated necrosis in addition to cytopathic effects. In the heart, allograft adenovirus infection was silent with no evidence of inflammatory changes. Immunohistochemical stain for adenovirus was positive in all grafts and in one native kidney. All patients were subsequently cleared of adenovirus infection, as evidenced by follow-up biopsies, with no loss of the grafts. Adenovirus infection can

  16. Lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns.

    PubMed

    Woods, L W; Hanley, R S; Chiu, P H; Lehmkuhl, H D; Nordhausen, R W; Stillian, M H; Swift, P K

    1999-03-01

    Adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California during the latter half of 1993. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. To study transmission of adenovirus infection in deer and susceptibility of black-tailed deer (Odocoileus hemionus columbianus) fawns to adenovirus infection, six 3-6-month-old black-tailed fawns were divided into two treatment groups. One group was inoculated intravenously and the other group was inoculated through the mucous membranes of the eyes, nose and mouth with purified adenovirus. Each treatment group also included two additional fawns (four total) that were not inoculated but were exposed to inoculated animals (contact animals). One fawn served as a negative control. Between 4 and 16 days postinoculation, 8/10 fawns developed systemic or localized infection with lesions identical to lesions seen in animals with natural disease that died during the epizootic. Transmission was by direct contact, and the route of inoculation did not affect the incubation period or the distribution of the virus (systemic or the localized infection). Immunohistochemical analysis using polyclonal antiserum against bovine adenovirus type 5 demonstrated staining in endothelial cells of vessels in numerous tissues in animals with systemic infection and endothelial staining only in vessels subtending necrotic foci in the upper alimentary tract in animals with the localized form of the disease. All inoculated or exposed animals had staining in the tonsillar epithelium. Transmission electron microscopic examination of lung and ileum from two fawns with pulmonary edema and hemorrhagic enteropathy demonstrated endothelial necrosis and

  17. Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom.

    PubMed

    Zeng, Zhiwei; Zhang, Jing; Jing, Shuping; Cheng, Zetao; Bofill-Mas, Silvia; Maluquer de Motes, Carlos; Hundesa, Ayalkibet; Girones, Rosina; Seto, Donald; Zhang, Qiwei

    2016-11-03

    The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

  18. Delivery of oncolytic adenovirus into the nucleus of tumorigenic cells by tumor microparticles for virotherapy.

    PubMed

    Ran, Li; Tan, Xiaohua; Li, Yanchun; Zhang, Huafeng; Ma, Ruihua; Ji, Tiantian; Dong, Wenqian; Tong, Tong; Liu, Yuying; Chen, Degao; Yin, Xiaonan; Liang, Xiaoyu; Tang, Ke; Ma, Jingwei; Zhang, Yi; Cao, Xuetao; Hu, Zhuowei; Qin, Xiaofeng; Huang, Bo

    2016-05-01

    Oncolytic viruses have been utilized for the treatment of various cancers. However, delivery of the viral particles to tumor cells remains a major challenge. Microparticles (MP) are vesicle forms of plasma membrane fragments of 0.1-1 μm in size that are shed by cells. We have previously shown the delivery of chemotherapeutic drugs using tumor cell-derived MPs (T-MP). Here we report that T-MPs can be utilized as a unique carrier system to deliver oncolytic adenoviruses to human tumors, leading to highly efficient cytolysis of tumor cells needed for in vivo treatment efficacy. This T-MP-mediated oncolytic virotherapy approach holds multiple advantages, including: 1) delivery of oncolytic adenovirus by T-MPs is able to avoid the antiviral effect of host antibodies; 2) delivery of oncolytic adenovirus by T-MPs is not limited by virus-specific receptor that mediates the entry of virus into tumor cells; 3) T-MPs are apt at delivering oncolytic adenoviruses to the nucleus of tumor cells as well as to stem-like tumor-repopulating cells for the desired purpose of killing them. These findings highlight a novel oncolytic adenovirus delivery system with highly promising clinical applications.

  19. Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

    PubMed Central

    San Martín, Carmen; Glasgow, Joel N.; Borovjagin, Anton; Beatty, Matthew S.; Kashentseva, Elena A.; T. Curiel, David; Marabini, Roberto; Dmitriev, Igor P.

    2008-01-01

    Summary Minor coat protein IIIa is conserved in all adenoviruses and required for correct viral assembly, but its precise function in capsid organization is unknown. The latest adenovirus capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant adenovirus variants with IIIa encoding six-histidine tag (6-His), 6-His and FLAG peptides, or 6-His linked to FLAG with a (Gly4Ser)3 linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the adenovirus encoding the 6-His-linker-FLAG-IIIa gene, the 6-His tag was present in light particles but not in mature virions. Immuno-electron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy (3DEM) and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between penton base and the peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in adenovirus capsid assembly and disassembly. PMID:18786542

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

    PubMed Central

    Sakudo, Akikazu; Toyokawa, Yoichi; Imanishi, Yuichiro

    2016-01-01

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

  1. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  2. Isolation of an adenovirus and an adeno-associated virus from goat kids with enteritis.

    PubMed

    Olson, Erik J; Haskell, Scott R R; Frank, Rodney K; Lehmkuhl, Howard D; Hobbs, Lea Ann; Warg, Janet V; Landgraf, John G; Wünschmann, Arno

    2004-09-01

    A dairy goat operation in Minnesota experienced a sudden, markedly increased mortality among its neonatal goats. Approximately 60 of 130 kids (46%) died. The animals had diarrhea and dyspnea of 1-2 days duration before death. Necropsy of 4 goat kids revealed marked, acute, catarrhal enteritis and fibrinous pleuropneumonia. Mannheimia haemolytica was isolated from the lungs. Basophilic inclusion bodies filling the entire nucleus were present in enterocytes of the ileum of 3 goats. Adenoviral particles were detected in the feces by electron microscopy and adenovirus was subsequently isolated from the intestinal content together with a parvo-like virus (dependovirus). Morphology, physicochemical characteristics, and neutralization tests indicated that the adenovirus resembled ovine adenovirus-2 (OAdV-2). However, the PstI restriction endonuclease pattern produced by the goat adenovirus was distinct from that of OAdV-2. This is the first report of enteritis in goats with an adenovirus antigenically related to OAdV-2 and with a parvo-like dependovirus.

  3. Hyperplastic stomatitis and esophagitis in a tortoise (Testudo graeca) associated with an adenovirus infection.

    PubMed

    Garcia-Morante, Beatriz; Pénzes, Judit J; Costa, Taiana; Martorell, Jaime; Martínez, Jorge

    2016-09-01

    A 2-year-old female, spur-thighed tortoise (Testudo graeca) was presented with poor body condition (1/5) and weakness. Fecal analysis revealed large numbers of oxyurid-like eggs, and radiographs were compatible with gastrointestinal obstruction. Despite supportive medical treatment, the animal died. At gross examination, an intestinal obstruction was confirmed. Histopathology revealed severe hyperplastic esophagitis and stomatitis with marked epithelial cytomegaly and enormous basophilic intranuclear inclusion bodies. Electron microscopy examination revealed a large number of 60-80 nm, nonenveloped, icosahedral virions arranged in crystalline arrays within nuclear inclusions of esophageal epithelial cells, morphologically compatible with adenovirus-like particles. PCR for virus identification was performed with DNA extracted from formalin-fixed, paraffin-embedded tissues. A nested, consensus pan-adenovirus PCR and sequencing analysis showed a novel adenovirus. According to phylogenetic calculations, it clustered to genus Atadenovirus in contrast with all other chelonian adenoviruses described to date. The present report details the pathologic findings associated with an adenovirus infection restricted to the upper digestive tract.

  4. A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

    PubMed Central

    Farrow, Anitra L.; Peng, Binghao J.; Gu, Linlin; Krendelchtchikov, Alexandre; Matthews, Qiana L.

    2016-01-01

    Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease. PMID:26978385

  5. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

    PubMed Central

    Henry, L J; Xia, D; Wilke, M E; Deisenhofer, J; Gerard, R D

    1994-01-01

    The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor. Images PMID:8035520

  6. Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

    PubMed Central

    Unzu, Carmen; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo

    2014-01-01

    The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors. PMID:24465560

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

    PubMed Central

    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

  8. Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary.

    PubMed

    Farkas, Szilvia L; Gál, János

    2009-07-02

    A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana- and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific Mycoplasma agassizii PCR targeting the 16S rRNA gene. Based on the analyses of partial sequences of the 16S rRNA gene, the Mycoplasma sp. of the ornate box turtle seemed to be identical with the recently described eastern box turtle (Terrapene carolina carolina) Mycoplasma sp. This is the first report of a novel chelonian adenovirus and a mycoplasma infection in an ornate box turtle (T. ornata ornata) in Europe.

  9. Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

    PubMed

    Lerondel, S; Le Pape, A; Sené, C; Faure, L; Bernard, S; Diot, P; Nicolis, E; Mehtali, M; Lusky, M; Cabrini, G; Pavirani, A

    2001-01-01

    Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

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

    PubMed

    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.

  11. Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

    PubMed Central

    Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

    2015-01-01

    Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

  12. Screening for adenoviruses in haematological neoplasia: High prevalence in mantle cell lymphoma.

    PubMed

    Kosulin, Karin; Rauch, Margit; Ambros, Peter F; Pötschger, Ulrike; Chott, Andreas; Jäger, Ulrich; Drach, Johannes; Nader, Alexander; Lion, Thomas

    2014-02-01

    Human adenoviruses possess oncogenic capacity which is well documented in mammalian animal models, but their possible implication in human malignancy has remained enigmatic. Following primary infection, adenoviruses can persist in a latent state in lymphocytes where the virus is apparently able to evade immune surveillance. In the present study, we have employed a broad-spectrum adenovirus polymerase chain reaction (PCR) assay to systematically screen more than 200 diagnostic specimens of different lymphoid malignancies including acute lymphocytic leukaemia (n=50), chronic lymphocytic leukaemia (n=50), various types of malignant lymphoma (n=100) and multiple myeloma (n=11) for the presence of adenoviral sequences. While most entities analysed revealed negative findings in virtually all specimens tested, adenoviral DNA was detected in 15/36 (42%) mantle cell lymphomas investigated. The most prevalent adenoviral species detected was C, and less commonly B. Adenovirus-positive findings in patients with mantle cell lymphoma were made at different sites including bone marrow (n=7), intestine (n=5), lymph nodes (n=2) and tonsillar tissue (n=1). The presence of adenoviral sequences identified by PCR was confirmed in individual cells by fluorescence in-situ hybridisation (FISH). The frequent observation of adenoviruses in mantle cell lymphoma is intriguings, and raises questions about their possible involvement in the pathogenesis of this lymphoid malignancy.

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    Unzu, Carmen; Melero, Ignacio; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo; Fontanellas, Antonio

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

    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.

  18. Phylogenomic characterization of California sea lion adenovirus-1.

    PubMed

    Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Waltzek, Thomas B; Salemi, Marco; Wellehan, James F X

    2015-04-01

    Significant adenoviral diversity has been found in humans, but in domestic and wild animals the number of identified viruses is lower. Here we present the complete genome of a recently discovered mastadenovirus, California sea lion adenovirus 1 (CSLAdV-1) isolated from California sea lions (Zalophus californianus), an important pathogen associated with hepatitis in pinnipeds. The genome of this virus has the typical mastadenoviral structure with some notable differences at the carboxy-terminal end, including a dUTPase that does not cluster with other mastadenoviral dUTPases, and a fiber that shows similarity to a trans-sialidase of Trypanosoma cruzi and choline-binding protein A (CbpA) of Streptococcus pneumoniae. The GC content is low (36%), and phylogenetic analyses placed the virus near the root of the clade infecting laurasiatherian hosts in the genus Mastadenovirus. These findings support the hypothesis that CSLAdV-1 in California sea lions represents a host jump from an unknown mammalian host in which it is endemic.

  19. The composed antigenic structure of the adenovirus hexon protein.

    PubMed

    Nász, I

    1988-01-01

    Three panels of MAbs were prepared against AV1, AV35, and BAV2 hexons, respectively. It was shown, that in all cases, antibodies are developed against the genus specific determinant of the adenovirus hexon. The other MAbs specified numerous identical or overlapping epitopes on the hexon types studied. The epitopes could be characterized as intertype-, intersubgenus-, subgenus- and type-specific ones beside the genus-specific determinant based on the RPs of the MAbs from indirect ELISA and passive HA results. The identical epitopes are present in more than one copy on the trimeric form of the hexon capsomer. The epitopes on the hexon molecule could be separated into three antigenic sites, of which one antigenic site is characterized by seven epitope clusters (antigenic site II). Monoclonal antibodies were able to precipitate different hexon types in gel diffusion tests by which the differentiation of the distinct epitopes seemed to be possible. With the help of monoclonal antibodies to AV1 hexon, 17 hours after the infection, hexons (or epitopes) were detected in the cytoplasm and in the nucleus of the infected cells showing different distribution patterns in indirect immunofluorescence assay.

  20. Adenovirus-mediated interleukin (IL)-24 immunotherapy for cancer.

    PubMed

    Ramesh, Rajagopal; Ioannides, Constantine G; Roth, Jack A; Chada, Sunil

    2010-01-01

    Interleukin-24 (IL-24) is a member of the IL-10 cytokine family. IL-24, also known as melanoma differentiation associated gene 7 (mda-7), is a unique cytokine in that it has cytokine properties and functions as a novel tumor suppressor gene. Studies by us and other investigators using viral and non-viral vectors have demonstrated IL-24 overexpression in human cancer cells inhibited tumor growth both in vitro and in vivo. A majority of these studies using immunodeficient animal models have focused on demonstrating the direct anticancer properties of IL-24. Very few studies have focused on studying the immunotherapeutic properties of IL-24 despite it being reported to function as a Th1 cytokine. A phase I clinical trial using an adenovirus vector expressing IL-24 (Ad-IL24/INGN241) reported Ad-IL24 treatment of cancer patients resulted in changes in cytokines and T cells. However, well-designed and detailed preclinical studies to support the clinical findings are warranted. Demonstrating immune modulation by IL-24 will provide a rationale for developing IL-24-based immunotherapeutic approaches for cancer treatment.In the present chapter, we provide experimental details for conducting IL-24-based immunotherapy studies. As it is not possible for the authors to cover all of the information the authors recommend reading other immunology-based literature and procedures for a better understanding of conducting preclinical studies.

  1. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    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 viral mRNA export, and cell cycle disruption. PMID:26561828

  2. Modification of Antigen Impacts on Memory Quality after Adenovirus Vaccination.

    PubMed

    Colston, Julia M; Bolinger, Beatrice; Cottingham, Matthew G; Gilbert, Sarah; Klenerman, Paul

    2016-04-15

    The establishment of robust T cell memory is critical for the development of novel vaccines for infections and cancers. Classical memory generated by CD8(+)T cells is characterized by contracted populations homing to lymphoid organs. T cell memory inflation, as seen for example after CMV infection, is the maintenance of expanded, functional, tissue-associated effector memory cell pools. Such memory pools may also be induced after adenovirus vaccination, and we recently defined common transcriptional and phenotypic features of these populations in mice and humans. However, the rules that govern which epitopes drive memory inflation compared with classical memory are not fully defined, and thus it is not currently possible to direct this process. We used our adenoviral model of memory inflation to first investigate the role of the promoter and then the role of the epitope context in determining memory formation. Specifically, we tested the hypothesis that conventional memory could be converted to inflationary memory by simple presentation of the Ag in the form of minigene vectors. When epitopes from LacZ and murine CMV that normally induce classical memory responses were presented as minigenes, they induced clear memory inflation. These data demonstrate that, regardless of the transgene promoter, the polypeptide context of a CD8(+)T cell epitope may determine whether classical or inflating memory responses are induced. The ability to direct this process by the use of minigenes is relevant to the design of vaccines and understanding of immune responses to pathogens.

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

    PubMed

    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.

  4. Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families.

    PubMed Central

    Mistchenko, A. S.; Huberman, K. H.; Gomez, J. A.; Grinstein, S.

    1992-01-01

    To examine the role of enteric adenoviruses (EAV) in an urban area of Buenos Aires (Argentina), we prospectively studied faecal samples from 49 families of newborns. These were monitored weekly for diarrhoea for 2 years. A total of 180 samples from cases of diarrhoea and 766 samples obtained during diarrhoea-free periods were studied by dot-blot hybridization with an EAV-specific DNA probe. EAV were found in 6/180 (3.3%) cases of diarrhoea and 6/766 (0.8%) asymptomatic samples (P < 0.015). Incidence of EAV was 3.9 cases per 100 person-years in children < 60 months old. EAV-related diarrhoeas were slight and of short duration. In addition, 129 faeces from hospital out-patients, 1-30 months old, were also studied. EAV was identified in 7/129 cases (5.4%). These cases were 9.5 +/- 3.5 months old and the diarrhoea was mild or severe, of 3 +/- 1.5 days of duration. We suggest that EAV are low-risk causes of diarrhoea under natural conditions, although a few children may develop more severe diarrhoea. The diagnosis of EAV needs to be considered in these patients. PMID:1468536

  5. Magnesium-Dependent Interaction of PKR with Adenovirus VAI

    SciTech Connect

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

    2011-12-31

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

  6. How I treat adenovirus in hematopoietic stem cell transplant recipients

    PubMed Central

    Lindemans, Caroline A.; Leen, Ann M.

