Sample records for adenovirus-2 major late

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 attenuatormore » 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.« less

  2. Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication.

    PubMed Central

    Grass, D S; Manley, J L

    1986-01-01

    A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box-containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed. Images PMID:3001338

  3. In vitro transcription of adenovirus.

    PubMed Central

    Fire, A; Baker, C C; Manley, J L; Ziff, E B; Sharp, P A

    1981-01-01

    A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters. Images PMID:7321101

  4. A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA.

    PubMed Central

    Zock, C; Iselt, A; Doerfler, W

    1993-01-01

    Human adenovirus type 12 (Ad12) cannot replicate in hamster cells, whereas human cells are permissive for Ad12. Ad12 DNA replication and late-gene and virus-associated RNA expression are blocked in hamster cells. Early Ad12 genes are transcribed, and the viral DNA can be integrated into the host genome. Ad12 DNA replication and late-gene transcription can be complemented in hamster cells by E1 functions of Ad2 or Ad5, for which hamster cells are fully permissive (for a review, see W. Doerfler, Adv. Virus Res. 39:89-128, 1991). We have previously demonstrated that a 33-nucleotide mitigator sequence, which is located in the downstream region of the major late promoter (MLP) of Ad12 DNA, is responsible for the inactivity of the Ad12 MLP in hamster cells (C. Zock and W. Doerfler, EMBO J. 9:1615-1623, 1990). A similar negative regulator has not been found in the MLP of Ad2 DNA. We have now studied the mechanism of action of this mitigator element. The results of nuclear run-on experiments document the absence of MLP transcripts in the nuclei of Ad12-infected BHK21 hamster cells. Surprisingly, the mitigator element cannot elicit its function in in vitro transcription experiments with nuclear extracts from both hamster BHK21 and human HeLa cells. Intact nuclear topology and/or tightly bound nuclear elements that cannot be eluted in nuclear extracts are somehow required for recognition of the Ad12 mitigator. Electrophoretic mobility shift assays have not revealed significant differences in the binding of proteins from human HeLa or hamster BHK21 cells to the mitigator sequence in the MLP of Ad12 DNA or to the corresponding sequence in Ad2 DNA. We have converted the sequence of the mitigator in the MLP of Ad12 DNA to the equivalent sequence in the MLP of Ad2 DNA by site-directed mutagenesis. This construct was not active in hamster cells. When the Ad12 mitigator, on the other hand, was inserted into the Ad2 MLP, the latter's function in hamster cells was not compromised

  5. Adenovirus 36, Adiposity, and Bone Strength in Late-Adolescent Females

    PubMed Central

    Laing, Emma M; Tripp, Ralph A; Pollock, Norman K; Baile, Clifton A; Della-Fera, Mary Anne; Rayalam, Srujana; Tompkins, Stephen M; Keys, Deborah A; Lewis, Richard D

    2017-01-01

    Adenovirus 36 (Ad36) is the only adenovirus to date that has been linked with obesity in humans. Our previous studies in late-adolescent females suggest that excess weight in the form of fat mass is associated with lower cortical bone strength. The purpose of this study was to assess the relationship between Ad36-specific antibodies, adiposity, and bone strength in our sample of late-adolescent females. A cross-sectional study of 115 females aged 18 to 19 years was performed. Participants were classified according to adiposity by dual-energy X-ray absorptiometry (body fat percentage as normal-fat [<32% body fat; n=93] or high-fat [≥ 32% body fat; n=22]), and according to the presence of Ad36-specific neutralizing antibodies. Peripheral quantitative computed tomography measured bone parameters at the 4% (trabecular bone) and 20% (cortical bone) site, and muscle cross-sectional area (MCSA) at the 66% site, from the distal metaphyses of the radius and the tibia. Bone strength was determined from volumetric bone mineral density and bone geometry to calculate bone strength index (BSI; trabecular site) and polar strength–strain index (SSI; cortical site). After adjustment for MCSA and limb length, radial SSI was lower in Ad36+ versus Ad36− subjects from the high-fat group (p<0.03), but not the normal-fat group. No significant differences were observed between groups in tibial SSI or BSI. These data support an association of adiposity and cortical bone strength at the radius with the presence of neutralizing antibodies to Ad36 in late-adolescent females. PMID:23296755

  6. Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

    PubMed Central

    Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

    1978-01-01

    Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure. Images PMID:207893

  7. Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

    PubMed

    Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

    1978-05-01

    Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

  8. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    PubMed Central

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

    2011-01-01

    Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

  9. Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome.

    PubMed Central

    Goldenberg, C J; Rosenthal, R; Bhaduri, S; Raskas, H

    1981-01-01

    Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection. Images PMID:6894621

  10. Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome.

    PubMed

    Goldenberg, C J; Rosenthal, R; Bhaduri, S; Raskas, H

    1981-06-01

    Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection.

  11. 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

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

    PubMed

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

    2013-12-03

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

  13. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing cell...

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

    PubMed

    Malouli, Daniel; Howell, Grant L; Legasse, Alfred W; Kahl, Christoph; Axthelm, Michael K; Hansen, Scott G; Früh, Klaus

    2014-09-01

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

  15. The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

    PubMed

    Lan, Susan; Kamel, Wael; Punga, Tanel; Akusjärvi, Göran

    2017-02-28

    The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Identification of a novel aviadenovirus, designated pigeon adenovirus 2 in domestic pigeons (Columba livia).

    PubMed

    Teske, L; Rubbenstroth, D; Meixner, M; Liere, K; Bartels, H; Rautenschlein, S

    2017-01-02

    The young pigeon disease syndrome (YPDS) affects mainly young pigeons of less than one year of age and leads to crop stasis, vomitus, diarrhea, anorexia and occasionally death. This disease is internationally a major health problem because of its seasonal appearance during competitions such as homing pigeon races or exhibitions of ornamental birds. While the etiology of YPDS is still unclear, adenoviruses are frequently discussed as potential causative agents. Electron microscopy of feces from a YPDS outbreak revealed massive shedding of adenovirus-like particles. Whole genome sequencing of this sample identified a novel adenovirus tentatively named pigeon adenovirus 2 (PiAdV-2). Phylogenetic and comparative genome analysis suggest PiAdV-2 to belong to a new species within the genus Aviadenovirus, for which we propose the name Pigeon aviadenovirus B. The PiAdV-2 genome shares 54.9% nucleotide sequence identity with pigeon adenovirus 1 (PiAdV-1). In a screening of further YPDS-affected flocks two variants of PiAdV-2 (variant A and B) were detected which shared 97.6% nucleotide identity of partial polymerase sequences, but only 79.7% nucleotide identity of partial hexon sequences. The distribution of both PiAdV-2 variants was further investigated in fecal samples collected between 2008 and 2015 from healthy or YPDS-affected racing pigeons of different lofts. Independent of their health status, approximately 20% of young and 13% of adult pigeon flocks harbored PiAdV-2 variants. Birds were free of PiAdV-1 or other aviadenoviruses as determined by PCRs targeting the aviadenovirus polymerase or the PiAdV-1 fiber gene, respectively. In conclusion, there is no indication of a correlation between YPDS outbreaks and the presence of PiAdV-2 or other aviadenoviruses, arguing against an causative role in this disease complex. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Development of replication-deficient adenovirus malaria vaccines.

    PubMed

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

    2017-03-01

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

  18. Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine.

    PubMed

    Berg, Michael G; Adams, Robert J; Gambhira, Ratish; Siracusa, Mark C; Scott, Alan L; Roden, Richard B S; Ketner, Gary

    2014-09-01

    Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Characterization of a fused protein specified by the adenovirus type 2-simian virus 40 hybrid Ad2+ND1 dp2.

    PubMed Central

    Fey, G; Lewis, J B; Grodzicker, T; Bothwell, A

    1979-01-01

    The adenovirus type 2-simian virus 40 (SV40) hybrid virus Ad2+ND1 dp2 (E. Lukanidin, manuscript in preparation) specified two proteins (molecular weights, 24,000 and 23,000) that are, in part, products of an insertion of SV40 early DNA sequences. This was demonstrated by translation in vitro from viral mRNA that had been selected by hybridization to SV40 DNA. These two phosphorylated, nonvirion proteins were produced late in infection in amounts similar to adenovirus 2 structural proteins and were closely related to each other in tryptic peptide composition. The portion of SV40 DNA (map units 0.17 to 0.22 on the SV40 genome) coding for these proteins was joined to sequences coding for the amino-terminal part of the adenovirus type 2 structural protein IV (fiber). The Ad2+ND1 dp2 23,000- and 24,000-molecular-weight proteins were hybrid polypeptides, with about two-thirds of their tryptic peptides contributed by the fiber protein and the remainder contributed by SV40 T-antigen. They shared with T-antigen (molecular weight, 96,000) a carboxy-terminal proline-rich tryptic peptide. Together, the tryptic peptide composition of these proteins and the known SV40 DNA sequences suggested the reading frame for the translation of T-antigen. The carboxy terminus for T-anigen would then be located on the SV40 genome map next to the TAA terminator triplet at position 0.175, 910 bases away from the cleavage site of the restriction endonuclease EcoRI. Seven host range mutants from Ad2+ND1 dp2 were isolated that had lost the capacity to propagate on monkey cells. They did not induce detectable levels of the hybrid proteins. Three of these mutants had lost the SV40 DNA insertion that codes in part for these proteins. Thus, in analogy to the Ad2+ND1 30,000-molecular-weight protein, the presence of these proteins correlates with the presence of the helper function for adenovirus replication on monkey cells. Images PMID:225516

  20. Identification of a New Human Adenovirus Protein Encoded by a Novel Late l-Strand Transcription Unit▿

    PubMed Central

    Tollefson, Ann E.; Ying, Baoling; Doronin, Konstantin; Sidor, Peter D.; Wold, William S. M.

    2007-01-01

    A short open reading frame named the “U exon,” located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein (DBP) intranuclear localization pattern and an apparent failure to organize replication centers during late infection. Mutants in which the U exon DNA is reconstructed have a reversed phenotype. Chow et al. (L. T. Chow et al., J. Mol. Biol. 134:265-303, 1979) described mRNAs initiating in the region of the U exon and spliced to downstream sequences in the late DBP mRNA leader and the DBP-coding region. We have cloned this mRNA (as cDNA) from Ad5 late mRNA; the predicted protein is 217 amino acids, initiating in the U exon and continuing in frame in the DBP leader and in the DBP-coding region but in a different reading frame from DBP. Polyclonal and monoclonal antibodies generated against the predicted U exon protein (UXP) showed that UXP is ∼24K in size by immunoblot and is a late protein. At 18 to 24 h postinfection, UXP is strongly associated with nucleoli and is found throughout the nucleus; later, UXP is associated with the periphery of replication centers, suggesting a function relevant to Ad DNA replication or RNA transcription. UXP is expressed by all four species C Ads. When expressed in transient transfections, UXP complements the aberrant DBP localization pattern of UXP-negative Ad5 mutants. Our data indicate that UXP is a previously unrecognized protein derived from a novel late l-strand transcription unit. PMID:17881437

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus...; 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 Virus...

  2. Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

    PubMed

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

    2012-06-01

    Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. 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 cell...

  4. 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 cell...

  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 cell...

  6. 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 cell...

  7. The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

    PubMed

    Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

    2005-12-20

    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

  8. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 Ad5more » (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).« less

  9. Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine ▿ †

    PubMed Central

    Page, Martin A.; Shisler, Joanna L.; Mariñas, Benito J.

    2010-01-01

    Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery. PMID:20305026

  10. 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

  11. Incidence of adenoviruses in raw and treated water.

    PubMed

    Van Heerden, Juanita; Ehlers, Marthie M; Van Zyl, Walda B; Grabow, Wilhelm O K

    2003-09-01

    Adenoviruses are of major public health importance and are associated with a variety of clinical manifestations, i.e. gastroenteritis, eye infections and respiratory infections. The importance of water in the epidemiology of adenoviruses and the potential health risks constituted by adenoviruses in water sources and supplies are widely recognised. This study was conducted to assess the incidence of human adenoviruses in raw and treated water systems. Various raw and treated water were routinely monitored for the presence of adenoviruses, over a 1-year period (July 2000-June 2001). The supplies were derived from acceptable quality surface water sources using treatment processes, which conform to international standards for the production of safe drinking water. Adenoviruses were detected by firstly amplifying the viruses in cell cultures and then amplifying the extracted nucleic acids of these viruses using molecular techniques (nested PCR). The results indicated human adenoviruses present in 13 (12.75%) of the raw and 9 (4.41%) of the treated water samples tested. The combination of cell culture and nested PCR has proved to be a quick and reliable method for the detection of adenoviruses in water environments.

  12. 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 Virus...

  13. 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 Virus...

  14. 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 Virus...

  15. 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 Virus...

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Hongxing, E-mail: Hongxing.Zhao@igp.uu.se; Chen, Maoshan; Lind, Sara Bergström

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

  17. Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

    PubMed Central

    Fang, Lei; Stevens, Jennitte L.; Berk, Arnold J.; Spindler, Katherine R.

    2004-01-01

    Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) encodes a virulence gene in viral infection of mice. To broaden our understanding of the functions of E1A in MAV-1 pathogenesis, an unbiased experimental approach, glutathione S-transferase (GST) pulldown, was used to screen for cellular proteins that interact with E1A protein. We identified mouse Sur2, a subunit of Mediator complex, as a protein that binds to MAV-1 E1A. The interaction between Sur2 and MAV-1 E1A was confirmed in virus-infected cells. Conserved region 3 (CR3) of MAV-1 E1A was mapped as the region required for Sur2-E1A interaction, as is the case for human adenovirus E1A. Although it has been proposed that human adenovirus E1A recruits the Mediator complex to transactivate transcription of viral early genes, Sur2 function in adenovirus replication has not been directly tested previously. Studies on the functions of Sur2 with mouse embryonic fibroblasts (MEFs) showed that there was a multiplicity-dependent growth defect of MAV-1 in Sur2−/− MEFs compared to Sur2+/+ MEFs. Comparison of the viral DNA and viral mRNA levels in Sur2+/+ and Sur2−/− MEFs confirmed that Sur2 was important for efficient viral replication. The viral replication defects in Sur2−/− MEFs appeared to be due at least in part to a defect in viral early gene transcription. PMID:15542641

  18. 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.

  19. 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.

  20. Suppression of RNA Interference by Adenovirus Virus-Associated RNA†

    PubMed Central

    Andersson, M. Gunnar; Haasnoot, P. C. Joost; Xu, Ning; Berenjian, Saideh; Berkhout, Ben; Akusjärvi, Göran

    2005-01-01

    We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3′ strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA. PMID:16014917

  1. Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay.

    PubMed

    Senoo, M; Matsubara, Y; Fujii, K; Nagasaki, Y; Hiratsuka, M; Kure, S; Uehara, S; Okamura, K; Yajima, A; Narisawa, K

    2000-04-01

    Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period. We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan-polymerase chain reaction (TaqMan-PCR) assay. We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus. Histochemical analysis showed limited transgene expression in fetal tissues. We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein. The highest beta-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland. The transgene expression was also present in kidney, intestine, and placenta to a lesser degree. No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs. Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver. The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues. The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer. Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.

  2. 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.

  3. Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum

    PubMed Central

    Harmon, Andrew W.; Moitra, Rituparna; Xu, Zhili

    2018-01-01

    Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors. PMID:29401488

  4. Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

    PubMed

    Freimuth, P; Anderson, C W

    1993-03-01

    The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei, Na; Fan, Jun Kai; Gu, Jin Fa

    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 lowermore » 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.« less

  6. A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine

    PubMed Central

    Qurei, Lina; Seto, Donald; Salah, Zaidoun; Azzeh, Maysa

    2012-01-01

    A molecular epidemiology survey was performed in order to establish and document the respiratory adenovirus pathogen profiles among children in Southern Palestine. Three hundred and thirty-eight hospitalized pediatric cases with adenovirus-associated respiratory tract infections were analyzed. Forty four cases out of the 338 were evaluated in more detail for the adenoviruses types present. All of the children resided in Southern Palestine, that is, in city, village and refugee camp environments within the districts of Hebron and Bethlehem. Human adenoviruses circulated throughout 2005–2010, with major outbreaks occurring in the spring months. A larger percent of the children diagnosed with adenoviral infections were male infants. DNA sequence analysis of the hexon genes from 44 samples revealed that several distinct adenovirus types circulated in the region; these were HAdV-C1, HAdV-C2, HAdV-B3 and HAdV-C5. However, not all of these types were detected within each year. This is the first study ever conducted in Palestine of the genetic epidemiology of respiratory adenovirus infections. PMID:22880092

  7. Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

    PubMed

    Yuan, Ziguo; Zhang, Xiuxiang; Zhang, Shoufeng; Liu, Ye; Gao, Shengyan; Zhang, Fei; Xu, Huijuan; Wang, Xiaohu; Hu, Rongliang

    2008-05-01

    Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

  8. Mapping of Adenovirus of serotype 3 fibre interaction to desmoglein 2 revealed a novel 'non-classical' mechanism of viral receptor engagement.

    PubMed

    Vassal-Stermann, Emilie; Mottet, Manon; Ducournau, Corinne; Iseni, Frédéric; Vragniau, Charles; Wang, Hongjie; Zubieta, Chloe; Lieber, André; Fender, Pascal

    2018-05-30

    High-affinity binding of the trimeric fibre protein to a cell surface primary receptor is a common feature shared by all adenovirus serotypes. Recently, a long elusive species B adenovirus receptor has been identified. Desmoglein 2 (DSG2) a component of desmosomal junction, has been reported to interact at high affinity with Human adenoviruses HAd3, HAd7, HAd11 and HAd14. Little is known with respect to the molecular interactions of adenovirus fibre with the DSG2 ectodomain. By using different DSG2 ectodomain constructs and biochemical and biophysical experiments, we report that the third extracellular cadherin domain (EC3) of DSG2 is critical for HAd3 fibre binding. Unexpectedly, stoichiometry studies using multi-angle laser light scattering (MALLS) and analytical ultra-centrifugation (AUC) revealed a non-classical 1:1 interaction (one DSG2 per trimeric fibre), thus differentiating 'DSG2-interacting' adenoviruses from other protein receptor interacting adenoviruses in their infection strategy.

  9. 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

  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. 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.

  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. Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

    PubMed Central

    Murali, V. K.; Ornelles, D. A.; Gooding, L. R.; Wilms, H. T.; Huang, W.; Tollefson, A. E.; Wold, W. S. M.

    2014-01-01

    The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection. PMID:24198418

  14. Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.

    PubMed

    Doronin, Konstantin; Toth, Karoly; Kuppuswamy, Mohan; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

    2003-01-20

    Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.

  15. Major element compositional variation within and between different late Eocene microtektite strewnfields

    NASA Astrophysics Data System (ADS)

    D'Hondt, S. L.; Keller, G.; Stallard, R. F.

    1987-03-01

    The major element composition of microspherules from all three late Eocene stratigraphic layers was analyzed using an electron microprobe. The results indicate a major element compositional overlap beween individual microspherules of different microtektite layers or strewn fields. However, multivariate factor analysis shows that the microtektites of the three late Eocene layers follow recognizably different compositional trends. The microtektite population of the North American strewn field is characterized by high concentrations of SiO2, Al2O3, and TiO2; the microspherules of an older layer, the Gl. cerroazulensis Zone, are relatively enriched in FeO and MgO and impoverished in SiO2 and TiO2; while those of the oldest layer in the uppermost G. semiinvoluta Zone are relatively enriched in CaO and impoverished in Al2O3 and Na2O.

  16. Immunogenicity of adenovirus vaccines expressing the PCV2 capsid protein in pigs.

    PubMed

    Li, Delong; Du, Qian; Wu, Bin; Li, Juejun; Chang, Lingling; Zhao, Xiaomin; Huang, Yong; Tong, Dewen

    2017-08-24

    Porcine circovirus type 2 (PCV2) is the main pathogen of porcine circovirus associated disease (PCVAD), causing great economic losses in pig industry. In previous study, we constructed adenovirus vector vaccines expressing PCV2 Cap either modified with Intron A and WPRE, or CD40L and GMCSF, and evaluated all of these vaccines in mice and in pigs. Although Ad-A-C-W and Ad-CD40L-Cap-GMCSF could induce stronger immune responses than Ad-Cap, neither of them was better than commercial inactivated vaccine PCV2 SH-strain. In this study, secretory recombinant adenoviruses (Ad-A-spCap-W and Ad-A-spCD40L-spCap-spGMCSF-W) and non-secretory recombinant adenovirus Ad-A-CD40L-Cap-GMCSF-W were constructed, and identified by western blot and confocal laser microscope observation. The results of ELISA and VN showed that humoral immune responses induced by Ad-A-spCap-W and Ad-A-CD40L-Cap-GMCSF-W were not significantly different from SH-strain, but Ad-A-spCD40L-spCap-spGMCSF-W could induce significantly higher humoral immune response than SH-strain. Lymphocytes proliferative and cytokines releasing levels of Ad-A-spCap-W and Ad-A-CD40L-Cap-GMCSF-W were not significantly different from SH-strain, but Ad-A-spCD40L-spCap-spGMCSF-W was significantly higher than SH-strain. PCV2-challenge experiment showed that virus loads were significantly reduced in Ad-A-spCD40L-spCap-spGMCSF-W vaccinated group, and no obviously clinical and microscopic lesions were observed in Ad-A-spCD40L-spCap-spGMCSF-W vaccinated group. Altogether, these results demonstrate that recombinant adenovirus vaccine Ad-A-spCD40L-spCap-spGMCSF-W induces stronger immune responses and provides better protection than commercial inactivated vaccine PCV2 SH-strain, and suggest that Ad-A-spCD40L-spCap-spGMCSF-W could be a potential vaccine candidate against PCVAD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. 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.

  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. Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

    PubMed Central

    Winnacker, E L

    1975-01-01

    Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA. PMID:1117487

  20. Viruses and interstitial cystitis: adenovirus genomes cannot be demonstrated in urinary bladder biopsies.

    PubMed

    Hukkanen, V; Haarala, M; Nurmi, M; Klemi, P; Kiilholma, P

    1996-01-01

    Microbes may be involved in the pathogenesis of interstitial cystitis (IC). Adenoviruses and BK virus (BKV) can infect epithelial cells in urinary bladder and they are causative agents for hemorrhagic cystitis. We therefore studied the presence of adenovirus and BKV genomes in urinary bladder tissue specimens of patients with IC using polymerase chain reaction (PCR) and in situ hybridization (ISH). Controls were specimens from cases with transitional cell carcinoma of the bladder. Nucleic acids were extracted from paraffin sections of the bladder tissue for PCR. Primers detecting all adenovirus types were used. In situ hybridization was carried out for the paraffin sections using digoxigenin-labeled DNA probes for adenovirus and BKV. The adenovirus DNA PCR was able to detect one to two infected cells/specimen. All the seven IC cases studied and six controls were negative for adenovirus DNA by PCR and ISH. The ISH test for BKV genomes was also considered negative in IC cases and controls. The specimens which were negative in PCR tests yielded a signal with beta-globin primers, thus being amplifiable. We conclude that adenovirus and BKV do not play a major pathogenetic role in interstitial cystitis.