    2010-01-01

    Adenovirus (AdV) infections are very common in the general pediatric population. The delayed clearance in young persons imposes a threat to immunocompromised patients after hematopoietic stem cell transplantation (HSCT), who can reactivate the virus, resulting in life-threatening disseminated disease. Although a definitive cure requires adequate immune reconstitution, 2 approaches appear to be feasible and effective to improve the outcomes of AdV infections. Strict monitoring with AdV quantitative polymerase chain reaction followed by preemptive treatment with low-dose (1 mg/kg) cidofovir 3 times a week, is effective in most cases to bridge the severely immunocompromised period shortly after HSCT, with acceptable toxicity rates. For centers who have the access, AdV-specific cytotoxic T cells can be the other important cornerstone of anti-AdV therapy with promising results so far. Methods to positively influence the reconstitution of the immune system after HSCT and optimizing new and currently available cellular immunotherapies will make HSCT safer against the threat of AdV infection/reactivation and associated disease. PMID:20837781

  7. Taxonomy proposal for Old World monkey adenoviruses: characterisation of several non-human, non-ape primate adenovirus lineages.

    PubMed

    Pantó, Laura; Podgorski, Iva I; Jánoska, Máté; Márkó, Orsolya; Harrach, Balázs

    2015-12-01

    A species classification regarding Old World monkey adenoviruses is proposed. We determined the nucleotide sequences of PCR-amplified fragments from the genes of the IVa2, DNA-dependent DNA polymerase, penton base, and hexon proteins from every simian adenovirus (SAdV) serotype that originated from Old World monkeys for which the full genome sequence had not yet been published. We confirmed that the majority of Old Word monkey SAdVs belong to two previously established species. Interestingly, one is the most recently established human AdV species, Human mastadenovirus G, which includes a single human virus, HAdV-52, as well as SAdV-1, -2, -7, -11, -12, and -15. The other approved species, Simian mastadenovirus A includes SAdV-3, -4, -6, -9, -10, -14, and -48. Several SAdVs (SAdV-5, -8, -49, -50) together with baboon AdV-1 and rhesus monkey AdV strains A1139, A1163, A1173, A1258, A1285, A1296, A1312, A1327 and A1335 have been proposed to be classified into an additional species, Simian mastadenovirus B. Another proposed species, Simian mastadenovirus C has been described for SAdV-19, baboon AdV-2/4 and -3. Our study revealed the existence of four additional AdV lineages. The corresponding new candidate species are Simian mastadenovirus D (for SAdV-13), Simian mastadenovirus E (for SAdV-16), Simian mastadenovirus F (for SAdV-17 and -18), and Simian mastadenovirus G (for SAdV-20). Several biological and genomic properties, such as the host origin, haemagglutination profile, number of fibre genes, and G+C content of the genome, strongly support this classification. Three SAdV strains originating from the American Type Culture Collection turned out to be mixtures of at least two virus types, either of the same species (SAdV-12 and -15 types from Human mastadenovirus G) or of two different species (SAdV-5 types from Simian mastadenovirus B and Human mastadenovirus G).

  8. Development and Pre-Clinical Evaluation of a Novel Prostate-Restricted Replication Competent Adenovirus-Ad-IU-1

    DTIC Science & Technology

    2005-05-01

    PSMA postivie LNCaP and C4-2 cells. 6 Task 3. Investigate the capability of m6 to drive adenovirus replication in a prostate cancer-specific manner...of PSES to direct adenovirus replication , and HSV-TK gene, a pro-drug enzyme gene, under the control of another copy of PSES enhancer to restrict

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibo...

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

    PubMed

    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.

  11. An adenovirus associated with intestinal impaction and mortality of male eiders (Somateria mollissima) in the Baltic Sea

    USGS Publications Warehouse

    Hollmén, Tuula E.; Franson, J Christian; Kilpi, Mikael; Docherty, Douglas E.; Myllys, V.

    2003-01-01

    We examined 10 common eider (Somateria mollissima) males found dead in 1998 during a die-off in the northern Baltic Sea off the southwestern coast of Finland. We diagnosed impaction of the posterior small intestine with mucosal necrosis as the cause of death in all 10 and isolated adenoviruses from cloacal samples of six birds. The adenovirus isolates were not neutralized by reference antisera to group I, II, or III avian adenoviruses. Cloacal swabs from 22 apparently healthy eider females nesting at the mortality area were negative for viruses. An adenovirus isolated from one of the eiders caused clinical signs of illness and gastrointestinal pathology in experimentally infected mallard (Anas platyrhynchos) ducklings. These findings suggest that the adenovirus contributed to the mortality of common eider males in the Finnish archipelago.

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

    SciTech Connect

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

    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.

  13. Attachment and Detachment Behaviour of Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material

    NASA Astrophysics Data System (ADS)

    Stevenson, Margaret; Blaschke, Alfred Paul; Kirschner, Alexander; Farnleitner, Andreas; Sommer, Regina; Sidhu, Jatinder

    2015-04-01

    Comparison of transport of virus surrogates to the pathogenic virus is necessary to understand the differences between the virus and surrogate. Since experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow through soil columns are used. Adenovirus, nanoparticles, PRD1 and MS2 bacteriophages were tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, Southern Australia. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material, although PRD1 did not mimic the detachment behaviour of adenovirus successfully under high pH conditions. It was also found that the charge of the colloid is not a dominant removal mechanism in this system. Implications from this study could influence how field tests using bacteriophages and nanoparticles are interpreted.

  14. An epizootic of adenovirus-induced hemorrhagic disease in captive black-tailed deer (Odocoileus hemionus).

    PubMed

    Boyce, W M; Woods, L W; Keel, M K; MacLachlan, N J; Porter, C O; Lehmkuhl, H D

    2000-09-01

    Ten fawns and four adult black-tailed deer (Odocoileus hemionus) in a captive herd died as a result of adenovirus-induced hemorrhagic disease. Acute, systemic infections were characterized by hemorrhagic enteropathy, pulmonary edema, and occasional ulceration of the upper alimentary tract. Localized infections were limited to the upper alimentary tract and included stomatitis, pharyngitis, mandibular osteomyelitis, and rumenitis. In deer with acute, systemic infections, a diagnosis was made by histopathology and immunohistochemistry. The serum neutralization test was useful for confirming a diagnosis in animals with prolonged, localized infections. Deer originating from herds with a history of adenovirus infection should not be transferred to other captive herds or released into free-ranging populations because they may serve as carriers of adenovirus.

  15. Adenoviruses as a model in the study of the effect of space flight factors

    NASA Astrophysics Data System (ADS)

    Nosach, L. M.; Povnitsa, O. Yu.; Zhovnovata, V. L.

    Simulated microgravity conditions, independently of multiplicity of infection, does not influence the reproduction of adenoviruses in cells which were clinorotated for 48 hours after adsorption of virus. The incubation of infected cells before clinorotation under static conditions at a temperature of 4 °C for three days (the conditions for keeping cells before the flight) does not change the number of infected cells relatively to control, but some changes of cell morphology are revealed, namely round off and aggregation of cells. The adenoviruses which were exposed in the medium keep infectivity under the conditions of clinorotation at 4 and 20-22 °C over prolonged periods (90 and 60 days, respectively). A model is elaborated for investigation of the influence of space flight factors on the interaction of the adenovirus and Epstein-Barr virus genomes at combined infection of limphoblastoid cells.

  16. Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

    PubMed Central

    Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

    1991-01-01

    Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

  17. Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

    PubMed

    Gao, Dong-sheng; Li, Xiao-jing; Wan, Wen-yan; Li, Hong-jie; Wang, Xiao-xue; Yang, Xia; Li, Yong-tao; Chang, Hong-tao; Chen, Lu; Wang, Chuan-qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

  18. Chapter eight--Oncolytic adenoviruses for cancer immunotherapy: data from mice, hamsters, and humans.

    PubMed

    Cerullo, Vincenzo; Koski, Anniina; Vähä-Koskela, Markus; Hemminki, Akseli

    2012-01-01

    Adenovirus is one of the most commonly used vectors for gene therapy and two products have already been approved for treatment of cancer in China (Gendicine(R) and Oncorine(R)). An intriguing aspect of oncolytic adenoviruses is that by their very nature they potently stimulate multiple arms of the immune system. Thus, combined tumor killing via oncolysis and inherent immunostimulatory properties in fact make these viruses in situ tumor vaccines. When further engineered to express cytokines, chemokines, tumor-associated antigens, or other immunomodulatory elements, they have been shown in various preclinical models to induce antigen-specific effector and memory responses, resulting both in full therapeutic cures and even induction of life-long tumor immunity. Here, we review the state of the art of oncolytic adenovirus, in the context of their capability to stimulate innate and adaptive arms of the immune system and finally how we can modify these viruses to direct the immune response toward cancer.

  19. Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

    PubMed Central

    Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-01

    In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

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

    PubMed

    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.

  1. Adenovirus types 40 and 41 and rotaviruses associated with diarrhea in children from Guatemala.

    PubMed Central

    Cruz, J R; Cáceres, P; Cano, F; Flores, J; Bartlett, A; Torún, B

    1990-01-01

    From March 1987 to February 1988, fecal excretion of adenovirus types 40 and 41 and rotavirus serotypes in 194 children (age, 0 to 3 years) from a rural community of Guatemala was monitored. In total, 458 samples taken during 385 episodes of diarrhea and 191 specimens obtained during symptom-free periods were examined by enzyme-linked immunosorbent assay. Fifty-seven children hospitalized because of diarrhea were also studied. Among the rural children, 43 (22.2%) excreted adenovirus types 40 and 41 and 20 (10.3%) shed rotaviruses. Adenovirus types 40 and 41 were associated with 54 (14.0%) illnesses, and rotaviruses were associated with 18 (4.7%) illnesses. Asymptomatic infections with adenovirus types 40 and 41 were documented in nine children and with rotaviruses in two children. Fifteen typeable rotaviruses were identified as serotype 2. In the hospital population, 36 (63.2%) children had viral infections. Rotaviruses were identified in 29 (50.9%) and adenovirus types 40 and 41 were identified in 15 (31.2%) of 48 subjects tested. Dual infections by these viruses were found in eight children. Of 22 typeable strains of rotaviruses, 9 (34.6%) were serotype 1, 12 (46.1%) were serotype 2, and 1 (3.8%) was serotype 3. All the children infected with serotype 2 rotavirus were coinfected with other enteric pathogens, while only three (37.5%) of those infected with rotavirus serotype 1 excreted another pathogen. Adenovirus types 40 and 41 are an important cause of gastroenteritis in both ambulatory and hospitalized Guatemalan children. There seems to be a difference in the pathogenicity among rotavirus serotypes. PMID:2168438

  2. Intraductal Delivery of Adenoviruses Targets Pancreatic Tumors in Transgenic Ela-myc Mice and Orthotopic Xenografts

    PubMed Central

    José, Anabel; Sobrevals, Luciano; Camacho-Sánchez, Juan Miguel; 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<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. PMID:23328228

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

    PubMed

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

    2004-12-01

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

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

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

  7. Molecular Epidemiology and Clinical Manifestations of Adenovirus Respiratory Infections in Taiwanese Children

    PubMed Central

    Wang, Ya-Fang; Shen, Fan-Ching; Wang, Shan-Li; Kuo, Pin-Hwa; Tsai, Huey-Pin; Liu, Ching-Chuan; Wang, Jen-Ren; Chi, Chia-Yu

    2016-01-01

    Abstract Human adenoviruses (HAdVs) are important causes of respiratory infections in children. They usually cause mild upper respiratory symptoms, but they can also produce severe pneumonia and other complications. The aims of this retrospective study were to better define the molecular epidemiology of respiratory adenoviruses circulating in Taiwanese children during 2002 and 2013, detect reinfections and co-infections, and characterize the clinical features and laboratory findings according to the causative genotypes. We collected a representative sample of 182 isolates of adenoviruses from 175 children during the 12-year study period. The most prevalent species was HAdV-B genotype 3 (HAdV-3) (92/182, 50.5%) followed by HAdV-C (HAdV-2) (38/182, 20.9%). A single outbreak of HAdV-E (6/182, 3.3%) was noted in 2007. The mean age of children with adenovirus infections was 3.7 ± 2.0 years, with a slight predominance of males (53.1%). Children with HAdV-B tended to be older, had more lower respiratory tract infections, gastrointestinal symptoms, and a higher rate of hospitalization than those with HAdV-C (P < 0.05). Adenovirus co-infections were noted in 25/175 (14.3%) of the children. The most frequent co-infections were with species B (HAdV-3) and C (HAdV-2) (14/25, 56.0%). Additional infections were noted in 23/175 (13.1%) of the children. Of these repeated infections, the initial isolates were always genotypes of HAdV-C. The second isolates were genotypes of HAdV-B or HAdV-E. The clinical features of the first HAdV-B infection and the reinfection of HAdV-B followed the HAdV-C were similar. In conclusion, HAdV-B, C, and E were the only adenovirus species that were isolated from children who were sufficiently ill with respiratory infections to require a visit to the hospital. Human adenovirus B (HAdV-3) accounted for half of these species. HAdV-B was more likely than other species to produce severe disease. The high incidence of adenovirus co-infection and

  8. Experimental study of Human Adenoviruses interactions with clays

    NASA Astrophysics Data System (ADS)

    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

  9. Adenovirus DNA synthesized in the presence of aphidicolin.

    PubMed Central

    Oguro, M; Yamashita, T; Ariga, H; Nagano, H

    1984-01-01

    Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals. Images PMID:6420772

  10. Pseudotyping Serotype 5 Adenovirus with the Fiber from Other Serotypes Uncovers a Key Role of the Fiber Protein in Adenovirus 5-Induced Thrombocytopenia.

    PubMed

    Raddi, Najat; Vigant, Frédéric; Wagner-Ballon, Oriane; Giraudier, Stéphane; Custers, Jerome; Hemmi, Silvio; Benihoud, Karim

    2016-02-01

    Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia.

  11. HUMAN ADENOVIRUS TYPE 37 AND THE BALB/C MOUSE: PROGRESS TOWARD A RESTRICTED ADENOVIRUS KERATITIS MODEL (AN AMERICAN OPHTHALMOLOGICAL SOCIETY THESIS)

    PubMed Central

    Chodosh, James

    2006-01-01

    Purpose To establish a mouse model of adenovirus keratitis in order to study innate immune mechanisms in the adenovirus-infected cornea. Methods Balb/c 3T3 fibroblasts were inoculated with human adenovirus (HAdV) serotypes 8, 19, or 37 and observed for cytopathic effect. Viral growth titers were performed, and apoptosis was measured by TUNEL assay. Viral and host cytokine gene expression was assessed by RT-PCR in cultured Balb/c 3T3 fibroblasts and in the corneas of virus-injected Balb/c mice. Western blot analysis was performed to detect cell signaling in the virus-infected cornea. Results Only HAdV37 induced cytopathic effect in mouse cells. Viral gene expression was limited, and viral replication was not detected. Apoptotic cell death in HAdV37-infected Balb/c cells was evident 48 and 72 hours postinfection (P < .01). MCP-1, IL-6, KC, and IP-10 mRNA levels were increased maximally by 8.4, 9.6, 10.5, and 20.0-fold, respectively, at 30 to 90 minutes after HAdV37 infection. Similar cytokine elevations were observed in the corneas of Balb/c mice 4 hours after stromal injection of HAdV37, when viral gene expression for the viral capsid protein IIIa was not detected. Western blot showed increased phosphorylation of ERK1/2 at 4 and 24 hours after corneal infection. Conclusions Despite limited viral gene expression, HAdV37 infection of Balb/c 3T3 fibroblasts results in increased proinflammatory gene expression. A similar pattern of cytokine expression in the corneas of HAdV37-infected Balb/c mice suggests the mouse adenoviral keratitis model may be useful for the study of early innate immune responses in the adenovirus-infected corneal stroma. PMID:17471351

  12. STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    PubMed Central

    Spurrell, Emma; Gangeswaran, Rathi; Wang, Pengju; Cao, Fengyu; Gao, Dongling; Feng, Baisui; Wold, William; Tollefson, Ann

    2014-01-01

    Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy. PMID:24335311

  13. Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

    PubMed Central

    Casto, Bruce C.

    1969-01-01

    Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 × 104 to 4 × 104, which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations. Images PMID:5786181

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

    PubMed Central

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

    2015-01-01

    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. PMID:25807051

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

    PubMed

    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.

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

    PubMed

    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.

  17. Influence of Inorganic Ions on Aggregation and Adsorption Behaviors of Human Adenovirus

    EPA Science Inventory

    In this study, we investigated the influence of inorganic ions on the aggregation and deposition (adsorption) behavior of human adenovirus (HAdV). Experiments were conducted to determine the surface charge and size of HAdV and viral adsorption capacity of sand in different salt c...