  1. 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

  2. Spatiotemporal Dynamics of Adenovirus Membrane Rupture and Endosomal Escape

    PubMed Central

    Maier, Oana; Marvin, Shauna A.; Wodrich, Harald; Campbell, Edward M.

    2012-01-01

    A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1+) early endosomes or LAMP-1+ late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3+ endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3+ endosomes, pVI appears to remain associated with the gal3+ ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events. PMID:22855481

  3. Proton magnetic resonance spectroscopy of late-life major depressive disorder.

    PubMed

    Chen, Cheng-Sheng; Chiang, I-Chan; Li, Chun-Wei; Lin, Wei-Chen; Lu, Chia-Ying; Hsieh, Tsyh-Jyi; Liu, Gin-Chung; Lin, Hsiu-Fen; Kuo, Yu-Ting

    2009-06-30

    The primary goal of this study was to examine the biochemical abnormalities of late-life major depression by using 3-tesla (3-T) proton magnetic resonance spectroscopy ((1)H-MRS). The antidepressant effects on the biochemical abnormalities were investigated as well. Study participants were 27 elderly patients with major depressive disorders (among which 9 were on antidepressant medication) and 19 comparison elderly subjects. (1)H-MRS spectra were acquired from voxels that were placed in the left frontal white matter, left periventricular white matter, and left basal ganglia. Ratios of N-acetylaspartate (NAA), choline (Cho) and myo-inositol to creatine were calculated. Patients with late-life major depressive disorder had a significantly lower NAA/creatine ratio in the left frontal white matter, and higher Cho/creatine and myo-inositol/creatine ratios in the left basal ganglia when compared with the control subjects. The myo-inositol correlated with global cognitive function among the patients. The biochemical abnormalities in late-life major depressive disorder were found on the left side of the frontal white matter and the basal ganglia. Neuron degeneration in the frontal white matter and second messenger system dysfunction or glial dysfunction in the basal ganglia are suggested to be associated with late-life depression.

  4. Cell transformation by human adenoviruses.

    PubMed

    Endter, C; Dobner, T

    2004-01-01

    The last 40 years of molecular biological investigations into human adenoviruses have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of their productive infection cycle in permissive host cells. Also, initial observations concerning the carcinogenic potential of human adenoviruses subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer, and established adenoviruses as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human adenoviruses is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in adenovirus-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, detailed studies on the tumorigenic potential of subgroup D adenovirus type 9 (Ad9) E4 have now revealed a new pathway that points to a novel, general mechanism of virus-mediated oncogenesis. In this chapter, we summarize the current state of knowledge about the oncogenes and oncogene products of human adenoviruses, focusing particularly on recent findings concerning the transforming and oncogenic properties of viral proteins encoded in the E1B and E4 transcription units.

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

    PubMed

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

    2007-06-01

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

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

    PubMed Central

    Hansen, Jacqueline J.; Warden, Paul S.; Margolin, Aaron B.

    2007-01-01

    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°C. In all R/O water trials, adenovirus type 5, rotavirus Wa and MS2 were below detectable levels (<100.5 TCID50/mL and <1 PFU/mL respectively) following 0.1-hr of liming. Adenovirus type 5, rotavirus Wa, and MS2, were inoculated into composted, raw and previously limed matrices, representative of sludge and biosolids, to achieve a final concentration of approximately 104 PFU or TCID50/mL. Each matrix was limed for 24-hr at 22 ± 5°C and 4 ± 2°C. In all trials virus was below detectable levels following a 24-hr incubation. The time required for viral inactivation varied depending on the temperature and sample matrix. This research demonstrates reduction of adenovirus type 5, rotavirus Wa, and male-specific bacteriophage, in water, sludge and biosolids matrices following addition of an 8% calcium hydroxide slurry to achieve a pH of 12 for 2-hr reduced to 11.5 for 22-hr by addition of 0.1 N HCl. In these trials, MS2 was a conservative indicator of the efficacy of lime stabilization of adenovirus Type 5 and rotavirus Wa and therefore is proposed as a useful indicator organism. PMID:17431317

  7. 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.

  8. Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

    PubMed Central

    Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

    2010-01-01

    Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined. PMID:19812395

  9. Proteomics analysis of the nucleolus in adenovirus-infected cells.

    PubMed

    Lam, Yun W; Evans, Vanessa C; Heesom, Kate J; Lamond, Angus I; Matthews, David A

    2010-01-01

    Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

  10. Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

    PubMed Central

    Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

    2011-01-01

    Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

  11. Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

    PubMed Central

    Kornfeld, Rosalind; Wold, William S. M.

    1981-01-01

    Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-3H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man9GlcNAc and Man8GlcNAc and small amounts of Man7GlcNAc and Man6GlcNAc. The pulse-chase sample had predominantly Man8GlcNAc and much less Man9GlcNAc, indicating that processing of the Man9GlcNAc to Man8GlcNAc had occurred during the chase period. Thus, Man8GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man9GlcNAc were identical to those of the lipid-linked Glc3Man9GlcNAc2 donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein. Images PMID:7321093

  12. Structures of the oligosaccharides of the glycoprotein coded by early region E3 of adenovirus 2.

    PubMed

    Kornfeld, R; Wold, W S

    1981-11-01

    Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-(3)H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-beta-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man(9)GlcNAc and Man(8)GlcNAc and small amounts of Man(7)GlcNAc and Man(6)GlcNAc. The pulse-chase sample had predominantly Man(8)GlcNAc and much less Man(9)GlcNAc, indicating that processing of the Man(9)GlcNAc to Man(8)GlcNAc had occurred during the chase period. Thus, Man(8)GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with alpha-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man(9)GlcNAc were identical to those of the lipid-linked Glc(3)Man(9)GlcNAc(2) donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell

  13. Late onset dysthymic disorder and major depression differ from early onset dysthymic disorder and major depression in elderly outpatients.

    PubMed

    Devanand, D P; Adorno, Elizabeth; Cheng, Jocelyn; Burt, Tal; Pelton, G H Gregory H; Roose, S P Steven P; Sackeim, H A Harold A

    2004-03-01

    Age of onset may affect clinical features and prognosis in elderly patients with major depression (MDD), but there is a lack of such data in elderly patients with dysthymic disorder (DD) and systematic comparisons of late onset MDD and DD have not been conducted. In a Late Life Depression Clinic, patients > or = 60 years old who met DSM-III-R or DSM-IV criteria for MDD or DD were studied. The 24-item Hamilton Rating Scale for Depression (HRSD) and SCID-P were completed, family history was obtained, and medical illnesses were assessed. In the total sample (n=370; 211 MDD and 159 DD), compared to early onset patients, late onset (onset > or =60 years) patients had a higher rate of cardiovascular disease (chi(2)=4.12, df=1, P<0.05), lower rate of anxiety disorder (chi(2)=4.19, df=1, P<0.05), and a lower rate of family history of affective disorder (chi(2)=9.37, df=1, P<0.002). Late onset DD patients were more likely to have cardiovascular disease than early onset DD patients (chi(2)=5.63, df=1, P<0.02), but the rate of cardiovascular disease did not differ between late and early onset MDD patients (chi(2)=0.35, df=1, P<0.6). Late onset MDD patients were less likely to have a family history of affective disorder than early onset MDD patients (chi(2)=10.71, df=1, P<0.001). Prevalence of anxiety disorders did not differ between the early and late onset MDD patients (chi(2)=0.07, df=1, P<0.79), but was more common in the early onset DD compared to the late onset DD patients (17.98% versus 4.29%, chi(2)=6.98, df=1, P<0.01). Late onset DD did not differ from late onset MDD in the rates of cardiovascular disease, anxiety disorders, and family history of affective disorder. Excluding patients with double depression (n=32) did not alter the cardiovascular or family history findings, but the difference in anxiety disorders between early and late onset DD patients was no longer significant. Academic clinic sample results may not generalize to community populations. In the

  14. 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

  15. Adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter for colorectal cancer.

    PubMed

    Wang, Zhao-Xia; Bian, Hai-Bo; Yang, Jing-Song; De, Wei; Ji, Xiao-Hui

    2009-08-01

    Colorectal cancer is a most frequent type of gastrointestinal tract cancers. The prognosis of patients with colorectal cancer remains poor despite intensive interventions. Tumor specific promoter-directed gene therapy and adenoviral technology can be promising strategies for such advanced disease. This study was conducted to explore the possible therapeutic approach of Cox-2 promoter-directed suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with adenoviral technology for advanced colorectal cancer. Firstly, the activity of Cox-2 promoter was assessed by dual luciferase and enhanced green fluorescent protein reporter gene assays in colorectal cancer cell lines and normal human intestinal epithelial cell line. Then, the expression of coxsackievirus and adenovirus receptor (CAR) was detected in colorectal cancer cell lines. The Cox-2 promoter-directed HSV-tk/ganciclovir (GCV) system mediated by adenovirus (Ad-Cp-TK) was developed (Ad-CMVp-TK, Ad-null and no Ad as controls). In vitro cytoxicity, colony formation and apoptosis assays were performed using Ad-Cp-TK. An animal study was carried out in which BALB/C nude mice bearing tumors were treated with Ad-Cp-TK and GCV treatments. Results showed that Cox-2 promoter possessed high transcriptional activity in a tumor-specific manner. All colorectal cancer cells were detected CAR-positive. In vitro cytotoxic and colony formation assays showed that colorectal cancer cells infected with Ad-Cp-TK became more sensitive to GCV but the sensitivity of normal cells infected with Ad-Cp-TK to GCV were not altered. Moreover, the Ad-Cp-TK system combined with GCV treatment could significantly induce apoptosis of colorectal cancer cells but not normal intestinal epithelial cells. Furthermore, this system also significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice. Thus, adenovirus primary receptor was positive in colorectal cancer cells and adenovirus

  16. Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells.

    PubMed

    Medina, Daniel J; Sheay, Wendy; Osman, Mona; Goodell, Lauri; Martin, John; Rabson, Arnold B; Strair, Roger K

    2005-11-01

    Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.

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

    PubMed Central

    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

    2014-01-01

    ABSTRACT 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. 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. Here 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. IMPORTANCE Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens. PMID:25410856

  18. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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

  20. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

    PubMed

    Tollefson, A E; Toth, K; Doronin, K; Kuppuswamy, M; Doronina, O A; Lichtenstein, D L; Hermiston, T W; Smith, C A; Wold, W S

    2001-10-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

  1. Inhibition of TRAIL-Induced Apoptosis and Forced Internalization of TRAIL Receptor 1 by Adenovirus Proteins

    PubMed Central

    Tollefson, Ann E.; Toth, Karoly; Doronin, Konstantin; Kuppuswamy, Mohan; Doronina, Oksana A.; Lichtenstein, Drew L.; Hermiston, Terry W.; Smith, Craig A.; Wold, William S. M.