  18. Relative transport of human adenovirus and MS2 in porous media

    EPA Science Inventory

    Human adenovirus (HAdV) is the most prevalent enteric virus found in the water environment by numerous monitoring studies and MS2 is the most common surrogate used for previous virus transport studies. However, the current knowledge on the transport behavior of HAdV in porous med...

  19. Influence of Inorganic Ions and Aggregation and Adsorption Behaviors of Human Adenovirus

    EPA Science Inventory

    In this study, influence of solution chemistries to the transport properties (aggregation and attachment behavior) of human adenovirus (HAdV) was investigated. Results showed isoelectric point (IEP) of HAdV in different salt conditions varied minimally, and it ranged from pH 3.5 ...

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

    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

  2. Establishment of higher passage PER.C6 cells for adenovirus manufacture.

    PubMed

    Berdichevsky, Marina; Gentile, Marie-Pierre; Hughes, Benjamin; Meis, Peter; Peltier, Joseph; Blumentals, Ilse; Auniņs, John; Altaras, Nedim Emil

    2008-01-01

    PER.C6 cells, an industrially relevant cell line for adenovirus manufacture, were extensively passaged in serum-free suspension cell culture to better adapt them to process conditions. The changes in cell physiology that occurred during this passaging were characterized by investigating cell growth, cell size, metabolism, and cultivation of replication-deficient adenovirus. The changes in cell physiology occurred gradually as the population doubling level, the number of times the cell population had doubled, increased. Higher passage PER.C6 (HP PER.C6) proliferated at a specific growth rate of 0.043 h(-1), 2-fold faster than lower passage PER.C6, and were capable of proliferation from lower inoculation cell densities. HP PER.C6 cell volume was 16% greater, and cellular yields on glucose, lactate, oxygen, and amino acids were greater as well. In batch cultures, HP PER.C6 cells volumetrically produced 3-fold more adenovirus, confirmed with three different constructs. The increase in productivity was also seen on a cell-specific basis. Although HP PER.C6 were more sensitive to the "cell density effect", requiring lower infection cell densities for optimal specific productivity, they proliferated more after infection than lower passage PER.C6, increasing the number of cells available for virus production. The extensive passaging established HP PER.C6 cells with several desirable attributes for adenovirus manufacture.

  3. Sequential and Simultaneous Applications of UV and Chlorine for Adenovirus Inactivation.

    PubMed

    Rattanakul, Surapong; Oguma, Kumiko; Takizawa, Satoshi

    2015-09-01

    Adenoviruses are water-borne human pathogens with high resistance to UV disinfection. Combination of UV treatment and chlorination could be an effective approach to deal with adenoviruses. In this study, human adenovirus 5 (HAdV-5) was challenged in a bench-scale experiment by separate applications of UV or chlorine and by combined applications of UV and chlorine in either a sequential or simultaneous manner. The treated samples were then propagated in human lung carcinoma epithelial cells to quantify the log inactivation of HAdV-5. When the processes were separate, a fluence of 100 mJ/cm(2) and a CT value of 0.02 mg min/L were required to achieve 2 log inactivation of HAdV-5 by UV disinfection and chlorination, respectively. Interestingly, synergistic effects on the HAdV-5 inactivation rates were found in the sequential process of chlorine followed by UV (Cl2-UV) (p < 0.05, ANCOVA) in comparison to the separate processes or the simultaneous application of UV/Cl2. This implies that a pretreatment with chlorine may increase the sensitivity of the virus to the subsequent UV disinfection. In conclusion, this study suggests that the combined application of UV and chlorine could be an effective measure against adenoviruses as a multi-barrier approach in water disinfection.

  4. Molecular Detection of Circovirus and Adenovirus in Feces of Fur Seals (Arctocephalus spp.).

    PubMed

    Chiappetta, Catarina Marcon; Cibulski, Samuel Paulo; Lima, Francisco Esmaile Sales; Varela, Ana Paula Muterle; Amorim, Derek Blaese; Tavares, Maurício; Roehe, Paulo Michel

    2017-03-01

    In some regions, little is known about exposure to viruses in coastal marine mammals. The present study aimed to detect viral RNA or DNA in 23 free-ranging fur seals on the northern coastline of Rio Grande do Sul State, Brazil. Polymerase chain reaction was used to detect nucleic acids of circoviruses, adenoviruses, morbilliviruses, vesiviruses, and coronaviruses in the feces from twenty-one South American fur seals (Arctocephalus australis) and two Subantarctic fur seals (A. tropicalis). Adenovirus DNA fragments were detected in two South American fur seals; nucleotide sequences of these fragments revealed a high degree of similarity to human adenovirus type C. Circovirus DNA fragments were detected in six animals of the same species. Two were phylogenetically similar to the Circovirus genus, whereas the other four nucleotide fragments showed no similarity to any of the known genera within the family Circoviridae. RNA fragments indicating the presence of coronavirus, vesivirus, and morbillivirus were not detected. These findings suggest that adenoviruses and circoviruses are circulating in fur seal populations found along the coast of Rio Grande do Sul State, Brazil.

  5. Challenges of Treating Adenovirus Infection: Application of a Deployable Rapid-Assembly Shelter Hospital.

    PubMed

    Bai, Song; Yu, Bao-Guo; Zhang, Yong-Zhong; Ding, Hui; Wu, Zhou-Wei; Fan, Bin; Fan, Hao-Jun; Hou, Shi-Ke; Chen, Feng

    2017-03-06

    This article outlines the evolution of a rescue team in responding to adenovirus prevention with a deployable field hospital. The local governments mobilized a shelter hospital and a rescue team consisting of 59 members to assist with rescue and response efforts after an epidemic outbreak of adenovirus. We describe and evaluate the challenges of preparing for deployment, field hospital maintenance, treatment mode, and primary treatment methods. The field hospital established at the rescue scene consisted of a medical command vehicle, a computed tomography shelter, an X-ray shelter, a special laboratory shelter, an oxygen and electricity supply vehicle, and epidemic prevention and protection equipment. The rescue team comprised paramedics, physicians, X-ray technicians, respiratory therapists, and logistical personnel. In 22 days, more than 3000 patients with suspected adenovirus infection underwent initial examinations. All patients were properly treated, and no deaths occurred. After emergency measures were implemented, the spread of adenovirus was eventually controlled. An emergency involving infectious diseases in less-developed regions demands the rapid development of a field facility with specialized medical personnel when local hospital facilities are either unavailable or unusable. An appropriate and detailed prearranged action plan is important for infectious diseases prevention. (Disaster Med Public Health Preparedness. 2017;page 1 of 6).

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

    PubMed Central

    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

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

    EPA Science Inventory

    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)...

  8. Biochemical studies on bovine adenovirus type 3. I. Purification and properties.

    PubMed Central

    Niiyama, Y; Igarashi, K; Tsukamoto, K; Kurokawa, T; Sugino, Y

    1975-01-01

    Bovine adenovirus type 3 (BAV3) was purified and its properties were studied. On productive infection of CKT1 cells (a cell line derived from calf kidney) with BAV3, it was observed that viral DNA synthesis was initiated after about 24 h and its rate was maximal after about 40 h. Maturation of the virus occurred several hours after this. Purified BAV3 was separated into four discrete bands by CsCl density gradient centrifugation (complete, incomplete, empty, and degraded viruses). The complete BAV3 was similar in size and structure to human and avian adenoviruses. Polyacrylamide gel electrophoresis showed that the complete BAV3 virion contained at least 10 polypeptides. The total structural proteins of the virion had a similar amino acid composition to those of human adenoviruses. DNA of the complete virus was a linear duplex and its contour length was 12.3 +/- 0.9 mum. The So20,w value of the DNA was 32.9S and its buoyant density in CsCl was 1.717 g/ml. There was about 25% homology between the DNAs of BAV3 and human adenovirus type 5 by filter hybridization. It was also noted that BAV3 produced incomplete virus. The incomplete virus was similar in morphology to the complete virus and contained almost all the structural polypeptides of the latter, but lacked infectivity. However, its DNA had a deletion(s) (13%) which seemed to locate near a terminal. Images PMID:1171993

  9. Complete Genome Sequence of Human Adenovirus 7 Associated with Fatal Adult Pneumonia.

    PubMed

    Yatsyshina, Svetlana B; Ageeva, Margarita R; Deviatkin, Andrey A; Pimkina, Ekaterina V; Markelov, Mikhail L; Dedkov, Vladimir G; Safonova, Marina V; Shumilina, Elena Y; Lukashev, Alexander N; Shipulin, German A

    2016-10-27

    Human adenovirus 7 (hAdv7) 19BOVLB/Volgograd/Rus/2014 was isolated from the autopsy material from an adult with fatal pneumonia in Volgograd, Russia, in March 2014. Whole-genome sequencing of the virus isolate was performed.

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

    DTIC Science & Technology

    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

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

    PubMed

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-01

    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.

  12. Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater

    EPA Science Inventory

    Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater. Therefore, wastewater is a source of a potentially diverse group of enteric viru...

  13. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells.

    PubMed Central

    Zorn, G A; Anderson, C W

    1981-01-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. Cells reconstructed from infected human karyoplasts and monkey cytoplasts expressed fiber, whereas cells reconstructed from infected monkey karyoplasts and human cytoplasts did not. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide. Furthermore, they suggest that the translational apparatus of monkey cells is competent to translate functional fiber mRNA synthesized in human cells. Images PMID:7218436

  14. Inactivation of human adenovirus by sequential disinfection with an alternative ultraviolet technology and monochloramine.

    PubMed

    Shin, Gwy-Am; Lee, Jung-Keun

    2010-07-01

    In an effort to reduce human exposure to adenoviruses through drinking water, we determined the effectiveness of sequential disinfection with an alternative ultraviolet (UV) technology (medium-pressure (MP) UV) and monochloramine. The results of this study showed that MP UV was much more effective than traditional UV technology (low-pressure (LP) UV) against human adenovirus 2 (Ad2). Specifically, an inactivation of approximately 3 log10 was achieved by a dose of 40 mJ/cm2 of MP UV compared to ~1 log10 by the same dose of LP UV. However, because of the ineffective inactivation of Ad2 by monochloramine, a very high dose (40 mJ/cm2) of MP UV and a very large Ct99 value (approximately 1200 mg/L.min) was still needed to achieve a significant inactivation (e.g., 4 log10) of Ad2. Also, it appears that the inactivation of Ad2 by monochloramine is not enhanced by prior exposure to MP UV. Overall, the results of this study indicated that, in spite of the enhanced effectiveness of alternative UV technologies on human adenoviruses, sequential disinfection with an alternative UV technology (MP UV) and monochloramine still may not provide adequate inactivation of human adenoviruses - especially at high pH and low temperature - in drinking water treatment processes.

  15. A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

    SciTech Connect

    Wei, Na; Fan, Jun Kai; Gu, Jin Fa; He, Ling Feng; Tang, Wen Hao; Cao, Xin; Liu, Xin Yuan

    2009-10-16

    Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.

  16. Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

    PubMed Central

    Roovers, D J; van der Lee, F M; van der Wees, J; Sussenbach, J S

    1993-01-01

    A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step. Images PMID:8416372

  17. The downstream regulatory sequence of the adenovirus type 2 major late promoter is functionally redundant.

    PubMed Central

    Li, X C; Huang, W L; Flint, S J

    1992-01-01

    Mutagenesis of promoter sequences and oligonucleotide competition assays have been used to demonstrate the late-phase-specific stimulation of the adenovirus type 2 major late promoter is mediated by functionally redundant elements located between positions +75 and +125. These octamer motif-related sequences are recognized by multiple factors. Images PMID:1501301

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

    PubMed Central

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

    2016-01-01

    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. PMID:27014840

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

    PubMed

    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.

  20. Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses

    PubMed Central

    Crosby, Catherine M.; Matchett, William E.; Anguiano-Zarate, Stephanie S.; Parks, Christopher A.; Weaver, Eric A.; Pease, Larry R.; Webby, Richard J.

    2016-01-01

    ABSTRACT Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines. IMPORTANCE Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold

  1. Identification of multiple genetic loci that regulate adenovirus gene therapy.

    PubMed

    Zhang, H-G; Hsu, H-C; Yang, P-A; Yang, X; Wu, Q; Liu, Z; Yi, N; Mountz, J D

    2004-01-01

    A key aspect of the immune response to adenovirus (Ad) gene therapy is the generation of a cytotoxic T-cell (CTL) response. To better understand the genetic network underlying these events, 20 strains of C57BL/6 x DBA/2 (BXD) recombinant inbred (RI) mice were administered with AdLacZ and analyzed at days 7, 21, 30, and 50 for liver beta-galactosidase (LacZ) expression and CTL response. Sera levels of interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were analyzed at different times after AdLacZ. There was a distinct strain-dependent expression of LacZ, which was strongly correlated with the CTL response. Among the five BXD RI strains that exhibited significantly prolonged LacZ expression, four also exhibited a marked defect in the production of Ad-specific CTL. There was a strong correlation between the sera levels of IFN-gamma, TNF-alpha, and IL-6, but cytokine responses were not significantly correlated with LacZ expression or the CTL response. Quantitative trait loci regulating LacZ on day 30 were found on chromosome (Chr) 19 (33 cM) and Chr 15 (42.8 cM). Cytotoxicity mapped to Chr 7 (41.0 and 57.4-65.2 cM), Chr 15 (61.7 cM), and Chr X (27.8 cM). IFN-gamma production mapped to Chr 18 (22, 27, and 32 cM) and Chr 11 (64.0 cM). TNF-alpha and IL-6 production mapped to Chr 6 (91.5 cM) Chr 9 (42.0 cM) and Chr 8 (52 and 73.0 cM). These results indicate that different strains of mice exhibit different pathways for effective clearance of AdLacZ depending on genetic polymorphisms and interactions at multiple genetic loci.

  2. Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

    PubMed Central

    Thurston-Enriquez, Jeanette A.; Haas, Charles N.; Jacangelo, Joseph; Gerba, Charles P.

    2005-01-01

    Chlorine dioxide (ClO2) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO2 multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct99.99% ranges and EFH model Ct99.99% values demonstrated that FCV is more resistant to ClO2 than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct99.99% values are higher than Ct99.99% values calculated from bench-scale experiments and from EFH model application. PMID:15933007

  3. p53/E1b58kDa complex regulates adenovirus replication.

    PubMed

    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.

  4. Experimental Cross-Species Infection of Common Marmosets by Titi Monkey Adenovirus

    PubMed Central

    Chen, Eunice C.; Liu, Maria; Brasky, Kathleen M.; Lanford, Robert E.; Kelly, Kristi R.; Bales, Karen L.; Schnurr, David P.; Canfield, Don R.; Patterson, Jean L.; Chiu, Charles Y.

    2013-01-01

    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. PMID:23894316

  5. Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors.

    PubMed

    Yamashita, Toshiharu; Okura, Masae; Ishii-Osai, Yasue; Hida, Tokimasa

    2016-10-01

    Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP-A to -G, and a variant type (XP-V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP-A, -B, -C, -D, -F or -G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV-C at 5-20 J/m(2) was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP-E and XP-V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co-infection of Ad-XPA with Ad-XPE increased survival rate of XP-E cells after UV-C exposure. When XP-V cell strains, including one derived from a Japanese patient, were infected with Ad-XPV, exposed to UV-B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP-V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP-E and XP-V.

  6. Use of macrophages to target therapeutic adenovirus to human prostate tumors.

    PubMed

    Muthana, Munitta; Giannoudis, Athina; Scott, Simon D; Fang, Hsin-Yu; Coffelt, Seth B; Morrow, Fiona J; Murdoch, Craig; Burton, Julian; Cross, Neil; Burke, Bernard; Mistry, Roshna; Hamdy, Freddie; Brown, Nicola J; Georgopoulos, Lindsay; Hoskin, Peter; Essand, Magnus; Lewis, Claire E; Maitland, Norman J

    2011-03-01

    New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells.

  7. Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus.

    PubMed

    Sanekata, Takeshi; Fukuda, Toshiaki; Miura, Takanori; Morino, Hirofumi; Lee, Cheolsung; Maeda, Ken; Araki, Kazuko; Otake, Toru; Kawahata, Takuya; Shibata, Takashi

    2010-06-01

    We evaluated the antiviral activity of a chlorine dioxide gas solution (CD) and sodium hypochlorite (SH) against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. CD at concentrations ranging from 1 to 100 ppm produced potent antiviral activity, inactivating >or= 99.9% of the viruses with a 15 sec treatment for sensitization. The antiviral activity of CD was approximately 10 times higher than that of SH.

  8. A Mortality Event in Elk ( Cervus elaphus nelsoni) Calves Associated with Malnutrition, Pasteurellosis, and Deer Adenovirus in Colorado, USA.

    PubMed

    Fox, Karen A; Atwater, Levi; Hoon-Hanks, Laura; Miller, Myrna

    2017-03-22

    This report describes clinical, necropsy, and ancillary diagnostic findings for a mortality event in Rocky Mountain elk ( Cervus elaphus nelsoni) calves attributed to malnutrition, pasteurellosis, and an alimentary presentation of adenovirus hemorrhagic disease.