    2001-01-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

  2. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

    PubMed Central

    Gorziglia, M I; Kadan, M J; Yei, S; Lim, J; Lee, G M; Luthra, R; Trapnell, B C

    1996-01-01

    A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy. PMID:8648763

  3. Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors.

    PubMed

    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

    2015-02-01

    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. 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. Here 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. Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Cancer gene therapy with targeted adenoviruses.

    PubMed

    Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

    2008-11-01

    Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

  5. 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.

  6. Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

    PubMed Central

    Ledinko, Nada; Fong, Caroline K. Y.

    1969-01-01

    Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of 3H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. 3H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much 3H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase. PMID:5806981

  7. Oral vaccination with an adenovirus-vectored vaccine protects against botulism

    PubMed Central

    Chen, Shan; Xu, Qingfu; Zeng, Mingtao

    2013-01-01

    We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (HC50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1×104 to 1×107 plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2×107 pfu adenovirus, has shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2×106 pfu adenovirus or greater were completely protected against challenge with 100×MLD50 of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism. PMID:23295065

  8. Core labeling of adenovirus with EGFP

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Le, Long P.; Le, Helen N.; Nelson, Amy R.

    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 expressionmore » 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.« less

  9. Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

    PubMed

    Ruch-Gallie, R; Moroff, S; Lappin, M R

    2016-01-01

    Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. Eight puppies from a research breeding facility negative for these pathogens. Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  10. 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

  11. Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

    PubMed Central

    Maluquer de Motes, Carlos; Clemente-Casares, Pilar; Hundesa, Ayalkibet; Martín, Margarita; Girones, Rosina

    2004-01-01

    In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies. PMID:15006765

  12. Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination.

    PubMed

    Maluquer de Motes, Carlos; Clemente-Casares, Pilar; Hundesa, Ayalkibet; Martín, Margarita; Girones, Rosina

    2004-03-01

    In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

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

    PubMed Central

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

    2015-01-01

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

  14. Effect of temperature and sunlight on the stability of human adenoviruses and MS2 as fecal contaminants on fresh produce surfaces.

    PubMed

    Carratalà, Anna; Rodriguez-Manzano, Jesús; Hundesa, Ayalkibet; Rusiñol, Marta; Fresno, Sandra; Cook, Nigel; Girones, Rosina

    2013-06-17

    Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Adenovirus sequences required for replication in vivo.

    PubMed Central

    Wang, K; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I. Images PMID:2991857

  16. 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

  17. Conserved Sequences at the Origin of Adenovirus DNA Replication

    PubMed Central

    Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

    1982-01-01

    The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

  18. Structure and Uncoating of Immature Adenovirus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Perez-Berna, A.J.; Mangel, W.; Marabini, R.

    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 particlesmore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C., E-mail: ertl@wistar.upenn.edu

    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 protectsmore » 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.« less

  20. [Construction and expression of a recombinant adenovirus with LZP3].

    PubMed

    Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

    2007-08-01

    To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

  1. 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

  2. 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

  3. 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

  4. Prevalence and Quantitation of Species C Adenovirus DNA in Human Mucosal Lymphocytes

    PubMed Central

    Garnett, C. T.; Erdman, D.; Xu, W.; Gooding, Linda R.

    2002-01-01

    The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 × 106 copies of the adenovirus genome/107 cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form. PMID:12368303

  5. Adenovirus-based genetic vaccines for biodefense.

    PubMed

    Boyer, Julie L; Kobinger, Gary; Wilson, James M; Crystal, Ronald G

    2005-02-01

    The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

  6. A Novel Adenovirus in Chinstrap Penguins (Pygoscelis antarctica) in Antarctica

    PubMed Central

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

    2014-01-01

    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. PMID:24811321

  7. 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.

  8. Evaluation of recombinant adenovirus vaccines based on glycoprotein D and truncated UL25 against herpes simplex virus type 2 in mice.

    PubMed

    Liu, Wei; Zhou, Yan; Wang, Ziyan; Zhang, Zeqiang; Wang, Qizhi; Su, Weiheng; Chen, Yan; Zhang, Yan; Gao, Feng; Jiang, Chunlai; Kong, Wei

    2017-05-01

    The high prevalence of herpes simplex virus 2 (HSV-2) infections in humans necessitates the development of a safe and effective vaccine that will need to induce vigorous T-cell responses to control viral infection and transmission. We designed rAd-gD2, rAd-gD2ΔUL25, and rAd-ΔUL25 to investigate whether recombinant replication-defective adenoviruses vaccine could induce specific T-cell responses and protect mice against intravaginal HSV-2 challenge compared with FI-HSV-2. In the present study, recombinant adenovirus-based HSV-2 showed higher reductions in mortality and stronger antigen-specific T-cell responses compared with FI-HSV-2 and the severity of genital lesions in mice immunized with rAd-gD2ΔUL25 was significantly decreased by eliciting IFN-γ-secreting T-cell responses compared with rAd-gD2 and rAd-ΔUL25 groups. Our results demonstrated the immunogenicity and protective efficacy of recombinant adenovirus vaccines in acute HSV-2 infection following intravaginal challenge in mice. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  9. A single exercise bout augments adenovirus-specific T-cell mobilization and function.

    PubMed

    Kunz, Hawley E; Spielmann, Guillaume; Agha, Nadia H; O'Connor, Daniel P; Bollard, Catherine M; Simpson, Richard J

    2018-04-30

    Adoptive transfer of virus-specific T-cells (VSTs) effectively treats viral infections following allogeneic hematopoietic stem cell transplantation (alloHSCT), but logistical difficulties have limited widespread availability of VSTs as a post-transplant therapeutic. A single exercise bout mobilizes VSTs specific for latent herpesviruses (i.e. CMV and EBV) to peripheral blood and augments their ex vivo expansion. We investigated whether exercise exerts similar effects on T-cells specific for a NON-latent virus such as adenovirus, which is a major contributor to infection-related morbidity and mortality after alloHSCT. Thirty minutes of cycling exercise increased circulating adenovirus-specific T-cells 2.0-fold and augmented their ex vivo expansion by ~33% compared to rest without altering antigen and MHC-specific autologous target cell killing capabilities. We conclude that exercise is a simple and economical adjuvant to boost the isolation and manufacture of therapeutic VSTs specific to latent and non-latent viruses from healthy donors. Copyright © 2018. Published by Elsevier Inc.

  10. 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.

  11. Conceptual model and experimental framework to determine the contributions of direct and indirect photoreactions to the solar disinfection of MS2, phiX174, and adenovirus.

    PubMed

    Mattle, Michael J; Vione, Davide; Kohn, Tamar

    2015-01-06

    Sunlight inactivates waterborne viruses via direct (absorption of sunlight by the virus) and indirect processes (adsorption of sunlight by external chromophores, which subsequently generate reactive species). While the mechanisms underlying these processes are understood, their relative importance remains unclear. This study establishes an experimental framework to determine the kinetic parameters associated with a virus' susceptibility to solar disinfection and proposes a model to estimate disinfection rates and to apportion the contributions of different inactivation processes. Quantum yields of direct inactivation were determined for three viruses (MS2, phiX174, and adenovirus), and second-order rate constants associated with indirect inactivation by four reactive species ((1)O2, OH(•), CO3(•-), and triplet states) were established. PhiX174 exhibited the greatest quantum yield (1.4 × 10(-2)), indicating that it is more susceptible to direct inactivation than MS2 (2.9 × 10(-3)) or adenovirus (2.5 × 10(-4)). Second-order rate constants ranged from 1.7 × 10(7) to 7.0 × 10(9) M(-1) s(-1) and followed the sequence MS2 > adenovirus > phiX174. A predictive model based on these parameters accurately estimated solar disinfection of MS2 and phiX174 in a natural water sample and approximated that of adenovirus within a factor of 6. Inactivation mostly occurred by direct processes, though indirect inactivation by (1)O2 also contributed to the disinfection of MS2 and adenovirus.

  12. Adenovirus small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells.

    PubMed

    Tao, Zhi-Wei; Zou, Ping-An

    2018-06-13

    Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent year. This study attempts to explore the effect of adenovirus-mediated small interfering RNA (siRNA) targeting ezrin on the proliferation, migration, invasion and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targeting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targeting ezrin elevated expression levels of Bax, P21, P53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2, and MMP-9. Furthermore, adenovirus-mediated siRNA targeting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targeting ezrin can induce apoptosis and inhibit the proliferation, migration and invasion of human osteosarcoma MG-63 cells. ©2018 The Author(s).

  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. 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.

  15. The complete genomic sequence of egg drop syndrome virus strain AAV-2.

    PubMed

    Jin, Q; Zeng, L; Yang, F; Li, M; Hou, Y

    1999-12-01

    In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restriction endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the complete genome nucleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on genomic structure and the distribution of open reading frame (ORF). There are no clear E1, E3 and E4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding ElA, pV and pIX, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group III Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus and will also help the search of all Adenoviruses.

  16. 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.

  17. Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes.

    PubMed

    Xu, Ning; Segerman, Bo; Zhou, Xiaofu; Akusjärvi, Göran

    2007-10-01

    Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.

  18. Comparison of different approaches to quantitative adenovirus detection in stool specimens of hematopoietic stem cell transplant recipients.

    PubMed

    Kosulin, K; Dworzak, S; Lawitschka, A; Matthes-Leodolter, S; Lion, T

    2016-12-01

    Adenoviruses almost invariably proliferate in the gastrointestinal tract prior to dissemination, and critical threshold concentrations in stool correlate with the risk of viremia. Monitoring of adenovirus loads in stool may therefore be important for timely initiation of treatment in order to prevent invasive infection. Comparison of a manual DNA extraction kit in combination with a validated in-house PCR assay with automated extraction on the NucliSENS-EasyMAG device coupled with the Adenovirus R-gene kit (bioMérieux) for quantitative adenovirus analysis in stool samples. Stool specimens spiked with adenovirus concentrations in a range from 10E2-10E11 copies/g and 32 adenovirus-positive clinical stool specimens from pediatric stem cell transplant recipients were tested along with appropriate negative controls. Quantitative analysis of viral load in adenovirus-positive stool specimens revealed a median difference of 0.5 logs (range 0.1-2.2) between the detection systems tested and a difference of 0.3 logs (range 0.0-1.7) when the comparison was restricted to the PCR assays only. Spiking experiments showed a detection limit of 10 2 -10 3 adenovirus copies/g stool revealing a somewhat higher sensitivity offered by the automated extraction. The dynamic range of accurate quantitative analysis by both systems investigated was between 10 3 and 10 8 virus copies/g. The differences in quantitative analysis of adenovirus copy numbers between the systems tested were primarily attributable to the DNA extraction method used, while the qPCR assays revealed a high level of concordance. Both systems showed adequate performance for detection and monitoring of adenoviral load in stool specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. EFFECT OF PROFLAVINE ON THE SYNTHESIS OF ADENOVIRUS, TYPE 5, AND ASSOCIATED SOLUBLE ANTIGENS

    PubMed Central

    Wilcox, Wesley C.; Ginsberg, Harold S.

    1962-01-01

    Wilcox, Wesley C. (University of Pennsylvania, Philadelphia) and Harold S. Ginsberg. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble antigens. J. Bacteriol. 84:526–533. 1962.—The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine, an acridine dye. In comparison, the processes leading to the production of soluble complement-fixing antigens and toxin were less sensitive to the action of this chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis of soluble antigens began prior to the first appearance of newly synthesized virus. This observation is compatible with the hypothesis that the soluble antigens may represent virus subunits or precursor materials. In addition, these data indicate that it is possible to interrupt the latter stages of the virus synthetic process by addition of proflavine late in the eclipse period. PMID:14000661

  20. Novel adenoviruses detected in British mustelids, including a unique Aviadenovirus in the tissues of pine martens (Martes martes)

    PubMed Central

    Gregory, William F.; Turnbull, Dylan; Rocchi, Mara; Meredith, Anna L.; Philbey, Adrian W.; Sharp, Colin P.

    2017-01-01

    Several adenoviruses are known to cause severe disease in veterinary species. Recent evidence suggests that canine adenovirus type 1 (CAV-1) persists in the tissues of healthy red foxes (Vulpes vulpes), which may be a source of infection for susceptible species. It was hypothesized that mustelids native to the UK, including pine martens (Martes martes) and Eurasian otters (Lutra lutra), may also be persistently infected with adenoviruses. Based on high-throughput sequencing and additional Sanger sequencing, a novel Aviadenovirus, tentatively named marten adenovirus type 1 (MAdV-1), was detected in pine marten tissues. The detection of an Aviadenovirus in mammalian tissue has not been reported previously. Two mastadenoviruses, tentatively designated marten adenovirus type 2 (MAdV-2) and lutrine adenovirus type 1 (LAdV-1), were also detected in tissues of pine martens and Eurasian otters, respectively. Apparently healthy free-ranging animals may be infected with uncharacterized adenoviruses with possible implications for translocation of wildlife. PMID:28749327

  1. The NGC 4013 tale: a pseudo-bulged, late-type spiral shaped by a major merger

    NASA Astrophysics Data System (ADS)

    Wang, Jianling; Hammer, Francois; Puech, Mathieu; Yang, Yanbin; Flores, Hector

    2015-10-01

    Many spiral galaxy haloes show stellar streams with various morphologies when observed with deep images. The origin of these tidal features is discussed, either coming from a satellite infall or caused by residuals of an ancient, gas-rich major merger. By modelling the formation of the peculiar features observed in the NGC 4013 halo, we investigate their origin. By using GADGET-2 with implemented gas cooling, star formation, and feedback, we have modelled the overall NGC 4013 galaxy and its associated halo features. A gas-rich major merger occurring 2.7-4.6 Gyr ago succeeds in reproducing the NGC 4013 galaxy properties, including all the faint stellar features, strong gas warp, boxy-shaped halo and vertical 3.6 μm luminosity distribution. High gas fractions in the progenitors are sufficient to reproduce the observed thin and thick discs, with a small bulge fraction, as observed. A major merger is able to reproduce the overall NGC 4013 system, including the warp strength, the red colour and the high stellar mass density of the loop, while a minor merger model cannot. Because the gas-rich model suffices to create a pseudo-bulge with a small fraction of the light, NGC 4013 is perhaps the archetype of a late-type galaxy formed by a relatively recent merger. Then late type, pseudo-bulge spirals are not mandatorily made through secular evolution, and the NGC 4013 properties also illustrate that strong warps in isolated galaxies may well occur at a late phase of a gas-rich major merger.

  2. Characterization of a new adenovirus isolated from black-tailed deer in California.

    PubMed

    Lehmkuhl, H D; Hobbs, L A; Woods, L W

    2001-01-01

    An adenovirus associated with systemic and localized vascular damage was demonstrated by transmission electron microscopy and immunohistochemistry in a newly recognized epizootic hemorrhagic disease in California black-tailed deer. In this study, we describe the cultural, physicochemical and serological characteristics of a virus isolated from lung using neonatal white-tail deer lung and turbinate cell cultures. The virus had the cultural, morphological and physicochemical characteristics of members of the Adenoviridae family. The virus would not replicate in low passage fetal bovine, caprine or ovine cells. Antiserum to the deer adenovirus, strain D94-2569, neutralized bovine adenovirus type-6 (BAdV-6), BAdV-7, and caprine adenovirus type-1 (GAdV-1). Antiserum to BAdV-6 did not neutralize the deer adenovirus but antiserum to BAdV-7 and GAdV-1 neutralized the deer adenovirus. Cross-neutralization with the other bovine, caprine and ovine adenovirus species was not observed. Restriction endonuclease patterns generated for the deer adenovirus were unique compared to those for the currently recognized bovine, caprine and ovine adenovirus types. Amino acid sequence alignments of the hexon gene from the deer adenovirus strain D94-2569 indicate that it is a member of the proposed new genus (Atadenovirus) of the Adenoviridae family. While closely related antigenically to BAdV-7 and GAdV-1, the deer adenovirus appears sufficiently distinct culturally and molecularly to justify consideration as a new adenovirus type.

  3. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Severe Infections with Human Adenovirus 7d in 2 Adults in Family, Illinois, USA, 2014

    PubMed Central

    Ison, Michael G.

    2016-01-01

    Human adenovirus 7d, a genomic variant with no reported circulation in the United States, was isolated from 2 adults with severe respiratory infections in Illinois. Molecular typing identified a close relationship with strains of the same genome type isolated from cases of respiratory disease in several provinces of China since 2009. PMID:26982199

  5. 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 Adenovirus...

  6. 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 Adenovirus...

  7. 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 Adenovirus...

  8. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 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 Adenovirus...

  9. 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 Adenovirus...

  10. Prolonged peritoneal gene expression using a helper-dependent adenovirus.

    PubMed

    Liu, Limin; Shi, Chang-Xin; Ghayur, Ayesha; Zhang, Claire; Su, Je Yen; Hoff, Catherine M; Margetts, Peter J

    2009-01-01

    Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis. The causes of EPS are not well defined and are likely multifactorial. A suitable animal model would facilitate research into the pathophysiology and treatment of EPS. We developed a helper-dependent adenovirus that expresses both green fluorescent protein (GFP) and active transforming growth factor-beta (TGF-beta1; HDAdTGF-beta1). Mice were administered HDAdTGF-beta1 via intraperitoneal injection and the response was compared with mice administered either first-generation adenovirus expressing TGF-beta1 (AdTGF-beta1) or control adenovirus (AdGFP). HDAdTGF-beta1-treated mice continued to express the GFP reporter transgene to day 74, the end of the observation period. Transgene expression lasted less than 28 days in the animals treated with first-generation adenoviruses. Animals treated with first-generation AdTGF-beta1 demonstrated submesothelial thickening and angiogenesis at day 7, with almost complete resolution by day 28. The HDAdTGF-beta1-treated mice demonstrated progressive peritoneal fibrosis with adhesion formation and encapsulation of bowels. Weight gain was significantly reduced in animals treated with HDAdTGF-beta1 compared to both the control-treated animals and the AdTGF-beta1-treated animals. Inflammation was not a major component of the fibroproliferative response. Peritoneal administration of a first-generation AdTGF-beta1 leads to transient gene expression, resulting in a resolving fibrotic response and histology similar to that seen in simple peritoneal sclerosis. Prolonged TGF-beta1 expression induced by the helper-dependent HDAdTGF-beta1 led to changes in peritoneal morphology resembling EPS. This suggests that TGF-beta1 may be a contributing factor in both simple peritoneal sclerosis and EPS. This model will be useful for elucidation of the mechanism of EPS and evaluation of potential treatment.

  11. E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus.

    PubMed

    Thomas, Michael A; Broughton, Robin S; Goodrum, Felicia D; Ornelles, David A

    2009-03-01

    Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

  12. Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes▿ §

    PubMed Central

    Xu, Ning; Segerman, Bo; Zhou, Xiaofu; Akusjärvi, Göran

    2007-01-01

    Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3′ strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes. PMID:17652395

  13. Homologous and heterologous recombination between adenovirus vector DNA and chromosomal DNA.

    PubMed

    Stephen, Sam Laurel; Sivanandam, Vijayshankar Ganesh; Kochanek, Stefan

    2008-11-01

    Adenovirus vector DNA is perceived to remain as episome following gene transfer. We quantitatively and qualitatively analysed recombination between high capacity adenoviral vector (HC-AdV) and chromosomal DNA following gene transfer in vitro. We studied homologous and heterologous recombination with a single HC-AdV carrying (i) a large genomic HPRT fragment with the HPRT CHICAGO mutation causing translational stop upon homologous recombination with the HPRT locus and (ii) a selection marker to allow for clonal selection in the event of heterologous recombination. We analysed the sequences at the junctions between vector and chromosomal DNA. In primary cells and in cell lines, the frequency of homologous recombination ranged from 2 x 10(-5) to 1.6 x 10(-6). Heterologous recombination occurred at rates between 5.5 x 10(-3) and 1.1 x 10(-4). HC-AdV DNA integrated via the termini mostly as intact molecules. Analysis of the junction sequences indicated vector integration in a relatively random manner without an obvious preference for particular chromosomal regions, but with a preference for integration into genes. Integration into protooncogenes or tumor suppressor genes was not observed. Patchy homologies between vector termini and chromosomal DNA were found at the site of integration. Although the majority of integrations had occurred without causing mutations in the chromosomal DNA, cases of nucleotide substitutions and insertions were observed. In several cases, deletions of even relative large chromosomal regions were likely. These results extend previous information on the integration patterns of adenovirus vector DNA and contribute to a risk-benefit assessment of adenovirus-mediated gene transfer.

  14. Removal of adenovirus, calicivirus, and bacteriophages by conventional drinking water treatment.

    PubMed

    Abbaszadegan, Morteza; Monteiro, Patricia; Nwachuku, Nena; Alum, Absar; Ryu, Hodon

    2008-02-01

    This study was conducted to evaluate the removal of adenovirus, feline calicivirus (FCV), and bacteriophages MS-2, fr, PRD-1, and Phi X-174 during conventional drinking water treatment using ferric chloride as a coagulant. Adenovirus and FCV were removed to a greater extent than PRD-1 and Phi X-174, indicating that these bacteriophages may be appropriate surrogates for both adenovirus and FCV. Of the four bacteriophages studied in the pilot plant, MS-2 was removed to the greatest extent (5.1 log), followed by fr (4.9 log), PRD-1 (3.5 log), and Phi X-174 (1.3 log). The virus removal trend in the pilot-scale testing was similar to the bench-scale testing; however, the bench-scale testing seemed to provide a conservative estimate of the pilot plant performance. In the pilot-scale testing, MS-2 and fr were removed with the greatest efficiency during filtration, whereas PRD-1 and Phi X-174 showed the greatest removal during sedimentation.

  15. 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

  16. [Adenovirus-mediated canine interferon-gamma expression and its antiviral activity against canine parvovirus].

    PubMed

    Zhang, Kao; Jin, Huijun; Zhong, Fei; Li, Xiujin; Neng, Changai; Chen, Huihui; Li, Wenyan; Wen, Jiexia

    2012-11-04

    To construct recombinant adenovirus containing canine interferon-gamma (cIFN-gamma) gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells (MDCK). [Methods] The cIFN-gamma gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-gamma expression vector, from which the cIFN-gamma expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-gamma. The pAd-cIFN-gamma plasmid was linearized by digestion and transfected into human embryonic kidney (HEK) 293T cells to generate the replication-defective cIFN-gamma recombinant adenovirus (Ad-cIFN-gamma). To analyze its anti-canine parvovirus activity, the MDCK cells were pre-infected by Ad-cIFN-gamma recombinant adenovirus, and then infected by canine parvovirus. The antiviral activity of the Ad-cIFN-gamma recombinant adenovirus against parvovirus was analyzed. The recombinant adenovirus containing cIFN-gamma gene was constructed by the ligation method. The recombinant adenovirus could mediates recombinant cIFN-gamma secretory expression in MDCK cells. The Ad-cIFN-gamma recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus. These results indicate that the Ad-cIFN-gamma recombinant adenovirus has the potent antiviral activity against canine parvovirus. The Ad-cIFN-gamma recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus.