  9. Detection of four adenovirus serotypes within water-isolated strains of Acanthamoeba in the Canary Islands, Spain.

    PubMed

    Lorenzo-Morales, Jacob; Coronado-Alvarez, Nieves; Martínez-Carretero, Enrique; Maciver, Sutherland K; Valladares, Basilio

    2007-10-01

    We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the Canary Islands, for the presence of human adenoviruses (HAdV) using a polymerase chain reaction (PCR)-based typing assay. A total of 34 of these strains were found to be positive for adenovirus belonging to four different HAdV serotypes (HAdV-1, 2, 8, and 37). We found that HAdV-2 was the most frequently encountered serotype amongst the Acanthamoeba strains, and their identification was confirmed by a nested PCR specific for this serotype. We showed that Acanthamoeba genotype T4 was highly associated with serotype HAdV-2, whereas Acanthamoeba genotype T3 was most often associated with adenovirus serotypes related to ocular diseases. Based on these data, we suggest that Acanthamoeba should be considered as a potential reservoir and perhaps even a transmitter of adenoviruses to human and other secondary hosts.

  10. DETECTION OF INFECTIOUS ADENOVIRUS IN TERTIARY TREATED AND UV DISINFECTED WASTEWATER DURING A UV DISINFECTION PILOT STUDY

    EPA Science Inventory

    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...

  11. 78 FR 3906 - Prospective Grant of a Co-Exclusive License: Adenovirus-Based Controls and Calibrators for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-17

    ... October 24, 2000, and entitled ``Replication Deficient Recombinant Adenovirus Vector'' to Life... recombinant constructs as controls and calibrators for molecular diagnostics for infectious disease agents...-0220; Email: Reichmau@mail.nih.gov . SUPPLEMENTARY INFORMATION: The invention relates to...

  12. Eradication of melanoma in vitro and in vivo via targeting with a Killer-Red-containing telomerase-dependent adenovirus.

    PubMed

    Takehara, Kiyoto; Yano, Shuya; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Narii, Nobuhiro; Mizuguchi, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2017-01-05

    Melanoma is a highly recalcitrant cancer and transformative therapy is necessary for the cure of this disease. We recently developed a telomerase-dependent adenovirus containing the fluorescent protein Killer-Red. In the present report, we first determined the efficacy of Killer-Red adenovirus combined with laser irradiation on human melanoma cell lines in vitro. Cell viability of human melanoma cells was reduced in a dose-dependent and irradiation time-dependent manner. We used an intradermal xenografted melanoma model in nude mice to determine efficacy of the Killer-Red adenovirus. Intratumoral injection of Killer-Red adenovirus, combined with laser irradiation, eradicated the melanoma indicating the potential of a new paradigm of cancer therapy.

  13. Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

    PubMed Central

    Lemarchand, P; Jaffe, H A; Danel, C; Cid, M C; Kleinman, H K; Stratford-Perricaudet, L D; Perricaudet, M; Pavirani, A; Lecocq, J P; Crystal, R G

    1992-01-01

    To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors. Images PMID:1631146

  14. [Circulation of parainfluenza viruses and adenoviruses in groups exposed to the action of noxious chemicals or not].

    PubMed

    Moisa, I; Bârnaure, F; Pârvu, C; Sîrbu, D

    1987-01-01

    Investigations were conducted during 1985 and 1986 years on the effect of some chemicals on the parainfluenza and adenovirus circulation in an industrial enterprise community. The presence of type 1, 2 and 3 parainfluenza virus and of adenovirus was revealed by immunofluorescence in exfoliated cells collected from nasopharynx. The kinetic of specific hemagglutination inhibiting and complement fixing antibodies was followed monthly by immunological tests. Meaning of the results is discussed from an epidemiological point of view.

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

    DTIC Science & Technology

    2006-06-01

    Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs Nancy J. Sullivan 1...Shedlock DJ, Xu L, et al. (2006) Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified...should be addressed. E-mail: gnabel@nih.gov [ These authors contributed equally to this work. A B S T R A C T Background Ebola virus causes a hemorrhagic

  16. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices

    EPA Pesticide Factsheets

    The data to support the evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matricesThis dataset is associated with the following publication:Rhodes , E., E. Huff, D. Hamilton, and J. Jones. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 228(2): 31-38, (2016).

  17. Severe conjunctivitis due to multidrug-resistant Neisseria gonorrhoeae and adenovirus 53 coinfection in a traveler returning from Thailand.

    PubMed

    Tappe, Dennis; Mueller, Andreas; Weißbrich, Benedikt; Schubert, Jörg; Schargus, Marc; Stich, August

    2013-01-01

    A male traveler returning from Thailand with severe bilateral conjunctivitis was tested for causative pathogens by culture and polymerase chain reaction in late 2010. The culturally grown Neisseria gonorrhoeae strain was resistant against penicillin, ciprofloxacin, and tetracycline. The patient was also found to have an eye infection with the unusual and likely recombinant adenovirus type 53. Besides multidrug-resistant gonococcal strains the unusual adenovirus strain is found circulating in Asia and both pathogens may be a risk for travelers.

  18. Functional characterization of a PEI-CyD-FA-coated adenovirus as delivery vector for gene therapy.

    PubMed

    Yao, Hong; Chen, Shih-Chi; Shen, Zan; Huang, Yun-Chao; Zhu, Xiao; Wang, Xiao-mei; Jiang, Wenqi; Wang, Zi-Feng; Bian, Xiu-Wu; Ling, Eng-Ang; Kung, Hsiang-fu; Lin, Marie C

    2013-01-01

    The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy.

  19. Human Adenovirus 36 Infection Increased the Risk of Obesity

    PubMed Central

    Xu, Mei-Yan; Cao, Bing; Wang, Dong-Fang; Guo, Jing-Hui; Chen, Kai-Li; Shi, Mai; Yin, Jian; Lu, Qing-Bin

    2015-01-01

    Abstract Human adenovirus 36 (HAdV-36), as the key pathogen, was supposed and discussed to be associated with obesity. We searched the references on the association between HAdV-36 infection and obesity with the different epidemiological methods, to explore the relationship with a larger sample size by meta-analysis and compare the differences of epidemiological methods and population subsets by the subgroup analyses. We conducted literature search on the association between HAdV-36 infections and obesity in English or Chinese published up to July 1, 2015. The primary outcome was the HAdV-36 infection rate in the obese and lean groups; the secondary outcomes were the BMI level and BMI z-score in the HAdV-36 positive and negative groups. The pooled odds ratio (OR) was calculated for the primary outcome; the standardized mean differences (SMDs) were calculated for the secondary and third outcomes. Prediction interval (PI) was graphically presented in the forest plot of the random effect meta-analyses. Metaregression analysis and subgroup analysis were performed. Finally 24 references with 10,191 study subjects were included in the meta-analysis. The obesity subjects were more likely to be infected with HAdV-36 compared to the lean controls (OR = 2.00; 95%CI: 1.46, 2.74; PI: 0.59, 6.76; P < 0.001) with a high heterogeneity (I2 = 80.1%; P < 0.001) estimated by the random effect model. Subgroup analysis demonstrated that the pooled OR of HAdV-36 infection for obesity were 1.77 (95%CI: 1.19, 2.63; PI: 0.44, 7.03; P = 0.005) and 2.26 (95%CI: 1.67, 3.07; PI: 1.45, 3.54; P < 0.001) in the adults and children, respectively. Compared to the HAdV-36 negative subjects, the SMD of BMI was 0.28 (95% CI: 0.08, 0.47; PI: −0.53, 1.08; P = 0.006) in the HAdV-36 positive subjects with a high heterogeneity (I2 = 86.5%; P < 0.001). The BMI z-score in the children with HAdV-36 infection was higher than those without HAdV-36 infection (SMD = 0

  20. Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.

    PubMed

    Subekti, D; Lesmana, M; Tjaniadi, P; Safari, N; Frazier, E; Simanjuntak, C; Komalarini, S; Taslim, J; Campbell, J R; Oyofo, B A

    2002-03-25

    Norwalk-like viruses (NLVs), rotavirus and adenovirus are reportedly responsible from 4 to 42% of non-bacterial acute sporadic gastroenteritis. The incidence of NLVs, adenovirus and rotavirus infections in Indonesia is unclear. A total of 402 symptomatic cases from Indonesian patients with acute gastroenteritis and 102 asymptomatic controls that tested negative for bacteria and parasites were screened for the presence of NLVs, rotavirus and adenovirus using the reverse transcriptase-polymerase chain reaction (RT-PCR), Rotaclone kits and Adenoclone kits. Specific prototype probes were used to ascertain which NLV prototypes were present in the area. NLVs were detected in 45/218 (21%), rotavirus was detected in 170/402 (42%) and adenovirus was detected in 11/273 (4%) samples examined. Genetic analysis of the RT-PCR products using specific prototype probes for NLVs indicated that the prototypes were 42% Taunton agent and 58% Hawaii/Snow Mountain agent. Comparative data on patients showed that the incidence of rotavirus infections was two times greater than the NLVs infections, and that adenovirus infections were the least prevalent. All of the control samples tested were negative for NLVs and adenoviruses, however 8/70 (11%) of the samples were positive for rotaviruses. The high incidence of enteric viral-related infections is a threat among acute diarrheic patients in Jakarta, Indonesia.

  1. Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter

    SciTech Connect

    Seiberg, M.; Aloni, Y. ); Levine, A.J. )

    1989-09-01

    Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuator RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.

  2. Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

    PubMed Central

    Zhou, Hongli; Wu, Chao; Paranhos-Baccalà, Gláucia; Vernet, Guy; Jin, Qi; Wang, Jianwei; Hung, Tao

    2013-01-01

    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 (χ2 =  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. PMID:23516396

  3. Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

    PubMed

    van der Vliet, P C; Kwant, M M

    1981-04-28

    In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

  4. Viral Pollution in the Environment and in Shellfish: Human Adenovirus Detection by PCR as an Index of Human Viruses

    PubMed Central

    Pina, Sonia; Puig, Montserrat; Lucena, Francisco; Jofre, Joan; Girones, Rosina

    1998-01-01

    A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of Bacteroides fragilis HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of B. fragilis HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable. PMID:9726885

  5. Molecular identification of adenovirus sequences: a rapid scheme for early typing of human adenoviruses in diagnostic samples of immunocompetent and immunodeficient patients.

    PubMed

    Madisch, Ijad; Wölfel, Roman; Harste, Gabi; Pommer, Heidi; Heim, Albert

    2006-09-01

    Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value and lead to immediate therapeutic intervention. A recently published molecular typing scheme consisting of two steps (sequencing of a generic PCR product closely adjacent to loop 1 of the main neutralization determinant epsilon, and for species HAdV-B, -C, and -D the sequencing of loop 2 [Madisch et al., 2005]) was applied to 119 clinical samples. HAdV DNA was typed unequivocally even in cases of culture negative samples, for example in immunodeficient patients before HAdV causes high virus loads and disseminated disease. Direct typing results demonstrated the predominance of HAdV-1, -2, -5, and -31 in immunodeficient patients suggesting the significance of the persistence of these viruses for the pathogenesis of disseminated disease. In contrast, HAdV-3 predominated in immunocompetent patients and cocirculation of four subtypes was demonstrated. Typing of samples from a conjunctivitis outbreak in multiple military barracks demonstrated various HAdV types (2, 4, 8, 19) and not the suspected unique adenovirus etiology. This suggests that our molecular typing scheme will be also useful for epidemiological investigations. In conclusion, our two-step molecular typing system will permit the precise and rapid typing of clinical HAdV isolates and even of HAdV DNA in clinical samples without the need of time-consuming virus isolation prior to typing.

  6. Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model

    PubMed Central

    Hurez, Vincent; Dzialo-Hatton, Robin; Oliver, James; Matthews, R James; Weaver, Casey T

    2002-01-01

    Background Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor. Results To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCARΔcyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCARΔcyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCARΔcyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCARΔcyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCARΔcyt with high efficiency. Expression of hCARΔcyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCARΔcyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization. Conclusion The DO11.hCARΔcyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation. PMID:12019030

  7. Transforming Region of Group A, B, and C Adenoviruses: DNA Homology Studies with Twenty-Nine Human Adenovirus Serotypes

    PubMed Central

    Mackey, Jesse K.; Wold, William S. M.; Rigden, Patricia; Green, Maurice

    1979-01-01

    The 31 human adenovirus (Ad) serotypes form five groups based upon DNA genome homologies: group A (Ad12, 18, 31), group B (Ad3, 7, 11, 14, 16, 21), group C (Ad1, 2, 5, 6), group D (Ad8, 9, 10, 13, 15, 17, 19, 20, 22-30), and group E (Ad4) (M. Green, J. Mackey, W. Wold, and P. Rigden, Virology, in press). Group A Ads are highly oncogenic in newborn hamsters, group B Ads are weakly oncogenic, and other Ads are nononcogenic. However, most or all Ads transform cultured cells. We have studied the homology of Ad5, Ad7, and Ad12 transforming restriction endonuclease DNA fragments with DNAs of 29 Ad types. Ad5 HindIII-G (map position 0-7.3), Ad7 XhoI-C (map position 0-10.8), and Ad12 (strain Huie) EcoRI-C (map position 0-16) and SalI-C (map position 0-10.6) fragments were purified, labeled in vitro (nick translation), and annealed with DNAs of Ad1 to Ad16, Ad18 to Ad24, and Ad26 to Ad31. Hybrids were assayed by using hydroxylapatite. Ad5 HindIII-G hybridized 98 to 100% with DNAs of group C Ads, but only 1 to 15% with DNAs of other types. Ad7 XhoI-C fragment hybridized 85 to 99% with DNAs of group B Ads, but only 6 to 21% with DNAs of other types. Ad12 (Huie) EcoRI-C hybridized 53 to 68% with DNAs of five other Ad12 strains, 53% with Ad18 DNA, 56% with Ad31 DNA, but only 3 to 13% with DNAs of other types. In vitro-labeled Ad12 (Huie) SalI-C hybridized 35 to 71% with DNAs of 6 other Ad12 strains, 44% with Ad18 DNA, 52% with Ad31 DNA, but only 2 to 7% with DNAs Ad7, Ad2, Ad26, or Ad4. When assayed using S-1 nuclease, SalI-C annealed 17 to 44% with DNAs of group A Ads. The melting temperatures of the hybrids of Ad5 HindIII-G with all group C Ad DNAs were 84°C in 0.12 M sodium phosphate (pH 6.8). The melting temperature of the Ad12 (Huie) EcoRI-C hybrid with Ad12 (Huie) DNA was 83°C, but was only 71 to 77°C with DNAs of other group A Ads. Thus, group C and group B Ads both have very homologous transforming regions that are not represented in DNAs of non-group C Ads or non

  8. 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

    PubMed Central

    Sibanda, Timothy; Okoh, Anthony I.

    2012-01-01

    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. PMID:23226986

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

    PubMed

    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.

  10. Association of the Adenovirus DNA-Binding Protein with RNA Both in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Cleghon, Vaughn G.; Klessig, Daniel F.

    1986-12-01

    The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain of DBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.

  11. Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells

    SciTech Connect

    Fender, P. . E-mail: fender@ibs.fr; Boussaid, A.; Mezin, P.; Chroboczek, J.

    2005-09-30

    Adenovirus penton is a non-covalent complex composed of the penton base and fiber proteins, localized at the twelve vertices of the icosahedral virus capsid. In cells infected by adenovirus serotype 3 (Ad3), penton is found not only in the virus capsid but also self-assembled in dodecahedra formed through interactions between the twelve penton bases. In this study, the intracellular trafficking of penton proteins from the cytoplasm to the nucleus has been followed, and the nuclear re-arrangement induced by viral infection has been observed by electron microscopy of ultrathin sections. The amount of dodecahedra has been assessed in relation to the number of Ad3 infectious virions produced during the Ad3 replication cycle. It appears that dodecahedra are produced in a large excess over viral infectious particles and that they are located intranuclearly along the nuclear membrane of Ad3-infected cells at late times of infection.

  12. Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells.

    PubMed

    Fender, P; Boussaid, A; Mezin, P; Chroboczek, J

    2005-09-30

    Adenovirus penton is a non-covalent complex composed of the penton base and fiber proteins, localized at the twelve vertices of the icosahedral virus capsid. In cells infected by adenovirus serotype 3 (Ad3), penton is found not only in the virus capsid but also self-assembled in dodecahedra formed through interactions between the twelve penton bases. In this study, the intracellular trafficking of penton proteins from the cytoplasm to the nucleus has been followed, and the nuclear re-arrangement induced by viral infection has been observed by electron microscopy of ultrathin sections. The amount of dodecahedra has been assessed in relation to the number of Ad3 infectious virions produced during the Ad3 replication cycle. It appears that dodecahedra are produced in a large excess over viral infectious particles and that they are located intranuclearly along the nuclear membrane of Ad3-infected cells at late times of infection.