  17. [Research advance on role of Coxsackie and adenovirus receptor (CAR) in tumor progression].

    PubMed

    Fan, Liang-Sheng; Chen, Gang; Ma, Ding

    2009-03-01

    Coxsackie and adenovirus receptor (CAR) is originally identified as the cellular receptor of 2-and 5-type adenoviruses. Many researches have suggested that CAR can affect the growth, adhesive ability and cytoskeleton of tumor cells, and has complicated functions in metastasis and invasion of tumors. Moreover, the expression of CAR has close relationship with tumor prognosis and cytoreduction mediated by adenoviruses. CAR has become a new hotspot in the research on mechanism of tumor progression and gene therapy. Our review focuses on the structure and function of CAR and its role in mediating occurrence and progression of tumor.

  18. Recent advances in genetic modification of adenovirus vectors for cancer treatment.

    PubMed

    Yamamoto, Yuki; Nagasato, Masaki; Yoshida, Teruhiko; Aoki, Kazunori

    2017-05-01

    Adenoviruses are widely used to deliver genes to a variety of cell types and have been used in a number of clinical trials for gene therapy and oncolytic virotherapy. However, several concerns must be addressed for the clinical use of adenovirus vectors. Selective delivery of a therapeutic gene by adenovirus vectors to target cancer is precluded by the widespread distribution of the primary cellular receptors. The systemic administration of adenoviruses results in hepatic tropism independent of the primary receptors. Adenoviruses induce strong innate and acquired immunity in vivo. Furthermore, several modifications to these vectors are necessary to enhance their oncolytic activity and ensure patient safety. As such, the adenovirus genome has been engineered to overcome these problems. The first part of the present review outlines recent progress in the genetic modification of adenovirus vectors for cancer treatment. In addition, several groups have recently developed cancer-targeting adenovirus vectors by using libraries that display random peptides on a fiber knob. Pancreatic cancer-targeting sequences have been isolated, and these oncolytic vectors have been shown by our group to be associated with a higher gene transduction efficiency and more potent oncolytic activity in cell lines, murine models, and surgical specimens of pancreatic cancer. In the second part of this review, we explain that combining cancer-targeting strategies can be a promising approach to increase the clinical usefulness of oncolytic adenovirus vectors. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  19. Immunizing Patients With Metastatic Melanoma Using Recombinant Adenoviruses Encoding MART-1 or gp100 Melanoma Antigens

    PubMed Central

    Rosenberg, Steven A.; Zhai, Yifan; Yang, James C.; Schwartzentruber, Douglas J.; Hwu, Patrick; Marincola, Francesco M.; Topalian, Suzanne L.; Restifo, Nicholas P.; Seipp, Claudia A.; Einhorn, Jan H.; Roberts, Bruce; White, Donald E.

    2008-01-01

    Background: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. Methods: In phase I studies, 54 patients received escalating doses (between 107 and 1011 plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. Results: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. Conclusions: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens. PMID:9862627

  20. 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.

  1. 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

  2. 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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Co-infection with human polyomavirus BK enhances gene expression and replication of human adenovirus.

    PubMed

    Bil-Lula, Iwona; Woźniak, Mieczysław

    2018-03-26

    Immunocompromised patients are susceptible to multiple viral infections. Relevant interactions between co-infecting viruses might result from viral regulatory genes which trans-activate or repress the expression of host cell genes as well as the genes of any co-infecting virus. The aim of the current study was to show that the replication of human adenovirus 5 is enhanced by co-infection with BK polyomavirus and is associated with increased expression of proteins including early region 4 open reading frame 1 and both the large tumor antigen and small tumor antigen. Clinical samples of whole blood and urine from 156 hematopoietic stem cell transplant recipients were tested. We also inoculated adenocarcinomic human alveolar basal epithelial cells with both human adenovirus 5 and BK polyomavirus to evaluate if co-infection of viruses affected their replication. Data showed that adenovirus load was significantly higher in the plasma (mean 7.5 x 10 3  ± 8.5 x 10 2 copies/ml) and urine (mean 1.9 x 10 3  ± 8.0 x 10 2 copies/ml) of samples from patients with co-infections, in comparison to samples from patients with isolated adenovirus infection. In vitro co-infection led to an increased (8.6 times) expression of the adenovirus early region 4 open reading frame gene 48 hours post-inoculation. The expression of the early region 4 open reading frame gene positively correlated with the expression of BK polyomavirus large tumor antigen (r = 0.90, p < 0.0001) and small tumor antigen (r = 0.83, p < 0.001) genes. The enhanced expression of the early region 4 open reading frame gene due to co-infection with BK polyomavirus was associated with enhanced adenovirus, but not BK polyomavirus, replication. The current study provides evidence that co-infection of adenovirus and BK polyomavirus contributes to enhanced adenovirus replication. Data obtained from this study may have significant importance in the clinical setting.

  4. 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

  5. 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).

  6. Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

    PubMed

    Chen, Weizao; Liu, Mingqiu; Jiao, Ye; Yan, Weiyao; Wei, Xuefeng; Chen, Jiulian; Fei, Liang; Liu, Yang; Zuo, Xiaoping; Yang, Fugui; Lu, Yonggan; Zheng, Zhaoxin

    2006-04-01

    Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.

  7. Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization ▿

    PubMed Central

    Frietze, Kathryn M.; Campos, Samuel K.; Kajon, Adriana E.

    2010-01-01

    Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism. PMID:20739542

  8. Detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of Benin, West Africa.

    PubMed

    Verheyen, Jens; Timmen-Wego, Monika; Laudien, Rainer; Boussaad, Ibrahim; Sen, Sibel; Koc, Aynur; Uesbeck, Alexandra; Mazou, Farouk; Pfister, Herbert

    2009-05-01

    Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions.

  9. 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

  10. 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

  11. Characterization of a New Species of Adenovirus in Falcons

    PubMed Central

    Schrenzel, Mark; Oaks, J. Lindsay; Rotstein, Dave; Maalouf, Gabriel; Snook, Eric; Sandfort, Cal; Rideout, Bruce

    2005-01-01

    In 1996, a disease outbreak occurred at a captive breeding facility in Idaho, causing anorexia, dehydration, and diarrhea or sudden death in 72 of 110 Northern aplomado falcons (Falco femoralis septentrionalis) from 9 to 35 days of age and in 6 of 102 peregrine falcons (Falco peregrinus) from 14 to 25 days of age. Sixty-two Northern aplomado and six peregrine falcons died. Epidemiologic analyses indicated a point source epizootic, horizontal transmission, and increased relative risk associated with cross-species brooding of eggs. Primary lesions in affected birds were inclusion body hepatitis, splenomegaly, and enteritis. The etiology in all mortalities was determined by molecular analyses to be a new species of adenovirus distantly related to the group I avian viruses, serotypes 1 and 4, Aviadenovirus. In situ hybridization and PCR demonstrated that the virus was epitheliotropic and lymphotropic and that infection was systemic in the majority of animals. Adeno-associated virus was also detected by PCR in most affected falcons, but no other infectious agents or predisposing factors were found in any birds. Subsequent to the 1996 epizootic, a similar disease caused by the same adenovirus was found over a 5-year period in orange-breasted falcons (Falco deiroleucus), teita falcons (Falco fasciinucha), a merlin (Falco columbarius), a Vanuatu peregrine falcon (Falco peregrinus nesiotes), and gyrfalcon × peregrine falcon hybrids (Falco rusticolus/peregrinus) that died in Wyoming, Oklahoma, Minnesota, and California. These findings indicate that this newly recognized adenovirus is widespread in western and midwestern North America and can be a primary pathogen in different falcon species. PMID:16000466

  12. Prime-boost immunization by both DNA vaccine and oncolytic adenovirus expressing GM-CSF and shRNA of TGF-β2 induces anti-tumor immune activation

    PubMed Central

    Choi, Hye Jin; Joo, Yeonsoo; Kim, Joo-Hang; Song, Jae J.

    2017-01-01

    A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-β2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-β2 (shmTGF-β2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-β2 (AdGshT) with that of oncolytic adenovirus expressing mGM-CSF only (AdG), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received AdGshT showed delayed tumor growth than those that received AdG. Heterologous prime-boost immunization was combined with oncolytic AdGshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-β2, tumor growth inhibition by shmTGF-β2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse

  13. Prime-boost immunization by both DNA vaccine and oncolytic adenovirus expressing GM-CSF and shRNA of TGF-β2 induces anti-tumor immune activation.

    PubMed

    Kim, So Young; Kang, Dongxu; Choi, Hye Jin; Joo, Yeonsoo; Kim, Joo-Hang; Song, Jae J

    2017-02-28

    A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-β2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-β2 (shmTGF-β2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-β2 (AdGshT) with that of oncolytic adenovirus expressing mGM-CSF only (AdG), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received AdGshT showed delayed tumor growth than those that received AdG. Heterologous prime-boost immunization was combined with oncolytic AdGshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-β2, tumor growth inhibition by shmTGF-β2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse

  14. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

    PubMed Central

    Harter, M L; Shanmugam, G; Wold, W S; Green, M

    1976-01-01

    (35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization. Images PMID:950686

  16. Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

    PubMed

    Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

    2008-04-01

    Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

  17. 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.

  18. Transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting.

    PubMed

    Russell, Kevin L; Broderick, Michael P; Franklin, Suzanne E; Blyn, Lawrence B; Freed, Nikki E; Moradi, Emily; Ecker, David J; Kammerer, Peter E; Osuna, Miguel A; Kajon, Adriana E; Morn, Cassandra B; Ryan, Margaret A K

    2006-10-01

    High levels of morbidity caused by adenovirus among US military recruits have returned since the loss of adenovirus vaccines in 1999. The transmission dynamics of adenovirus have never been well understood, which complicates prevention efforts. Enrollment and end-of-study samples were obtained and active surveillance for febrile respiratory illnesses (FRIs) was performed for 341 recruits and support personnel. Environmental samples were collected simultaneously. Classic and advanced diagnostic techniques were used. Seventy-nine percent (213/271) of new recruits were seronegative for either adenovirus serotype 4 (Ad-4) or adenovirus serotype 7 (Ad-7). FRI caused by Ad-4 was observed in 25% (67/271) of enrolled recruits, with 100% of them occurring in individuals with enrollment titers <1 : 4. The percentage of recruits seropositive for Ad-4 increased from 34% at enrollment to 97% by the end of the study. Adenovirus was most commonly detected in the environment on pillows, lockers, and rifles. Potential sources of adenovirus transmission among US military recruits included the presence of adenovirus on surfaces in living quarters and extended pharyngeal viral shedding over the course of several days. The introduction of new recruits, who were still shedding adenovirus, into new training groups was documented. Serological screening could identify susceptible recruits for the optimal use of available vaccines. New high-throughput technologies show promise in providing valuable data for clinical and research applications.

  19. 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).

  20. Covalent decoration of adenovirus vector capsids with the carbohydrate epitope αGal does not improve vector immunogenicity, but allows to study the in vivo fate of adenovirus immunocomplexes.

    PubMed

    Kratzer, Ramona F; Espenlaub, Sigrid; Hoffmeister, Andrea; Kron, Matthias W; Kreppel, Florian

    2017-01-01

    Adenovirus-based vectors are promising tools for genetic vaccination. However, several obstacles have to be overcome prior to a routine clinical application of adenovirus-based vectors as efficacious vectored vaccines. The linear trisaccharide epitope αGal (alpha-Gal) with the carbohydrate sequence galactose-α-1,3-galactosyl-β-1,4-N-acetylglucosamine has been described as a potent adjuvant for recombinant or attenuated vaccines. Humans and α-1,3-galactosyltransferase knockout mice do not express this epitope. Upon exposure of α-1,3-galactosyltransferase-deficient organisms to αGal in the environment, large amounts of circulating anti-Gal antibodies are produced consistently. Immunocomplexes formed between recombinant αGal-decorated vaccines and anti-Gal antibodies exhibit superior immunogenicity. We studied the effects of the trisaccharide epitope on CD8 T cell responses that are directed specifically to vector-encoded transgenic antigens. For that, covalently αGal-decorated adenovirus vectors were delivered to anti-Gal α-1,3-galactosyltransferase knockout mice. We generated replication-defective, E1-deleted adenovirus type 5 vectors that were decorated with αGal at the hexon hypervariable regions 1 or 5, at fiber knob, or at penton base. Surprisingly, none of the adenovirus immunocomplexes being formed from αGal-decorated adenovirus vectors and anti-Gal immunoglobulins improved the frequencies of CD8 T cell responses against the transgenic antigen ovalbumin. Humoral immunity directed to the adenovirus vector was neither increased. However, our data indicated that decoration of Ad vectors with the αGal epitope is a powerful tool to analyze the fate of adenovirus immunocomplexes in vivo.

  1. Targeted delivery of CYP2E1 recombinant adenovirus to malignant melanoma by bone marrow-derived mesenchymal stem cells as vehicles.

    PubMed

    Wang, Jishi; Ma, Dan; Li, Yan; Yang, Yuan; Hu, Xiaoyan; Zhang, Wei; Fang, Qin

    2014-03-01

    The aim of this study was to explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) as intermediate carriers on targeting of P450 gene recombinant adenovirus to malignant melanoma in vitro and in vivo. BMSCs were transduced with pAd5-CMV-CYP2E1 recombinant adenovirus. BMSC migration was detected by Transwell plates in vitro and by superparamagnetic iron oxide particles in vivo. Growth-inhibitory effect and apoptosis were determined by MTT and immunity fluorescence staining. Anticancer effects were examined by a human melanoma nude mouse model in vivo. BMSCs moved toward A375 cells in Transwell plates. Numerous superparamagnetic MSCs labeled with iron oxide were identified in the peripheral areas of the tumor, but were detected in primary organs by Prussian blue staining. BMSC-CYP2E1 cells mediated a bystander killing effect on CYP2E1-negative A375 cells during coculture (IC50 values for A375 cells cocultured with BMSC-EGFP and BMSC-CYP2E1 were 4.08 and 2.68 mmol/l, respectively). Intravenously injecting CYP2E1 recombinant adenovirus-loaded BMSCs in mice with established human melanoma managed to target the tumor site, and BMSCs with forced expression of CYP2E1 inhibited the growth of malignant cells in vivo by activating 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. BMSCs may serve as a platform of P450 gene-directed enzyme prodrug therapy for the delivery of chemotherapeutic prodrugs to tumors.

  2. The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

    PubMed Central

    Martinez, Ruben; Schellenberger, Pascale; Vasishtan, Daven; Aknin, Cindy; Austin, Sisley; Dacheux, Denis; Rayne, Fabienne; Siebert, Alistair; Ruzsics, Zsolt; Gruenewald, Kay

    2014-01-01

    ABSTRACT Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. PMID:25473051

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

    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)more » 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.« less

  4. Hexon and fiber of adenovirus type 14 and 55 are major targets of neutralizing antibody but only fiber-specific antibody contributes to cross-neutralizing activity.

    PubMed

    Feng, Ying; Sun, Xikui; Ye, Xianmiao; Feng, Yupeng; Wang, Jinlin; Zheng, Xuehua; Liu, Xinglong; Yi, Changhua; Hao, Mingli; Wang, Qian; Li, Feng; Xu, Wei; Li, Liang; Li, Chufang; Zhou, Rong; Chen, Ling; Feng, Liqiang

    2018-05-01

    Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Developing Novel Oncolytic Adenoviruses through Bioselection

    PubMed Central

    Yan, Wen; Kitzes, Galila; Dormishian, Farid; Hawkins, Lynda; Sampson-Johannes, Adam; Watanabe, Josh; Holt, Jenny; Lee, Vivian; Dubensky, Thomas; Fattaey, Ali; Hermiston, Terry; Balmain, Allan; Shen, Yuqiao

    2003-01-01

    Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed. PMID:12552003

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

    PubMed Central

    Gallaher, Sean D.; Berk, Arnold J.

    2013-01-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity. PMID:23624118

  7. Molecular Typing of Clinical Adenovirus Specimens by an Algorithm which Permits Detection of Adenovirus Coinfections and Intermediate Adenovirus Strains

    PubMed Central

    McCarthy, Troy; Lebeck, Mark G.; Capuano, Ana W.; Schnurr, David P.; Gray, Gregory C.

    2009-01-01

    Background Epidemiological data suggest that clinical outcomes of human adenovirus (HAdV) infection may be influenced by virus serotype, coinfection with multiple strains, or infection with novel intermediate strains. In this report, we propose a clinical algorithm for detecting HAdV coinfection and intermediate strains. Study Design We PCR amplified and sequenced subregions of the hexon and fiber genes of 342 HAdV positive clinical specimens obtained from 14 surveillance laboratories. Sequences were then compared with those from 52 HAdV prototypic strains. HAdV positive specimens that showed nucleotide sequence identity with a corresponding prototype strain were designated as being of that strain. When hexon and fiber gene sequences disagreed, or sequence identity was low, the specimens were further characterized by viral culture, plaque purification, repeat PCR with sequencing, and genome restriction enzyme digest analysis. Results Of the 342 HAdV-positive clinical specimens, 328 (95.9%) were single HAdV strain infections, 12 (3.5%) were coinfections, and 2 (0.6%) had intermediate strains. Coinfected specimens and intermediate HAdV strains considered together were more likely to be associated with severe illness compared to other HAdv-positive specimens (OR=3.8; 95% CI = 1.2–11.9). Conclusions The majority of severe cases of HAdV illness cases occurred among immunocompromised patients. The analytic algorithm we describe here can be used to screen clinical specimens for evidence of HAdV coinfection and novel intermediate HAdV strains. This algorithm may be especially useful in investigating HAdV outbreaks and clusters of unusually severe HAdV disease. PMID:19577957

  8. Effect of Exposure to UV-C Irradiation and Monochloramine on Adenovirus Serotype 2 Early Protein Expression and DNA Replication▿

    PubMed Central

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

    2008-01-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20°C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants. PMID:18424543

  9. Pre-Transplant Screening for Latent Adenovirus in Donors and Recipients

    PubMed Central

    Piatti, Gabriella

    2016-01-01

    Human adenoviruses are frequent cause of slight self-limiting infections in immune competent subjects, while causing life-threatening and disseminated diseases in immunocompromised patients, particularly in the subjects affected by acquired immunodeficiency syndrome and in bone marrow and organ transplant recipients. Here, infections interest lungs, liver, encephalon, heart, kidney and gastro enteric tract. To date, human adenoviruses comprise 51 serotypes grouped into seven species, among which species C especially possesses the capability to persist in infected tissues. From numerous works, it emerges that in the recipient, because of loss of immune-competence, both primary infection, via the graft or from the environment, and reactivated endogenous viruses can be responsible for transplantation related adenovirus disease. The transplants management should include the evaluation of anti-adenovirus pre-transplant screening similar to that concerning cytomegalovirus. The serological screening on cytomegalovirus immunity is currently performed to prevent viral reactivation from grafts and recipient, the viral spread and dissemination to different organs and apparatus, and potentially lethal outcome. PMID:27006724

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

    PubMed

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

    2017-01-15

    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. 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 promise to be more potent

  11. 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

  12. 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

  13. Species-Specific Identification of Human Adenoviruses in Sewage.

    PubMed

    Wieczorek, Magdalena; Krzysztoszek, Arleta; Witek, Agnieszka

    2015-01-01

    Human adenovirus (HAdV) diversity in sewage was assessed by species-specific molecular methods. Samples of raw sewage were collected in 14 sewage disposal systems from January to December 2011, in Poland. HAdVs were detected in 92.1% of the analysed sewage samples and was significantly higher at cities of over 100 000 inhabitants. HAdV DNA was detected in sewage during all seasons. The most abundant species identified were HAdV-F (average 89.6%) and -A (average 19.6%), which are associated with intestine infections. Adenoviruses from B species were not detected. The result of the present study demonstrate that human adenoviruses are consistently present in sewage in Poland, demonstrating the importance of an adequate treatment before the disposal in the environment. Multiple HAdV species identified in raw sewage provide new information about HAdV circulation in the Polish population.

  14. High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines.

    PubMed

    Wright, N; Jackson, F R; Niezgoda, M; Ellison, J A; Rupprecht, C E; Nel, L H

    2013-08-28

    Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

    PubMed

    Kuboki, T; Nakanishi, T; Kanyama, M; Sonoyama, W; Fujisawa, T; Kobayashi, K; Ikeda, T; Kubo, T; Yamashita, A; Takigawa, M

    1999-09-01

    Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint

  16. Getting genetic access to natural adenovirus genomes to explore vector diversity.

    PubMed

    Zhang, Wenli; Ehrhardt, Anja

    2017-10-01

    Recombinant vectors based on the human adenovirus type 5 (HAdV5) have been developed and extensively used in preclinical and clinical studies for over 30 years. However, certain restrictions of HAdV5-based vectors have limited their clinical applications because they are rather inefficient in specifically transducing cells of therapeutic interest that lack the coxsackievirus and adenovirus receptor (CAR). Moreover, enhanced vector-associated toxicity and widespread preexisting immunity have been shown to significantly hamper the effectiveness of HAdV-5-mediated gene transfer. However, evolution of adenoviruses in the natural host is driving the generation of novel types with altered virulence, enhanced transmission, and altered tissue tropism. As a consequence, an increasing number of alternative adenovirus types were identified, which may represent a valuable resource for the development of novel vector types. Thus, researchers are focusing on the other naturally occurring adenovirus types, which are structurally similar but functionally different from HAdV5. To this end, several strategies have been devised for getting genetic access to adenovirus genomes, resulting in a new panel of adenoviral vectors. Importantly, these vectors were shown to have a host range different from HAdV5 and to escape the anti-HAdV5 immune response, thus underlining the great potential of this approach. In summary, this review provides a state-of-the-art overview of one essential step in adenoviral vector development.

  17. Therapeutic Effect of Recombinant Adenovirus Encoding Interferon-γ in a Murine Model of Progressive Pulmonary Tuberculosis.