  13. Use of dodecahedron "VLPs" as an alternative to the whole adenovirus.

    PubMed

    Fender, Pascal

    2014-01-01

    During human adenovirus type 3 (Ad3) infection, an excess of penton base and fiber proteins are produced. These form dodecahedral particles composed of 12 pentamers of penton base and 12 trimers of fiber protein. Beside this "natural" expression, the adenovirus dodecahedron can be expressed in the heterologous baculovirus system in two forms: a fiber-devoid dodecahedron made only of 12 penton bases (called base-dodecahedron: Bs-Dd) and the fiber-containing dodecahedron (called penton dodecahedron: Pt-Dd). These particles partly mimic the adenoviral cellular entry pathway but are devoid of genetic information making them an unusual tool for basic research or applications. We report here how these particles are expressed and purified, the labeling method for trafficking studies as well as their use in molecular interaction studies. The potential of these particles for biotechnological applications is under evaluation, making their study a "niche" along side traditional adenoviral vectors.

  14. Influenza recombinant vaccine: matrix protein M1 on the platform of the adenovirus dodecahedron.

    PubMed

    Naskalska, A; Szolajska, E; Chaperot, L; Angel, J; Plumas, J; Chroboczek, J

    2009-12-09

    We propose a novel influenza vaccine composed of the adenovirus dodecahedron (Dd) as delivery platform carrying an internal influenza matrix protein M1. To attach the antigen to the vector we used WW domains interacting with Dd. Successful internalization of the Dd-M1WW complex was observed using biochemical and cell biology techniques. We show here that the complex of Dd with antigen is a potent activator of human myeloid dendritic cells (MDC), and that it is efficiently presented by MDC to M1-specific CD8+ T lymphocytes. These results show that proposed vaccine model is feasible and that adenovirus dodecahedron is a potent delivery platform for foreign antigens to human cells.

  15. Crystallization of the head and galectin-like domains of porcine adenovirus isolate NADC-1 fibre

    PubMed Central

    Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Fox, Gavin C.; Glasgow, Joel N.; van Raaij, Mark J.

    2009-01-01

    The porcine adenovirus NADC-1 isolate, a strain of porcine adenovirus type 4, has a fibre with an atypical architecture. In addition to a classical virus-attachment region, shaft and head domains, it contains an additional galectin-like domain C-­terminal to the head domain and connected to the head domain by a long RGD-containing loop. The galectin-like domain contains two putative carbohydrate-recognition domains. The head and galectin-like domains have been independently crystallized. Diffraction data have been obtained to 3.2 Å resolution from crystals of the head domain and to 1.9 Å resolution from galectin-like domain crystals. PMID:19923738

  16. A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.

    PubMed

    Buitrago, Dolores; Cano-Gómez, Cristina; Agüero, Montserrat; Fernandez-Pacheco, Paloma; Gómez-Tejedor, Concepción; Jiménez-Clavero, Miguel Angel

    2010-09-01

    In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.

  17. Adenovirus DNA template for late transcription is not a replicative intermediate.

    PubMed Central

    Brison, O; Kédinger, C; Chambon, P

    1979-01-01

    The relationship between adenovirus replication and late transcription has been investigated using viral replication and transcription complexes isolated from infected HeLa cell nuclei. These two types of complexes extracted from adenovirus type 2-infected cell nuclei did not sediment at the same rate on sucrose gradients. Viral replicative intermediates were quantitatively precipitated by immunoglobulins raised against purified 72,000-dalton DNA-binding protein, whereas viral transcription complexes remained in the supernatant. These results show that late transcription does not occur on active replication complexes or on 72,000-dalton DNA-binding protein-containing replicative intermediates inactive in DNA synthesis. Additional evidence is presented indicating that it is very unlikely that replicative intermediates lacking the 72,000-dalton DNA-binding protein could be the template for late transcription. PMID:232191

  18. Establishment of an agamid cell line and isolation of adenoviruses from central bearded dragons (Pogona vitticeps).

    PubMed

    Ball, Inna; Hoferer, Marc; Marschang, Rachel E

    2014-03-01

    A cell line was established from whole 6-8-week-old central bearded dragon (Pogona vitticeps) embryos. Cells were mid-sized and showed an elongated and polymorphic form. The cell line grew in a monolayer and has been serially passaged for 17 passages at time of publication. This cell line has been used with samples from adenovirus polymerase chain reaction (PCR)-positive bearded dragons, and 2 virus isolates have been obtained so far. The isolates show a clear cytopathic effect in inoculated cells. Both virus isolates have been serially passaged on this cell line, and have been identified by PCR amplification and sequencing of a portion of the DNA-dependent DNA polymerase gene and show 100% nucleotide identity to the corresponding region of an agamid adenovirus. Electron microscopic examination of supernatant from infected cells demonstrated the presence of nonenveloped particles, with a diameter of approximately 80 nm in both virus isolates.

  19. Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley Fever vaccine in mice

    PubMed Central

    2013-01-01

    Background Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. Methods Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. Results A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. Conclusions Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials. PMID:24304565

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

    PubMed

    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.

  1. MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

    PubMed Central

    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. PMID:23349911

  2. Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

    PubMed Central

    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. PMID:22916129

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

    PubMed

    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.

  4. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells*

    PubMed Central

    Luo, Xu-wei; Liu, Kang; Chen, Zhu; Zhao, Ming; Han, Xiao-wei; Bai, Yi-guang; Feng, Gang

    2016-01-01

    Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells. PMID:26739524

  5. Vasculature-Specific Adenovirus Vectors for Gene Therapy of Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    translation and, thus, never become available to the disulfide isomerases, the ER-localized enzymes that facilitate the formation of the disulfide bonds...duplexes and cloned into Bael-cut expression vectors. Upon transformation of Ecoli , colonies containing recombinant plasmids were identified by PCR...R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-3. 6

  6. Design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain

    PubMed Central

    Sun, Guoyong; Hsueh, Pang-Yu; Janib, Siti M.; Hamm-Alvarez, Sarah; MacKay, J. Andrew

    2011-01-01

    Hepatocytes and acinar cells exhibit high-efficiency, fiber-dependent internalization of adenovirus; however, viral capsids have unpredictable immunological effects and are challenging to develop into targeted drug carriers. To exploit this internalization pathway and minimize the use of viral proteins, we developed a simple gene product that self assembles nanoparticles decorated with the knob domain of adenovirus serotype 5 fiber protein. The most significant advantages of this platform include: (i) compatibility with genetic engineering; (ii) no bioconjugate chemistry is required to link fusion proteins to the nanoparticle surface; and (iii) it can direct the reversible assembly of large nanoparticles, which are monodisperse, multivalent, and biodegradable. These particles are predominantly composed from diblock copolymers of elastin-like polypeptide (ELP). ELPs have unique phase transition behavior, whereby they self-assemble above a transition temperature that is simple to control. The diblock ELP described contains two motifs with distinct transition temperatures, which assemble nanoparticles at physiological temperatures. Analysis by non-denaturing-PAGE demonstrated that the purified knob-ELP formed trimers or dimers, which is a property of the native knob/fiber protein. Dynamic light scattering indicated that the diblock copolymer, with or without knob, is able to self assemble into nanoparticles ~40 nm in diameter. To examine the functionality of knob-ELP, their uptake was assessed in a hepatocyte cell-line that expresses the receptor for adenovirus serotype 5 fiber and knob, the coxsackievirus and adenovirus receptor (CAR). Both plain ELP and knob-ELP were bound to the outside of hepatocytes; however, the knob-ELP fusion protein exhibits more internalization and localization to lysosomes of hepatocytes. These findings suggest that functional fusion proteins may only minimally influence the assembly temperature and diameter of ELP nanoparticles. These

  7. Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells

    PubMed Central

    Fillat, Cristina

    2015-01-01

    Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3′UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3′UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses. PMID:25714032

  8. Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

    PubMed Central

    Guardado Calvo, Pablo; Llamas-Saiz, Antonio L.; Langlois, Patrick; van Raaij, Mark J.

    2006-01-01

    Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579–793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10 000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kα radiation to at least 1.9 Å resolution and a complete data set was collected from a single crystal to 2.2 Å resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress. PMID:16682773

  9. Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity.

    PubMed

    Fernández-Ulibarri, Inés; Hammer, Katharina; Arndt, Michaela A E; Kaufmann, Johanna K; Dorer, Dominik; Engelhardt, Sarah; Kontermann, Roland E; Hess, Jochen; Allgayer, Heike; Krauss, Jürgen; Nettelbeck, Dirk M

    2015-05-01

    Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.

  10. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    SciTech Connect

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  11. Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    PubMed Central

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

    2002-01-01

    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. PMID:11932420

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

    PubMed

    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.

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

    PubMed

    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.

  14. A rapid generation of adenovirus vector with a genetic modification in hexon protein.

    PubMed

    Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

    2012-02-10

    The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy.

  15. The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

    PubMed Central

    Rebelo, L; Almeida, F; Ramos, C; Bohmann, K; Lamond, A I; Carmo-Fonseca, M

    1996-01-01

    The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble. Images PMID:8862526

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

    NASA Astrophysics Data System (ADS)

    Han, Jianfeng; Zhao, Dong; Zhong, Zhirong; Zhang, Zhirong; Gong, Tao; Sun, Xun

    2010-03-01

    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.

  17. Immobilization of 293 cells using porous support particles for adenovirus vector production

    PubMed Central

    Morishita, Naoya; Katsuda, Tomohisa; Kubo, Shuji; Gotoh, Akinobu

    2010-01-01

    Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 107 cells cm−3-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment. PMID:20140496

  18. Development of Multiplexed Real-Time Quantitative PCR Assay for Detecting Human Adenoviruses

    PubMed Central

    Huang, Meei-Li; Nguy, Long; Ferrenberg, James; Boeckh, Michael; Cent, Anne; Corey, Lawrence

    2008-01-01

    Adenoviruses (AdV) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Due to the large number of existing Adv types, few real-time quantitative AdV PCR assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into two separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (Types 1-49) available from ATCC. We then subsequently multiplexed all the primers and probes into one reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/ml plasma). In a retrospective evaluation we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplant (HSCT) between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for adenovirus testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real time PCR assay allows rapid, sensitive and specific quantification of all currently defined adenoviruses into either two or one multiplex assay for clinical samples. PMID:18707838

  19. Distribution of DNA-condensing protein complexes in the adenovirus core

    PubMed Central

    Pérez-Berná, Ana J.; Marion, Sanjin; Chichón, F. Javier; Fernández, José J.; Winkler, Dennis C.; Carrascosa, José L.; Steven, Alasdair C.; Šiber, Antonio; San Martín, Carmen

    2015-01-01

    Genome packing in adenovirus has long evaded precise description, since the viral dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the virus protein coating (capsid). We show that useful insights regarding the organization of the core can be inferred from the analysis of spatial distributions of the DNA and condensing protein units (adenosomes). These were obtained from the inspection of cryo-electron tomography reconstructions of individual human adenovirus particles. Our analysis shows that the core lacks symmetry and strict order, yet the adenosome distribution is not entirely random. The features of the distribution can be explained by modeling the condensing proteins and the part of the genome in each adenosome as very soft spheres, interacting repulsively with each other and with the capsid, producing a minimum outward pressure of ∼0.06 atm. Although the condensing proteins are connected by DNA in disrupted virion cores, in our models a backbone of DNA linking the adenosomes is not required to explain the experimental results in the confined state. In conclusion, the interior of an adenovirus infectious particle is a strongly confined and dense phase of soft particles (adenosomes) without a strictly defined DNA backbone. PMID:25820430

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

    PubMed

    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.

  1. The product of the adenovirus intermediate gene IX is a transcriptional activator.

    PubMed Central

    Lutz, P; Rosa-Calatrava, M; Kedinger, C

    1997-01-01

    We have investigated the functional properties of the product of the adenovirus type 5 gene IX. This gene, which is expressed at intermediate times postinfection, encodes a small polypeptide (pIX) of 140 residues that has previously been shown to be incorporated into the viral capsid. Here, we show that pIX, in addition to its structural contribution, exhibits transcriptional properties. In transient transfection experiments, expression of pIX stimulated adenovirus major late promoter activity. The effect was independent of other viral proteins, but the level of promoter activation appeared strongly pIX dose dependent; similar levels of induction were observed with other cellular or viral TATA-containing (but not with TATA-less) promoters. This promoter specificity could be reproduced in a cell-free transcription system by the addition of purified recombinant pIX, further stressing the transcriptional nature of the phenomenon. A preliminary structural analysis of pIX indicated that the integrity of a putative leucine zipper at the carboxy-terminal end of the molecule, as well as elements within the amino-terminal half, was critical for pIX transcriptional activity. The relevance of these findings in adenovirus infection is discussed. PMID:9188576

  2. A 5-year study of adenoviruses causing conjunctivitis in Izmir, Turkey.

    PubMed

    Erdin, Begüm Nalça; Pas, Suzan D; Durak, İsmet; Schutten, Martin; Sayıner, A Arzu

    2015-03-01

    Adenoviruses are a common cause of conjunctivitis. Genotypes are diverse and differ according to population and geographical distribution of the virus. There is limited data regarding ocular adenoviral infections and genotype distribution in Turkey. This study aimed to determine the adenovirus genotypes and their epidemiological features among patients with conjunctivitis between 2006 and 2010, in Izmir, Turkey. Adenoviral DNA was detected by PCR in 213 of 488 (44%) of the ocular samples collected from patients with viral conjunctivitis during the 5-year study period. Of these, 101 (47%) were randomly chosen and genotyped by sequence analysis. Seven genotypes were identified, including 3, 4, 8, 11, 19, 37, and 53. Genotype 8 and 4 were the dominant types detected in 67 (66.3%) and 25 (24.7%) of the samples, respectively. Other five genotypes (3, 11, 19, 37, 53) were detected in 9 (8.9%) samples. Genotype and seasonal differences observed throughout the study. Human adenoviruse (HAdV)-8 was the most frequent type, except 2008. The prevalence of genotype 4 increased starting from 2006, became dominant in 2008 and decreased in the following years. The peak season was mostly spring months, although it was possible to detect positive samples throughout the year. In conclusion, genotype 8 followed by genotype 4 was the most frequent adenoviral types causing conjunctivitis during the 5-year study period. Findings suggest that there is a slow shift between genotypes throughout the years.

  3. Regulation of transcription of the adenovirus EII promoter by gene products: Absence of sequence specificity

    SciTech Connect

    Kingston, R.E.; Kaufman, R.J.; Sharp, P.A.

    1984-10-01

    During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. The authors have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutaant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. They suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.

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

    PubMed Central

    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

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

    PubMed

    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.

  6. POU domain transcription factors from different subclasses stimulate adenovirus DNA replication.

    PubMed Central

    Verrijzer, C P; Strating, M; Mul, Y M; van der Vliet, P C

    1992-01-01

    POU domain proteins constitute a family of eukaryotic transcription factors that exert critical functions during development. They contain a conserved 160 amino acids DNA binding domain, the POU domain. Genetic data have demonstrated that some POU domain proteins are essential for the proliferation of specific cell types, suggesting a possible role in DNA replication. In addition, the ubiquitous POU transcription factor Oct-1 or its isolated POU domain enhances adenovirus DNA replication. Here we compared the DNA binding specificities of POU domain proteins from different subclasses. They exhibit overlapping, yet distinct binding site preferences. Furthermore, purified Pit-1, Oct-1, Oct-2, Oct-6, Oct-4 and zebrafish POU[C] could all stimulate adenovirus DNA replication in a reconstituted in vitro system. Thus, activation appears to depend on a property common to most POU domain proteins. Adenovirus DNA replication is also stimulated by the transcription factor NFI/CTF. In contrast to NFI, the POU domain did not enhance binding of precursor terminal protein-DNA polymerase to the origin nor did it stabilize the preinitiation complex. These results suggest that the POU domain acts on a rate limiting step after formation of the preinitiation complex. Images PMID:1475198

  7. Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

    SciTech Connect

    Li, Xiaohua; Fan, Rui; Zou, Xue; Gao, Lin; Jin, Haifeng; Du, Rui; Xia, Lin; Fan, Daiming . E-mail: fandaim@yahoo.com.cn

    2007-06-29

    Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.

  8. Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

    PubMed Central

    van Bergen, B G; van der Ley, P A; van Driel, W; van Mansfeld, A D; van der Vliet, P C

    1983-01-01

    Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication. Images PMID:6300787

  9. Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

    PubMed

    Kwant, M M; van der Vliet, P C

    1980-09-11

    There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.

  10. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

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

    PubMed Central

    Bulut, Oya; Yapici, Orhan; Avci, Oguzhan; Simsek, Atilla; Atli, Kamil; Dik, Irmak; Yavru, Sibel; Hasircioglu, Sibel; Kale, Mehmet; Mamak, Nuri

    2013-01-01

    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. PMID:24223508

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

    PubMed

    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.

  13. Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

    PubMed Central

    Mul, Y M; van Miltenburg, R T; De Clercq, E; van der Vliet, P C

    1989-01-01

    The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity. Images PMID:2587248

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

    PubMed

    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.

  15. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  16. Platelet-adenovirus vs. inert particles interaction: effect on aggregation and the role of platelet membrane receptors.

    PubMed

    Gupalo, Elena; Kuk, Cynthia; Qadura, Mohammad; Buriachkovskaia, Liudmila; Othman, Maha

    2013-01-01

    Platelets are involved in host defense via clearance of bacteria from the circulation, interaction with virus particles, and uptake of various size particulates. There is a growing interest in micro- and nanoparticles for drug delivery and there is evidence that the properties of these particles critically influence their interaction and uptake by various tissues and cells including platelets. Virus mediated gene therapy applications are still challenged by the resultant thrombocytopenia and the mechanism(s) of platelet-foreign particles interaction remains unclear. We studied the specifics of platelet interaction with an active biological agent (adenovirus) and inert latex microspheres (MS) and investigated the role of platelet proteins in this interaction. We show that activated and not resting platelets internalize MS, without influencing platelet aggregation. In contrast, adenovirus induces and potentiates ADP-induced platelet aggregation and results in rapid expression of P-selectin. Platelets then internalize adenovirus and viral particles appear inside the open canalicular system. Inhibition of platelet αIIbβ3, GPIbα, and P-selectin decreases both platelet aggregation and internalization of MS. Inhibition of αIIbβ3 and αVβ3 does not abolish adenovirus platelet internalization and adenovirus-induced platelet activation is maintained. Our study demonstrates that platelets react differentially with foreign particles and that αIIbβ3 is a key player in platelet engulfing of foreign particles but not in mediating adenovirus internalization. Other platelet candidate molecules remain to be investigated as potential targets for management of adenovirus-induced thrombocytopenia.

  17. Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine

    PubMed Central

    Tran, Kaylie N.; Croyle, Maria A.; Strong, James E.; Feldmann, Heinz; Kobinger, Gary P.

    2009-01-01

    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 previous generation

  18. Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

    SciTech Connect

    Guardado Calvo, Pablo; Llamas-Saiz, Antonio L.; Langlois, Patrick; Raaij, Mark J. van

    2006-05-01

    Avian adenovirus long-fibre head trimers were expressed, purified and crystallized. The crystals belong to space group C2 (unit-cell parameters a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°). A complete highly redundant data set was collected to 2.2 Å resolution at 100 K using a rotating-anode X-ray source. Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579–793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10 000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kα radiation to at least 1.9 Å resolution and a complete data set was collected from a single crystal to 2.2 Å resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress.

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

    PubMed Central

    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. PMID:23455436

  20. Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle.

    PubMed

    Fender, Pascal; Schoehn, Guy; Perron-Sierra, Françoise; Tucker, Gordon C; Lortat-Jacob, Hugues

    2008-02-05

    The adenovirus penton base is a strategic protein involved in the virus internalisation pathway through interaction between its RGD sequences and integrin. In some human adenovirus serotypes, this pentameric protein features the ability of interacting together by twelve, leading to the formation of a symmetric nanoparticle called dodecahedron (Dd). This non-infectious adenovirus-like particle exhibiting sixty RGD sequences interacts with integrin but also with heparan sulfate proteoglycans (HSPGs) expressed at the cell surface. In this study, we discriminate the respective importance of HSPGs and integrin on human adenovirus serotype 3 dodecahedron attachment and entry. Using different cell lines and a specific integrin inhibitor, we have determined that HSPGs are mainly responsible for particle attachment to the cell surface, favouring a strictly required interaction with integrin that triggers internalisation. No other receptors are involved in Dd entry and integrins on their own can mediate the particle entry in HSPGs-deficient cells. Moreover, integrin recognition by Dd is highly susceptible to cations and particularly to manganese that enhances particle binding by 4- to 7-fold compared to calcium. Interestingly, investigations on Dd receptors along the cell cycle revealed an enhanced particle targeting to mitotic cells and a loss of internalisation at this stage. This phenomenon observed with both HeLa- and HSPGs-deficient cells, depends on integrin remodelling during mitosis. This provides new clues for the use of this adenovirus nanoparticle as a delivery vector and sheds light on the integrin and HSPGs relationship in both resting and dividing cells.

  1. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

    PubMed Central

    Fasbender, A; Lee, J H; Walters, R W; Moninger, T O; Zabner, J; Welsh, M J

    1998-01-01

    Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo. PMID:9649572

  2. Broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses.

    PubMed

    Wang, Zheng; Malanoski, Anthony P; Lin, Baochuan; Long, Nina C; Leski, Tomasz A; Blaney, Kate M; Hansen, Christian J; Brown, Jason; Broderick, Michael; Stenger, David A; Tibbetts, Clark; Russell, Kevin L; Metzgar, David

    2010-05-01

    Military recruits experience a high incidence of febrile respiratory illness (FRI), leading to significant morbidity and lost training time. Adenoviruses, group A Streptococcus pyogenes, and influenza virus are implicated in over half of the FRI cases reported at recruit training center clinics, while the etiology of the remaining cases is unclear. In this study, we explore the carriage rates and disease associations of adenovirus, enterovirus, rhinovirus, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis in military recruits using high-density resequencing microarrays. The results showed that rhinoviruses, adenoviruses, S. pneumoniae, H. influenzae, and N. meningitidis were widely distributed in recruits. Of these five agents, only adenovirus showed significant correlation with illness. Among the samples tested, only pathogens associated with FRI, such as adenovirus 4 and enterovirus 68, revealed strong temporal and spatial clustering of specific strains, indicating that they are transmitted primarily within sites. The results showed a strong negative association between adenoviral FRI and the presence of rhinoviruses in recruits, suggesting some form of viral interference.

  3. Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

    PubMed

    Ding, Ning; Craik, Stephen A; Pang, Xiaoli; Lee, Bonita; Neumann, Norman F

    2017-04-01

      Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

  4. Sulawesi tortoise adenovirus-1 in two impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota).

    PubMed

    Schumacher, Vanessa L; Innis, Charles J; Garner, Michael M; Risatti, Guillermo R; Nordhausen, Robert W; Gilbert-Marcheterre, Kelly; Wellehan, James F X; Childress, April L; Frasca, Salvatore

    2012-09-01

    Sulawesi tortoise adenovirus-1 (STAdV-1) is a newly discovered virus infecting endangered and threatened tortoises. It was initially described from a confiscated group of 105 Sulawesi tortoises (Indotestudo forsteni) obtained by the Turtle Survival Alliance and distributed to five sites with available veterinary care across the United States. In a 3-yr period from the initial outbreak, one multi-species collection that rehabilitated and housed adenovirus-infected Sulawesi tortoises experienced deaths in impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota). Impressed tortoises that died had evidence of systemic viral infection with histopathologic features of adenovirus. Adenovirus was identified by consensus nested polymerase chain reaction (PCR) testing and subsequent sequencing of PCR products. Sequencing indicated that the adenovirus infecting these impressed tortoises and Burmese star tortoise was STAdV-1. In one impressed tortoise, viral infection was confirmed using transmission electron microscopy. In situ hybridization using a semiautomated protocol and fluorescein-labeled riboprobe identified STAdV-1 inclusions in spleen, liver, kidney, and testis of one impressed tortoise. The impact of this virus on captive and wild populations of tortoises is unknown; however, these findings indicate that STAdV-1 can be transmitted to and can infect other tortoise species, the impressed tortoise and Burmese star tortoise, when cohabitated with infected Sulawesi tortoises.

  5. Investigation of an Acute Respiratory Disease Outbreak Due to Adenovirus Type 4 Among Recruits Fort Benning, Georgia, April-May 2000

    DTIC Science & Technology

    2000-05-01

    proposed for new respiratory tract viruses. Science 1956; 124:119-20. 8. Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Adenoviruses. In: Medical ... Microbiology , 3r edition. Mosby, MO, 1998; 412-418. 9. Sanchez MP, Erdman DD, Torok TJ, Freeman CJ, Matyas BT. Outbreak of adenovirus 35 pneumonia among

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

    PubMed

    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.

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

    DOEpatents

    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.

  8. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

  9. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

    PubMed

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-09-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment.

  10. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population

    PubMed Central

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment. PMID:27485384

  11. Recombinant Human Adenovirus: Targeting to the Human Transferrin Receptor Improves Gene Transfer to Brain Microcapillary Endothelium

    PubMed Central

    Xia, Haibin; Anderson, Brian; Mao, Qinwen; Davidson, Beverly L.

    2000-01-01

    Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of β-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR. PMID:11070036

  12. Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system.

    PubMed

    Venkatraman, Giri; Behrens, Maik; Pyrski, Martina; Margolis, Frank L

    2005-09-01

    Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.

  13. Endemic Infection of Stranded Southern Sea Otters (Enhydra lutris nereis) with Novel Parvovirus, Polyomavirus, and Adenovirus.

    PubMed

    Siqueira, Juliana D; Ng, Terry; Miller, Melissa; Li, Linlin; Deng, Xutao; Dodd, Erin; Batac, Francesca; Delwart, Eric

    2017-02-13

    Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened species under federal law and a fully protected species under California law. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter (SO) polyomavirus 1 and the complete coding sequence of SO parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

  14. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    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.

  15. Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival

    PubMed Central

    Miller, Daniel L; Myers, Chad L; Rickards, Brenden; Coller, Hilary A; Flint, S Jane

    2007-01-01

    Background Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications. Results We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins. Conclusion These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors. PMID:17430596

  16. Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

    PubMed Central

    Schwarzenberger, P; Hunt, J D; Robert, E; Theodossiou, C; Kolls, J K

    1997-01-01

    To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA. PMID:9343214

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

    NASA Astrophysics Data System (ADS)

    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.

  18. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

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

    PubMed

    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.

  20. Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

    PubMed Central

    Fahnestock, M L; Lewis, J B

    1989-01-01

    A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation. Images PMID:2523001

  1. Adenovirus-mediated double suicide gene selectively kills gastric cancer cells.

    PubMed

    Luo, Xian-Run; Li, Jian-Sheng; Niu, Ying; Miao, Li

    2012-01-01

    The aim of this study was to evaluate the effect of the adenovirus-mediated double suicide gene (CD/TK) for selective killing of gastric cancer cells. Gastric cancer cells SCG7901 and normal gastric epithelial cell lines were infected by adenoviruses Ad-survivin/GFP and Ad-survivin/CD/TK. GFP expression and CD-TK were detected by fluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. After treatment of the infected cells with the pro-drugs ganciclovir (GCV) and/or 5-FC, the cell growth status was evaluated by methyl thiazolyl tetrazolium assay. Cell cycle changes were detected using flow cytometry. In nude mice bearing human gastric cancer, the recombinant adenovirus vector was injected directly into the tumor followed by an intraperitoneal injection of GCV and/or 5-FC. The subsequent tumor growth was then observed. The GFP gene driven by survivin could be expressed within the gastric cancer line SCG7901, but not in normal gastric epithelial cells. RT-PCR demonstrated the presence of the CD/TK gene product in the infected SCG7901 cells, but not in the infected normal gastric epithelial cells. The infected gastric cancer SCG7901, but not the gastric cells, was highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide genes in killing the target cells (P<0.01). Treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G0-Gl phase and decreased percentage in S phase. In nude mice bearing SCG7901 cells, treatment with the double suicide gene system significantly inhibited tumor growth, showing much stronger effects than either of the single suicide genes (P<0.01). The adenovirus-mediated CD/TK double suicide gene driven by survivin promoter combined with GCV an 5-FC treatment could be an effective therapy against experimental gastric cancer with much greater efficacy than the single suicide gene CD/TK combined

  2. Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors

    PubMed Central

    Qiu, Qi; Xu, Zhili; Tian, Jie; Moitra, Rituparna; Gunti, Sreenivasulu; Notkins, Abner L.

    2014-01-01

    Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice. PMID:25552715

  3. A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly

    PubMed Central

    Moyer, Crystal L.; Besser, Eli S.

    2015-01-01

    ABSTRACT Proteolytic maturation drives the conversion of stable, immature virus particles to a mature, metastable state primed for cell infection. In the case of human adenovirus, this proteolytic cleavage is mediated by the virally encoded protease AVP. Protein VI, an internal capsid cement protein and substrate for AVP, is cleaved at two sites, one of which is near the N terminus of the protein. In mature capsids, the 33 residues at the N terminus of protein VI (pVIn) are sequestered inside the cavity formed by peripentonal hexon trimers at the 5-fold vertex. Here, we describe a glycine-to-alanine mutation in the N-terminal cleavage site of protein VI that profoundly impacts proteolytic processing, the generation of infectious particles, and cell entry. The phenotypic effects associated with this mutant provide a mechanistic framework for understanding the multifunctional nature of protein VI. Based on our findings, we propose that the primary function of the pVIn peptide is to mediate interactions between protein VI and hexon during virus replication, driving hexon nuclear accumulation and particle assembly. Once particles are assembled, AVP-mediated cleavage facilitates the release of the membrane lytic region at the amino terminus of mature VI, allowing it to lyse the endosome during cell infection. These findings highlight the importance of a single maturation cleavage site for both infectious particle production and cell entry and emphasize the exquisite spatiotemporal regulation governing adenovirus assembly and disassembly. IMPORTANCE Postassembly virus maturation is a cornerstone principle in virology. However, a mechanistic understanding of how icosahedral viruses utilize this process to transform immature capsids into infection-competent particles is largely lacking. Adenovirus maturation involves proteolytic processing of seven precursor proteins. There is currently no information for the role of each independent cleavage event in the generation of

  4. Protection of Non-Human Primates against Rabies with an Adenovirus Recombinant Vaccine

    PubMed Central

    Xiang, Z.Q.; Greenberg, L.; Ertl, H. C.; Rupprecht, C.E.

    2014-01-01

    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. PMID:24503087

  5. Isolation and Characterization of the DNA and Protein Binding Activities of Adenovirus Core Protein V

    PubMed Central

    Pérez-Vargas, Jimena; Vaughan, Robert C.; Houser, Carolyn; Hastie, Kathryn M.; Kao, C. Cheng

    2014-01-01

    ABSTRACT The structure of adenovirus outer capsid was revealed recently at 3- to 4-Å resolution (V. Reddy, S. Natchiar, P. Stewart, and G. Nemerow, Science 329:1071–1075, 2010, http://dx.doi.org/10.1126/science.1187292); however, precise details on the function and biochemical and structural features for the inner core still are lacking. Protein V is one the most important components of the adenovirus core, as it links the outer capsid via association with protein VI with the inner DNA core. Protein V is a highly basic protein that strongly binds to DNA in a nonspecific manner. We report the expression of a soluble protein V that exists in monomer-dimer equilibrium. Using reversible cross-linking affinity purification in combination with mass spectrometry, we found that protein V contains multiple DNA binding sites. The binding sites from protein V mediate heat-stable nucleic acid associations, with some of the binding sites possibly masked in the virus by other core proteins. We also demonstrate direct interaction between soluble proteins V and VI, thereby revealing the bridging of the inner DNA core with the outer capsid proteins. These findings are consistent with a model of nucleosome-like structures proposed for the adenovirus core and encapsidated DNA. They also suggest an additional role for protein V in linking the inner nucleic acid core with protein VI on the inner capsid shell. IMPORTANCE Scant knowledge exists of how the inner core of adenovirus containing its double-stranded DNA (dsDNA) genome and associated proteins is organized. Here, we report a purification scheme for a recombinant form of protein V that allowed analysis of its interactions with the nucleic acid core region. We demonstrate that protein V exhibits stable associations with dsDNA due to the presence of multiple nucleic acid binding sites identified both in the isolated recombinant protein and in virus particles. As protein V also binds to the membrane lytic protein VI molecules

  6. Intranasal immunization with an adenovirus vaccine protects guinea pigs from Ebola virus transmission by infected animals.

    PubMed

    Wong, Gary; Richardson, Jason S; Cutts, Todd; Qiu, Xiangguo; Kobinger, Gary P

    2015-04-01

    Experimental Ebola virus (EBOV) vaccines have previously been shown to protect animals against a high dose intramuscular (IM) challenge, which is seen as a stringent challenge model. However, the protective efficacy against other modes of infection, such as contact with infectious hosts, is unknown. Using a previously established EBOV transmission animal model, we evaluated the efficacy of an adenovirus-based EBOV vaccine given to guinea pigs (gps) 4weeks before direct contact with untreated, infectious animals. Prior vaccination resulted in robust levels of EBOV-specific antibodies and conferred complete protection in gps. These results support the use of vaccines to prevent EBOV transmission between hosts.

  7. Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene.

    PubMed

    Esandi, M C; van Someren, G D; Vincent, A J; van Bekkum, D W; Valerio, D; Bout, A; Noteboom, J L

    1997-04-01

    Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local

  8. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

    PubMed Central

    Pascual, E.; Avia, M.; Rangel, G.; de Molina, A.; Alejo, A.; Sevilla, N.

    2016-01-01

    Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. PMID:26739058

  9. Comparative Studies on the Structure of Human Adenovirus Genomes 4, 7 and 21.

    DTIC Science & Technology

    1980-02-01

    PADMANABHAN UNCLASSIFIED 01 FEB 80 DAMD17-79-C-9102 F/G 6/1 NL IEI!ONhuEhhhEIIIIIIIIEEI EN 136 11111 I1.8 1(11(125 .4I’* (fi.6 MICROCOPY RESOLUTION TEST ...the DNA was tested by labeling the protein moiety with [ I ] using chloramine T a t e oxidizing agent (11). This procedure labels the tyrosine residue...the DNA molecules from the human adenovirus serotypes so far tested possess this unique property as observed by EM. Our laboratory has been interested

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

    PubMed

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

    1984-06-04

    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, ribonuclease-sensitive fraction from the cytosol of uninfected cells. This fraction stimulated the initiation about 3-fold and the replication of origin fragments 5-10-fold. Sedimentation analysis indicated the presence of a fast-sedimenting and a slow-sedimenting component which complemented each other. At least part of the stimulation was caused by low-molecular-mass RNA.

  11. Analysis of the DNA-terminal protein from different serotypes of human adenovirus.

    PubMed Central

    Rekosh, D

    1981-01-01

    The DNA-terminal proteins from adenovirus type 12, type 3, and type 5 were analyzed after labeling in vitro with 125I by the chloramine T method, and were shown to be serotype specific. We also studied the kinetics of cleavage by alkali of the terminal protein-DNA linkage and showed that the half-time of cleavage with either 0.1 M NaOH or 0.5 M piperidine at 37 degree C is about 15 to 30 min. The substitution of the volatile base piperidine for NaOH in this procedure provided a useful tool for rapid analysis of the labeled protein. Images PMID:6895236

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

    PubMed Central

    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

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

    PubMed

    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.

  14. The nucleotide sequence at the termini of adenovirus type 5 DNA.

    PubMed Central

    Steenbergh, P H; Maat, J; van Ormondt, H; Sussenbach, J S

    1977-01-01

    The sequences of the first 194 base pairs at both termini of adenovirus type 5 (Ad5) DNA have been determined, using the chemical degradation technique developed by Maxam and Gilbert (Proc. Nat. Acad. Sci. USA 74 (1977), pp. 560-564). The nucleotide sequences 1-75 were confirmed by analysis of labeled RNA transcribed from the terminal HhaI fragments in vitro. The sequence data show that Ad5 DNA has a perfect inverted terminal repetition of 103 base pairs long. Images PMID:600799

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

    PubMed

    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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  19. Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

    PubMed Central

    Belousova, Natalya; Mikheeva, Galina; Xiong, Chiyi; Stagg, Loren J.; Gagea, Mihai; Fox, Patricia S.; Bassett, Roland L.; Ladbury, John E.; Braun, Michael B.; Stehle, Thilo; Li, Chun; Krasnykh, Victor

    2016-01-01

    Unique molecular properties of species D adenoviruses (Ads)—the most diverse yet underexplored group of Ads—have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies. PMID:27462785

  20. Tamoxifen improves cytopathic effect of oncolytic adenovirus in primary glioblastoma cells mediated through autophagy

    PubMed Central

    Ulasov, Ilya V.; Shah, Nameeta; Kaverina, Natalya V.; Lee, Hwahyang; Lin, Biaoyang; Lieber, Andre; Kadagidze, Zaira G.; Yoon, Jae-Guen; Schroeder, Brett; Hothi, Parvinder; Ghosh, Dhimankrishna; Baryshnikov, Anatoly Y.; Cobbs, Charles S.

    2015-01-01

    Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy. PMID:25738357

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

    PubMed

    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.

  2. Adenovirus-Mediated FKHRL1/TM Sensitizes Melanoma Cells to Apoptosis Induced by Temozolomide

    PubMed Central

    Egger, Michael E.; McNally, Lacey R.; Nitz, Jonathan; McMasters, Kelly M.

    2014-01-01

    Abstract Melanoma exhibits variable resistance to the alkylating agent temozolomide (TMZ). We evaluated the potential of adenovirus expressing forkhead human transcription factor like 1 triple mutant (Ad-FKHRL1/TM) to sensitize melanoma cells to TMZ. Four melanoma cell lines were treated with Ad-FKHRL1/TM and TMZ, alone or in combination. Apoptosis was assessed by activation and inhibition of caspase pathway, nuclei fragmentation, and annexin V staining. The potential therapeutic efficacy of Ad-FKHRL1/TM with TMZ was also assessed in a mouse melanoma xenograft model. Combination therapy of Ad-FKHRL1/TM and TMZ resulted in greater cell killing (<20% cell viability) compared with single therapy and controls (p<0.05). Combination indices of Ad-FKHRL1/TM and TMZ therapy indicated significant (p<0.05) synergistic killing effect. Greater apoptosis induction was found in cells treated with Ad-FKHRL1/TM and TMZ than with Ad-FKHRL1/TM or TMZ-treated cells alone. Treatment with TMZ enhanced adenovirus transgene expression in a cell type-dependent manner. In an in vivo model, combination therapy of Ad-FKHRL1/TM with TMZ results in greater tumor growth reduction in comparison with single treatments. We suggest that Ad-FKHRL1/TM is a promising vector to sensitize melanoma cells to TMZ, and that a combination of both approaches would be effective in the clinical setting. PMID:25238278

  3. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    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. PMID:26848680

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

    PubMed

    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.

  5. cis and trans requirements for the selective packaging of adenovirus type 5 DNA.

    PubMed Central

    Gräble, M; Hearing, P

    1992-01-01

    Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome. Images PMID:1731109

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

    PubMed

    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.

  7. Expression of coxsackievirus and adenovirus receptor and its cellular localization in myocardial tissues of dilated cardiomyopathy

    PubMed Central

    Kaur, Tripta; Mishra, Baijayantimala; Saikia, Uma Nahar; Sharma, Mirnalini; Bahl, Ajay; Ratho, Radha Kanta

    2012-01-01

    BACKGROUND: Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children and adults. Recently, the human coxsackievirus and adenovirus receptor (CAR), a common receptor for coxsackieviruses and adenoviruses, was discovered and its increased expression has been reported in patients with DCM and myocarditis. OBJECTIVE: To measure the expression of CAR in myocardial tissues of patients with DCM and its cellular localization in DCM cases. METHODS: Formalin-fixed myocardial tissues collected during autopsy from 26 cases of DCM, and 20 cases each of noncardiac disease and cardiac disease other than DCM were included as the test group, and control groups A and B, respectively. Expression of CAR was studied using immunohistochemical staining of myocardial tissue with a CAR-specific rabbit polyclonal antibody. CAR messenger RNA was semiquantified by reverse transcription polymerase chain reaction followed by agarose gel analysis and measurement of band intensity. RESULTS: CAR positivity in DCM cases was found to be 96% (25 of 26) compared with 30% in control group A and 40% in control group B. CAR was found to be expressed in myocytes, endothelial and interstitial cells; however, positivity in myocytes was significantly higher than in other cells in all groups. The site of CAR expression was predominantly the sarcolemma along with cytoplasm in cardiomyocytes. CONCLUSIONS: The present study highlighted the increased expression of CAR in DCM cases, with localization in myocytes and endothelial cells. PMID:23592932

  8. Novel coronaviruses, astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China.

    PubMed

    Han, H-J; Wen, H-L; Zhao, L; Liu, J-W; Luo, L-M; Zhou, C-M; Qin, X-R; Zhu, Y-L; Liu, M-M; Qi, R; Li, W-Q; Yu, H; Yu, X-J

    2017-04-01

    Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long-footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat-borne viruses.

  9. Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

    PubMed

    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.

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

    PubMed

    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.

  11. Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro

    PubMed Central

    Lasek, Witold; Janyst, Michał; Młynarczyk, Grażyna

    2016-01-01

    There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells. PMID:26862302

  12. Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

    PubMed

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2015-04-07

    Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

  13. Aggregation of Adenovirus 2 in Source Water and Impacts on Disinfection by Chlorine.

    PubMed

    Kahler, Amy M; Cromeans, Theresa L; Metcalfe, Maureen G; Humphrey, Charles D; Hill, Vincent R

    2016-06-01

    It is generally accepted that viral particles in source water are likely to be found as aggregates attached to other particles. For this reason, it is important to investigate the disinfection efficacy of chlorine on aggregated viruses. A method to produce adenovirus particle aggregation was developed for this study. Negative stain electron microscopy was used to measure aggregation before and after addition of virus particles to surface water at different pH and specific conductance levels. The impact of aggregation on the efficacy of chlorine disinfection was also examined. Disinfection experiments with human adenovirus 2 (HAdV2) in source water were conducted using 0.2 mg/L free chlorine at 5 °C. Aggregation of HAdV2 in source water (≥3 aggregated particles) remained higher at higher specific conductance and pH levels. However, aggregation was highly variable, with the percentage of particles present in aggregates ranging from 43 to 71 %. Upon addition into source water, the aggregation percentage dropped dramatically. On average, chlorination CT values (chlorine concentration in mg/L × time in min) for 3-log10 inactivation of aggregated HAdV2 were up to three times higher than those for dispersed HAdV2, indicating that aggregation reduced the disinfection rate. This information can be used by water utilities and regulators to guide decision making regarding disinfection of viruses in water.

  14. Aggregation of Adenovirus 2 in Source Water and Impacts on Disinfection by Chlorine

    PubMed Central

    Cromeans, Theresa L.; Metcalfe, Maureen G.; Humphrey, Charles D.; Hill, Vincent R.

    2016-01-01

    It is generally accepted that viral particles in source water are likely to be found as aggregates attached to other particles. For this reason, it is important to investigate the disinfection efficacy of chlorine on aggregated viruses. A method to produce adenovirus particle aggregation was developed for this study. Negative stain electron microscopy was used to measure aggregation before and after addition of virus particles to surface water at different pH and specific conductance levels. The impact of aggregation on the efficacy of chlorine disinfection was also examined. Disinfection experiments with human adenovirus 2 (HAdV2) in source water were conducted using 0.2 mg/L free chlorine at 5 °C. Aggregation of HAdV2 in source water (≥3 aggregated particles) remained higher at higher specific conductance and pH levels. However, aggregation was highly variable, with the percentage of particles present in aggregates ranging from 43 to 71 %. Upon addition into source water, the aggregation percentage dropped dramatically. On average, chlorination CT values (chlorine concentration in mg/L × time in min) for 3-log10 inactivation of aggregated HAdV2 were up to three times higher than those for dispersed HAdV2, indicating that aggregation reduced the disinfection rate. This information can be used by water utilities and regulators to guide decision making regarding disinfection of viruses in water. PMID:26910058

  15. Transcription control region within the protein-coding portion of adenovirus E1A genes.

    PubMed Central

    Osborne, T F; Arvidson, D N; Tyau, E S; Dunsworth-Browne, M; Berk, A J

    1984-01-01

    A single-base deletion within the protein-coding region of the adenovirus type 5 early region 1A (E1A) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an E1A protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress E1A mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of E1A which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated E1A protein does not produce the cis-acting defect in E1A transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 E1A. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for E1A transcription during the late phase of infection. Images PMID:6334230

  16. Pathological and in situ hybridisation findings in calves experimentally infected with bovine adenovirus type 4.

    PubMed

    Giusti, A M; Luini, M; Benkö, M; Scanziani, E

    1998-04-01

    Three colostrum deprived calves were inoculated intravenously with a strain of bovine adenovirus type 4 (BAV-4) isolated from a calf with acute fibrinous enteritis. The calves were monitored clinically and submitted to post mortem examination at 5, 6 and 10 days post inoculation (p. i.) respectively. The 3 animals showed raise in temperature and diarrhoea. Acute enteritis was observed in calves submitted to post mortem examination at 5 and 6 days p. i. In these animals viral DNA was specifically detected on histological sections by an in situ hybridization (ISH) technique; BAV-4 was isolated from most of the organs and numerous adenovirus-positive endothelial cells were present in the gastrointestinal tract, mesenteric lymph nodes and liver. In the calf examined 10 days p. i. no lesions were observed and rare ISH positive cells were detected. These results indicate that in experimental conditions our strain of BAV-4 is capable to induce clinical signs and lesions similar to those reported in field cases.

  17. T-cell subsets in peripheral blood and tumors of patients treated with oncolytic adenoviruses.

    PubMed

    Kristian, Taipale; Ilkka, Liikanen; Juuso, Juhila; Aila, Karioja-Kallio; Minna, Oksanen; Riku, Turkki; Nina, Linder; Johan, Lundin; Ari, Ristimäki; Anna, Kanerva; Anniina, Koski; Timo, Joensuu; Markus, Vähä-Koskela; Akseli, Hemminki

    2015-05-01

    The quality of the antitumor immune response is decisive when developing new immunotherapies for cancer. Oncolytic adenoviruses cause a potent immunogenic stimulus and arming them with costimulatory molecules reshapes the immune response further. We evaluated peripheral blood T-cell subsets of 50 patients with refractory solid tumors undergoing treatment with oncolytic adenovirus. These data were compared to changes in antiviral and antitumor T cells, treatment efficacy, overall survival, and T-cell subsets in pre- and post-treatment tumor biopsies. Treatment caused a significant (P < 0.0001) shift in T-cell subsets in blood, characterized by a proportional increase of CD8(+) cells, and decrease of CD4(+) cells. Concomitant treatment with cyclophosphamide and temozolomide resulted in less CD4(+) decrease (P = 0.041) than cyclophosphamide only. Interestingly, we saw a correlation between T-cell changes in peripheral blood and the tumor site. This correlation was positive for CD8(+) and inverse for CD4(+) cells. These findings give insight to the interconnections between peripheral blood and tumor-infiltrating lymphocyte (TIL) populations regarding oncolytic virotherapy. In particular, our data suggest that induction of T-cell response is not sufficient for clinical response in the context of immunosuppressive tumors, and that peripheral blood T cells have a complicated and potentially misleading relationship with TILs.

  18. Adenovirus 3 penton dodecahedron exhibits structural changes of the base on fibre binding.

    PubMed

    Schoehn, G; Fender, P; Chroboczek, J; Hewat, E A

    1996-12-16

    It was recently shown that co-expression of adenovirus type 3 (Ad3) penton base and fibre in the baculovirus system produces dodecahedral particles, as does the expression of the penton base alone. The structure of both of these dodecahedral particles, with and without fibre, has been determined by cryoelectron microscopy and 3-dimensional reconstruction techniques to a resolution of 25 and 20 A, respectively. The general form of the penton base resembles that of the base protein in the recent reconstruction of adenovirus type 2. There is a remarkable difference in the penton base structure with and without the fibre. The five small protuberances on the outer surface of each base move away from the 5-fold axis by approximately 15 A when the fibre is present. These protuberances are of relatively low density and most probably represent a flexible loop possibly containing the RGD site involved in integrin binding. The fibre is apparently bound to the outer surface of the penton base, rather than inserted into it. The fibre is flexible and the shaft contains two distinct globular regions 26 A in diameter. The volume of the inner cavity of the dodecahedron is 350 +/- 100 nm3. This small volume precludes the use of the inner cavity to house genetic information for gene therapy; however, the possibility remains of linking the gene to the dodecahedron surface in the hope that it will be internalized with the dodecahedron.

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

    SciTech Connect

    Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin; Xiong, Wei; Sun, Wei; Yang, Chongwen; Zhang, Kai; Wang, Ying; Liu, Hongrong; Huang, Xiaojun; Ji, Gang; Sun, Fei; Zheng, Congyi; Zhu, Ping

    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.

  20. Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression

    PubMed Central

    Anders, M; Vieth, M; Röcken, C; Ebert, M; Pross, M; Gretschel, S; Schlag, P M; Wiedenmann, B; Kemmner, W; Höcker, M

    2009-01-01

    Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R0-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor. PMID:19142187

  1. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma

    PubMed Central

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy. PMID:27895465

  2. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma.

    PubMed

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-Jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy.

  3. A quasi-atomic model of human adenovirus type 5 capsid

    PubMed Central

    Fabry, Céline M S; Rosa-Calatrava, Manuel; Conway, James F; Zubieta, Chloé; Cusack, Stephen; Ruigrok, Rob W H; Schoehn, Guy

    2005-01-01

    Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 Å resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon–hexon and hexon–penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1. PMID:15861131

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

    PubMed

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

    2016-08-01

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

  5. Lack of evidence for the role of human adenovirus-36 in obesity in a European cohort.

    PubMed

    Goossens, Valère J; deJager, Steve A; Grauls, Gert E; Gielen, Marij; Vlietinck, Robert F; Derom, Catherine A; Loos, Ruth J F; Rensen, Sander S; Buurman, Wim A; Greve, Jan W; van Baak, Marleen A; Wolffs, Petra F; Bruggeman, Cathrien A; Hoebe, Christian J P A

    2011-01-01

    Adenovirus infection has been shown to increase adiposity in chickens, mice, and nonhuman primates. Adenovirus type 36 (Ad-36) DNA was detected in adipose tissues in these animal trials. In the United States, Ad-36 significantly correlates with obesity as illustrated by an Ad-36 seroprevalence of 30% in obese individuals and 11% in nonobese individuals. We investigated the possibility of a similar correlation of Ad-36 in Dutch and Belgian persons. In total, 509 serum samples were analyzed for Ad-36 antibodies using a serum neutralization assay. In addition, PCR was used to detect adenoviral DNA in visceral adipose tissue of 31 severely obese surgical patients. Our results indicated an overall Ad-36 seroprevalence of 5.5% increasing with age. BMI of Ad-36 seropositive humans was not significantly different from seronegative humans. No adenoviral DNA could be found using PCR on visceral adipose tissue. In conclusion, this first Ad-36 study in the Netherlands and in Belgium indicates that Ad-36 does not play a role as a direct cause of BMI increase and obesity in humans in Western Europe.

  6. Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

    PubMed Central

    Callegari, Elisa; Elamin, Bahaeldin K.; D’Abundo, Lucilla; Falzoni, Simonetta; Donvito, Giovanna; Moshiri, Farzaneh; Milazzo, Maddalena; Altavilla, Giuseppe; Giacomelli, Luciano; Fornari, Francesca; Hemminki, Akseli; Di Virgilio, Francesco; Gramantieri, Laura; Negrini, Massimo; Sabbioni, Silvia

    2013-01-01

    The down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3’ UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicity. PMID:24069256

  7. Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

    PubMed Central

    2013-01-01

    Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer. PMID:23937994

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27901019

  9. Coinfection of a bearded dragon, Pogona vitticeps, with adenovirus- and dependovirus-like viruses.

    PubMed

    Jacobson, E R; Kopit, W; Kennedy, F A; Funk, R S

    1996-05-01

    Four neonate bearded dragons, Pogona vitticeps, from two collections became ill and died. Multiple tissues were collected and processed for light microscopy. In hematoxylin and eosin-stained sections of liver of one lizard, numerous basophilic intranuclear inclusions were observed. In three lizards, intranuclear inclusions were primarily seen within enterocytes in the small intestine. A portion of paraffin-embedded liver of one lizard and small intestine of a second lizard were removed, deparaffinized, and examined by electron microscopy. For the most part, inclusions in the liver consisted of nonenveloped viral particles 60-66 nm in diameter. Smaller nonenveloped virions 15-17 nm in diameter were occasionally seen in association with these particles. In the intestine, inclusions consisted only of 60-70 nm particles. Based on morphology and location, the larger particles were consistent with an adenovirus. Based on size and presence within nuclei of host cells coinfected with the adenovirus-like virus, the smaller viral agent was consistent with members of the genus Dependovirus.

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

    PubMed

    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.

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

    PubMed Central

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

    2012-01-01

    The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. PMID:23093934

  12. A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence.

    PubMed

    Lam, E; Ramke, M; Groos, S; Warnecke, G; Heim, A

    2011-12-01

    The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established. These primary cells proved to be fully permissive for HAdV-C5 infection in submerged culture, demonstrating efficient HAdV genome replication, infectious viral particle release (1.07×10(8) TCID(50)/ml±6.63×10(7)) and development of cytopathic effect (CPE). Differentiation of porcine bronchial epithelial cells was achieved at the air-liquid interface on collagen I coated 0.4μm polyester membranes. Morphology, expression of tubulin and occludin, the development of tight-junctions and cilia were similar to human bronchial epithelial cells. Infection with HAdV-C5 from the basolateral side resulted in release of infectious virus progeny (2.05×10(7) TCID(50)/ml±2.39×10(7)) to the apical surface as described recently in human bronchial epithelial cells, although complete CPE was not observed. Differentiated porcine bronchial epithelial cells hold promise as a novel method for studying the virulence and pathophysiology of pneumonia associated HAdV types.

  13. Tetracycline-inducible promoter-based conditionally replicative adenoviruses for the control of viral replication.

    PubMed

    Zhang, H; Takayama, K; Zhang, L; Uchino, J; Harada, A; Harada, T; Hisasue, J; Nakagaki, N; Zhou, C; Nakanishi, Y

    2009-05-01

    The use of conditionally replicative adenoviruses (CRAds) as a promising strategy for cancer gene therapy has been developed to overcome inefficient transduction of solid tumor masses by replication-deficient adenoviruses. Many modifications have been made to CRAds to enlarge tropism, increase selectivity and lytic ability, and improve safety. However, safety is still a concern in the context of future clinical application of CRAds. Particularly, after injection into the body, viral replication cannot be controlled externally. Therefore, we constructed a novel CRAd using a tetracycline-inducible promoter system to realize external pharmacological control of its replication. The effect of this CRAd in vitro was measured at the levels of viral DNA replication, cell death and progeny production. We showed that CRAd replication was tightly controlled by the presence or absence of doxycycline (Dox). Moreover, this system showed a significant gene expression in vivo, in which the viral replication was controlled by the oral administration of Dox. This strategy may help improve the safety of cancer gene therapy.

  14. Adenovirus DNA is associated with the nuclear matrix of infected cells.

    PubMed Central

    Younghusband, H B; Maundrell, K

    1982-01-01

    Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops. Images PMID:6287038

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

    PubMed

    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 < 0.001), granulocyte macrophage colony-stimulating factor (GM-CSF) signaling (P < 0.001), and leukocyte extravasation signaling (P < 0.001), in patients surviving a shorter time than their controls. In immunohistochemical analysis, markers CD4 and CD163 were significantly elevated (P = 0.020 and P = 0.016 respectively), in patients with shorter than expected survival. Interestingly, T-cell exhaustion marker TIM-3 was also found to be significantly upregulated (P = 0.006) in patients with poor prognosis. Collectively, these data suggest that activation of several functions of the innate 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.

  16. Attachment and Detachment Behavior of Human Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material.

    PubMed

    Stevenson, Margaret E; Sommer, Regina; Lindner, Gerhard; Farnleitner, Andreas H; Toze, Simon; Kirschner, Alexander K T; Blaschke, Alfred P; Sidhu, Jatinder P S

    2015-09-01

    The transport of human adenovirus, nanoparticles, and PRD1 and MS2 bacteriophages was tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, South Australia. Comparison of transport and removal of virus surrogates with the pathogenic virus is necessary to understand the differences between the virus and surrogate. Because experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow-through soil columns were used. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material but not under high ionic strength or high pH conditions. It was also found that straining due to size and the charge of the colloid were not dominant removal mechanisms in this system. Implications of this study indicate that a certain surrogate may not represent a specific pathogen solely based on similar size, morphology, and/or surface charge. Moreover, if a particular surrogate is representative of a pathogen in one aquifer system, it may not be the most appropriate surrogate in another porous media system. This was apparent in the inferior performance of MS2 as a surrogate, which is commonly used in virus transport studies.

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

    PubMed

    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.

  18. Immunological effects of low-dose cyclophosphamide in cancer patients treated with oncolytic adenovirus.

    PubMed

    Cerullo, Vincenzo; Diaconu, Iulia; Kangasniemi, Lotta; Rajecki, Maria; Escutenaire, Sophie; Koski, Anniina; Romano, Valentina; Rouvinen, Noora; Tuuminen, Tamara; Laasonen, Leena; Partanen, Kaarina; Kauppinen, Satu; Joensuu, Timo; Oksanen, Minna; Holm, Sirkka-Liisa; Haavisto, Elina; Karioja-Kallio, Aila; Kanerva, Anna; Pesonen, Sari; Arstila, Petteri T; Hemminki, Akseli

    2011-09-01

    Patients with advanced solid tumors refractory to and progressing after conventional therapies were treated with three different regimens of low-dose cyclophosphamide (CP) in combination with oncolytic adenovirus. CP was given with oral metronomic dosing (50 mg/day, N = 21), intravenously (single 1,000 mg dose, N = 7) or both (N = 7). Virus was injected intratumorally. Controls (N = 8) received virus without CP. Treatments were well tolerated and safe regardless of schedule. Antibody formation and virus replication were not affected by CP. Metronomic CP (oral and oral + intravenous schedules) decreased regulatory T cells (T(regs)) without compromising induction of antitumor or antiviral T-cell responses. Oncolytic adenovirus given together with metronomic CP increased cytotoxic T cells and induced Th1 type immunity on a systemic level in most patients. All CP regimens resulted in higher rates of disease control than virus only (all P < 0.0001) and the best progression-free (PFS) and overall survival (OS) was seen in the oral + intravenous group. One year PFS and OS were 53 and 42% (P = 0.0016 and P < 0.02 versus virus only), respectively, both which are unusually high for chemotherapy refractory patients. We conclude that low-dose CP results in immunological effects appealing for oncolytic virotherapy. While these first-in-human data suggest good safety, intriguing efficacy and extended survival, the results should be confirmed in a randomized trial.

  19. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

    PubMed

    Toth, Karoly; Lee, Sang R; Ying, Baoling; Spencer, Jacqueline F; Tollefson, Ann E; Sagartz, John E; Kong, Il-Keun; Wang, Zhongde; Wold, William S M

    2015-08-01

    Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.

  20. Adenovirus E1B 19-Kilodalton Protein Modulates Innate Immunity through Apoptotic Mimicry

    PubMed Central

    Grigera, Fernando; Ucker, David S.; Cook, James L.

    2014-01-01

    ABSTRACT Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. IMPORTANCE We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not

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

    PubMed

    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.

  2. Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues

    PubMed Central

    Koski, Anniina; Bramante, Simona; Kipar, Anja; Oksanen, Minna; Juhila, Juuso; Vassilev, Lotta; Joensuu, Timo; Kanerva, Anna; Hemminki, Akseli

    2015-01-01

    In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients. PMID:26156245

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  4. Adenovirus and herpesvirus diversity in free-ranging great apes in the Sangha region of the Republic Of Congo.

    PubMed

    Seimon, Tracie A; Olson, Sarah H; Lee, Kerry Jo; Rosen, Gail; Ondzie, Alain; Cameron, Kenneth; Reed, Patricia; Anthony, Simon J; Joly, Damien O; Karesh, William B; McAloose, Denise; Lipkin, W Ian

    2015-01-01

    Infectious diseases have caused die-offs in both free-ranging gorillas and chimpanzees. Understanding pathogen diversity and disease ecology is therefore critical for conserving these endangered animals. To determine viral diversity in free-ranging, non-habituated gorillas and chimpanzees in the Republic of Congo, genetic testing was performed on great-ape fecal samples collected near Odzala-Kokoua National Park. Samples were analyzed to determine ape species, identify individuals in the population, and to test for the presence of herpesviruses, adenoviruses, poxviruses, bocaviruses, flaviviruses, paramyxoviruses, coronaviruses, filoviruses, and simian immunodeficiency virus (SIV). We identified 19 DNA viruses representing two viral families, Herpesviridae and Adenoviridae, of which three herpesviruses had not been previously described. Co-detections of multiple herpesviruses and/or adenoviruses were present in both gorillas and chimpanzees. Cytomegalovirus (CMV) and lymphocryptovirus (LCV) were found primarily in the context of co-association with each other and adenoviruses. Using viral discovery curves for herpesviruses and adenoviruses, the total viral richness in the sample population of gorillas and chimpanzees was estimated to be a minimum of 23 viruses, corresponding to a detection rate of 83%. These findings represent the first description of DNA viral diversity in feces from free-ranging gorillas and chimpanzees in or near the Odzala-Kokoua National Park and form a basis for understanding the types of viruses circulating among great apes in this region.

  5. Expression of the Coxsackievirus and Adenovirus Receptor in Cultured Human Umbilical Vein Endothelial Cells: Regulation in Response to Cell Density

    PubMed Central

    Carson, Steven D.; Hobbs, Justin T.; Tracy, Steven M.; Chapman, Nora M.

    1999-01-01

    Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B. PMID:10400813

  6. The adenovirus L4-22K protein is multifunctional and is an integral component of crucial aspects of infection.

    PubMed

    Wu, Kai; Orozco, Diana; Hearing, Patrick

    2012-10-01

    A variety of cellular and viral processes are coordinately regulated during adenovirus (Ad) infection to achieve optimal virus production. The Ad late gene product L4-22K has been associated with disparate activities during infection, including the regulation of late gene expression, viral DNA packaging, and infectious virus production. We generated and characterized two L4-22K mutant viruses to further explore L4-22K functions during viral infection. Our results show that L4-22K is indeed important for temporal control of viral gene expression not only because it activates late gene expression but also because it suppresses early gene expression. We also show that the L4-22K protein binds to viral packaging sequences in vivo and is essential to recruit two other packaging proteins, IVa2 and L1-52/55K, to this region. The elimination of L4-22K gave rise to the production of only empty virus capsids and not mature virions, which confirms that the L4-22K protein is required for Ad genome packaging. Finally, L4-22K contributes to adenovirus-induced cell death by regulating the expression of the adenovirus death protein. Thus, the adenovirus L4-22K protein is multifunctional and an integral component of crucial aspects of infection.

  7. RGD-modifided oncolytic adenovirus exhibited potent cytotoxic effect on CAR-negative bladder cancer-initiating cells.

    PubMed

    Yang, Y; Xu, H; Shen, J; Yang, Y; Wu, S; Xiao, J; Xu, Y; Liu, X-Y; Chu, L

    2015-05-14

    Cancer-initiating cell (CIC) is critical in cancer development, maintenance and recurrence. The reverse expression pattern of coxsackie and adenovirus receptor (CAR) and αν integrin in bladder cancer decreases the infection efficiency of adenovirus. We constructed Arg-Gly-Asp (RGD)-modified oncolytic adenovirus, carrying EGFP or TNF-related apoptosis-inducing ligand (TRAIL) gene (Onco(Ad).RGD-hTERT-EGFP/TRAIL), and applied them to CAR-negative bladder cancer T24 cells and cancer-initiating T24 sphere cells. Onco(Ad).RGD-hTERT-EGFP had enhanced infection ability and cytotoxic effect on T24 cells and T24 sphere cells, but little cytoxicity on normal urothelial SV-HUC-1 cells compared with the unmodified virus Onco(Ad).hTERT-EGFP. Notably, Onco(Ad).RGD-hTERT-TRAIL induced apoptosis in T24 cells and T24 sphere cells. Furthermore, it completely inhibited xenograft initiation established by the oncolytic adenovirus-pretreated T24 sphere cells, and significantly suppressed tumor growth by intratumoral injection. These results provided a promising therapeutic strategy for CAR-negative bladder cancer through targeting CICs.

  8. Broad Spectrum Respiratory Pathogen Analysis of Throat Swabs from Military Recruits Reveals Interference Between Rhinoviruses and Adenoviruses

    DTIC Science & Technology

    2010-03-09

    this study, we explore the carriage rates and disease associations of adenovirus, enterovirus, rhinovirus, Streptococcus pneumoniae , Haemophilus...rhinovirus, H Haemophilus influenzae, N Neisseria meningitidis, Sp Streptococcus pneumoniae , Sa Staphylococcus aureus 626 Z. Wang et al. Benning and three...rhinovirus, HEV human enterovirus, Hi Haemophilus influenzae, Nm Neisseria meningitidis, Sp Streptococcus pneumoniae , Sa Staphylococcus aureus, FRI febrile

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

    Chen, Carl Z.; Gorham, Ronald D.; Gaieb, Zied; Morikis, Dimitrios

    2015-01-01

    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. PMID:26357573

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

    PubMed

    Chen, Carl Z; Gorham, Ronald D; Gaieb, Zied; Morikis, Dimitrios

    2015-01-01

    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.

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. A Novel Psittacine Adenovirus Identified During an Outbreak of Avian Chlamydiosis and Human Psittacosis: Zoonosis Associated with Virus-Bacterium Coinfection in Birds

    PubMed Central

    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

    2014-01-01

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

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

    PubMed

    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

  16. Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

    PubMed Central

    Lam, Eric

    2014-01-01

    ABSTRACT The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. IMPORTANCE This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions

  17. Chemical Modification with High Molecular Weight Polyethylene Glycol Reduces Transduction of Hepatocytes and Increases Efficacy of Intravenously Delivered Oncolytic Adenovirus

    PubMed Central

    Doronin, Konstantin; Shashkova, Elena V.; May, Shannon M.; Hofherr, Sean E.

    2009-01-01

    Abstract Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites. PMID:19469693

  18. Inactivation of adenovirus type 5, rotavirus Wa and male specific coliphage (MS2) in biosolids by lime stabilization.

    PubMed

    Hansen, Jacqueline J; Warden, Paul S; Margolin, Aaron B

    2007-03-01

    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 degrees 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 degrees C and 4 +/- 2 degrees 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.

  19. Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

    PubMed

    Guzman, Efrain; Taylor, Geraldine; Hope, Jayne; Herbert, Rebecca; Cubillos-Zapata, Carolina; Charleston, Bryan

    2016-10-01

    Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

  20. Novel Adenoviruses in Wild Primates: a High Level of Genetic Diversity and Evidence of Zoonotic Transmissions ▿ †

    PubMed Central

    Wevers, Diana; Metzger, Sonja; Babweteera, Fred; Bieberbach, Marc; Boesch, Christophe; Cameron, Kenneth; Couacy-Hymann, Emmanuel; Cranfield, Mike; Gray, Maryke; Harris, Laurie A.; Head, Josephine; Jeffery, Kathryn; Knauf, Sascha; Lankester, Felix; Leendertz, Siv Aina J.; Lonsdorf, Elizabeth; Mugisha, Lawrence; Nitsche, Andreas; Reed, Patricia; Robbins, Martha; Travis, Dominic A.; Zommers, Zinta; Leendertz, Fabian H.; Ehlers, Bernhard

    2011-01-01

    Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events. PMID:21835802