    PubMed

    Mata-Espinosa, Dulce A; Mendoza-Rodríguez, Valentin; Aguilar-León, Diana; Rosales, Ricardo; López-Casillas, Fernando; Hernández-Pando, Rogelio

    2008-06-01

    We constructed recombinant adenoviruses encoding murine interferon-γ (AdIFNγ) and tested its therapeutic efficiency in a well characterized model of progressive pulmonary tuberculosis (TB) in Balb/c mice, infected through the trachea with the laboratory drug-susceptible H37Rv strain or multidrug-resistant (MDR) clinical isolate. When the disease was in a late phase, 2 months after infection, we administered by intratracheal cannulation a single dose [1.7 × 10 9 plaque forming units (pfu)] of AdIFNγ or the control adenovirus. Groups of mice were killed at different time-points and the lungs were examined to determine bacilli colony forming units (CFU), cytokine/chemokine gene expression, and CD4/CD8 subpopulations, and also subjected to automated histomorphometry. In comparison with the control group, after 2 weeks of treatment and during the next 6 months, AdIFNγ-treated animals infected with either the H37Rv strain or the MDR strain showed significantly lower bacilli loads and tissue damage (pneumonia), higher expressions of IFN-γ, tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS), and bigger granulomas. When compared with the results from conventional chemotherapy or AdIFNγ treatment alone, the combined treatment with AdIFNγ plus conventional chemotherapy shortened the time taken for reduction of bacillary load. This shows that gene therapy with AdIFNγ efficiently reconstituted the protective immune response and controlled the progress of pulmonary TB produced by MDR or non-MDR strains. Copyright © 2008 The American Society of Gene Therapy. Published by Elsevier Inc. All rights reserved.

  18. 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

  19. Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

    PubMed Central

    Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

    2000-01-01

    We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

  20. 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.

  1. 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

  2. Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

    PubMed

    Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

    2013-10-17

    Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

  3. Adenovirus Core Protein VII Downregulates the DNA Damage Response on the Host Genome

    PubMed Central

    Avgousti, Daphne C.; Della Fera, Ashley N.; Otter, Clayton J.; Herrmann, Christin; Pancholi, Neha J.

    2017-01-01

    ABSTRACT Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin. IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam

    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 studymore » 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.« less

  5. The climatic context of major plague outbreaks in late medieval England

    NASA Astrophysics Data System (ADS)

    Pribyl, Kathleen

    2017-04-01

    The climatological triggers of major plague outbreaks in late medieval and early modern Europe remain unclear; recent studies have been inconclusive. Plague is primarily a rodent disease and due to the involvement of rodent hosts and insect vectors, the epidemiology of plague is complicated, but research on outbreaks in the Third Pandemic, which began in the late nineteenth century, has shown that in central and eastern Asia plague is linked to specific meteorological conditions. The disease adapts to a varied spectrum of ecological and climatological settings, which influence the development of plague waves, and due to Europe's geographical diversity, this paper focuses on one region, England, in its search for meteorological parameters contributing to plague outbreaks. The study period of this paper is defined by the arrival of Yersinia pestis in the British Isles in 1348 and the end of the fifteenth century. During this time, England's population dynamics were mortality-driven due to recurrent epidemic disease; and public health measures, such as quarantining, had not yet been introduced, hence the influence of social factors on the formation of major plague waves was very limited. The geographical and temporal focus of this study allows for the combination of the series of English major plague outbreaks, verified in the original texts, with the high-quality climate reconstructions based on both documentary sources and proxy data available for this region. The detailed analysis of the mechanisms contributing to English plague waves presented in this paper, reveals a complex interplay of time-lag responses and concurrent conditions involving temperature and precipitation parameters.

  6. Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells.

    PubMed

    Turner, Mallory A; Middha, Sumit; Hofherr, Sean E; Barry, Michael A

    2015-12-01

    Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development. A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head

  7. Atomic Structures of Minor Proteins VI and VII in the Human Adenovirus.

    PubMed

    Dai, Xinghong; Wu, Lily; Sun, Ren; Zhou, Z Hong

    2017-10-04

    Human adenoviruses (Ad) are dsDNA viruses associated with infectious diseases, yet better known as tools for gene delivery and oncolytic anti-cancer therapy. Atomic structures of Ad provide the basis for the development of antivirals and for engineering efforts towards more effective applications. Since 2010, atomic models of human Ad5 have been independently derived from photographic film cryoEM and X-ray crystallography, but discrepancies exist concerning the assignment of cement proteins IIIa, VIII and IX. To clarify these discrepancies, here we have employed the technology of direct electron-counting to obtain a cryoEM structure of human Ad5 at 3.2 Å resolution. Our improved structure unambiguously confirmed our previous cryoEM models of proteins IIIa, VIII and IX and explained the likely cause of conflict in the crystallography models. The improved structure also allows the identification of three new components in the cavities of hexons - the cleaved N-terminus of precursor protein VI (pVIn), the cleaved N-terminus of precursor protein VII (pVIIn2), and mature protein VI. The binding of pVIIn2--by extension that of genome-condensing pVII--to hexons is consistent with the previously proposed dsDNA genome-capsid co-assembly for adenoviruses, which resembles that of ssRNA viruses but differs from the well-established mechanism of pumping dsDNA into a preformed protein capsid, as exemplified by tailed bacteriophages and herpesviruses. IMPORTANCE Adenovirus is a double-edged sword to humans - as a widespread pathogen and a bioengineering tool for anti-cancer and gene therapy. Atomic structure of the virus provides the basis for antiviral and application developments, but conflicting atomic models from conventional/film cryoEM and X-ray crystallography for important cement proteins IIIa, VIII, and IX have caused confusion. Using the cutting-edge cryoEM technology with electron counting, we improved the structure of human adenovirus type 5 and confirmed our

  8. Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells

    PubMed Central

    Hidalgo, Paloma; Anzures, Lourdes; Hernández-Mendoza, Armando; Guerrero, Adán; Wood, Christopher D.; Valdés, Margarita; Dobner, Thomas

    2016-01-01

    ABSTRACT Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCE RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the

  9. Production of recombinant adenovirus containing human interlukin-4 gene.

    PubMed

    Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

    2011-11-01

    Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

  10. Cancer-Targeted Oncolytic Adenoviruses for Modulation of the Immune System.

    PubMed

    Cerullo, Vincenzo; Capasso, Cristian; Vaha-Koskela, Markus; Hemminki, Otto; Hemminki, Akseli

    2018-01-01

    Adenovirus is one of the most commonly used vectors for gene therapy and it is the first approved virus-derived drug for treatment of cancer. As an oncolytic agent, it can induce lysis of infected cells, but it can also engage the immune system, promoting activation and maturation of antigen- presenting cells (APCs). In essence, oncolysis combined with the associated immunostimulatory actions result in a "personalized in situ vaccine" for each patient. In order to take full advantage of these features, we should try to understand how adenovirus interacts with the immune system, what are the receptors involved in triggering subsequent signals and which kind of responses they elicit. Tackling these questions will give us further insight in how to manipulate adenovirus-mediated immune responses for enhancement of anti-tumor efficacy. In this review, we first highlight how oncolytic adenovirus interacts with the innate immune system and its receptors such as Toll-like receptors, nucleotide-binding and oligomerization domain (NOD)- like receptors and other immune sensors. Then we describe the effect of these interactions on the adaptive immune system and its cells, especially B and T lymphocytes. Finally, we summarize the most significant preclinical and clinical results in the field of gene therapy where researchers have engineered adenovirus to manipulate the host immune system by expressing cytokines and signalingmediators. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Adenovirus, MS2 and PhiX174 interactions with drinking water biofilms developed on PVC, cement and cast iron.

    PubMed

    Helmi, K; Menard-Szczebara, F; Lénès, D; Jacob, P; Jossent, J; Barbot, C; Delabre, K; Arnal, C

    2010-01-01

    Biofilms colonizing pipe surfaces of drinking water distribution systems could provide habitat and shelter for pathogenic viruses present in the water phase. This study aims (i) to develop a method to detect viral particles present in a drinking water biofilm and (ii) to study viral interactions with drinking water biofilms. A pilot scale system was used to develop drinking water biofilms on 3 materials (7 cm(2) discs): PVC, cast iron and cement. Biofilms were inoculated with viral model including MS2, PhiX174 or adenovirus. Five techniques were tested to recover virus from biofilms. The most efficient uses beef extract and glycine at pH = 9. After sonication and centrifugation, the pH of the supernatant is neutralized prior to viral analysis. The calculated recovery rates varied from 29.3 to 74.6% depending on the virus (MS2 or PhiX174) and the material. Applying this protocol, the interactions of virus models (MS2 and adenovirus) with drinking water biofilms were compared. Our results show that adsorption of viruses to biofilms depends on their isoelectric points, the disc material and the hydrodynamic conditions. Applying hydrodynamic conditions similar to those existing in drinking water networks resulted in a viral adsorption corresponding to less than 1% of the initial viral load.

  12. p53-dependent cell death/apoptosis is required for a productive adenovirus infection.

    PubMed

    Hall, A R; Dix, B R; O'Carroll, S J; Braithwaite, A W

    1998-09-01

    The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity. One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters. This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process. Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication. However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells. These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication. However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein. These studies indicate that the hypothesis may be an oversimplification. Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect. This correlates with the ability of adenovirus to induce cell death. Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection. In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy.

  13. 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

  14. Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus.

    PubMed

    Zhi, Yan; Figueredo, Joanita; Kobinger, Gary P; Hagan, Heather; Calcedo, Roberto; Miller, James R; Gao, Guangping; Wilson, James M

    2006-05-01

    Replication-deficient human adenovirus type 5 (AdH5) vectors can induce strong transgene product-specific cellular and humoral responses. However, many adult humans have neutralizing antibodies (NAbs) against AdH5 as a result of natural infection with this virus. Therefore, a chimpanzee adenovirus C7 (AdC7) vector was developed to circumvent interference by preexisting immunity to AdH5. This study evaluated the impact of preexisting immunity to human adenovirus on the efficacy of adenovirus-based vaccines against the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). Efficacy was assessed after intramuscular injection of the vector into mice and was measured as the frequency of SARS-CoV-specific T cells and NAbs against SARS-CoV. Immunogenicity of the AdH5-based vaccine was significantly attenuated or completely abolished when the preexisting anti-AdH5 NAb titer was higher than 40. Because 27% of human serum samples from the United States tested so far have an anti-AdH5 NAb titer higher than 40, our results suggested that a significant percentage of humans with preexisting anti-AdH5 immunity would not be candidates for vaccination with an AdH5-based genetic vaccine. In contrast, preexisting anti-AdH5 NAbs have a minimal effect on the potency of the AdC7-based genetic vaccine. Taken together, our studies warrant the further development of AdC7 as a vaccine carrier for human trials.

  15. Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

    PubMed Central

    Cheetham, B F; Shaw, D C; Bellett, A J

    1982-01-01

    Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation. PMID:7177112

  16. Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

    PubMed

    Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

    2017-08-15

    MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

  17. [Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

    PubMed

    Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

    2015-08-01

    To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

  18. Spread of Adenovirus to Geographically Dispersed Military Installations, May-October 2007

    DTIC Science & Technology

    2010-05-01

    and enterovirus after additional evaluation. Tube cultures were examined for 10 days for cytopathologic effects, and cells from the shell vial...respiratory syncytial virus (2 [0.4%]), and enterovirus (1 [0.2%]). Among the specimens that were culture posi- tive for adenovirus, 358 (86.7%) were

  19. Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA.

    PubMed

    Liu, Xiongtao; Yang, Jian; Shang, Fujun; Hong, Changming; Guo, Wangang; Wang, Bing; Zheng, Qiangsun

    2009-07-01

    GIRK4 has been shown to be a subunit of I(KACh), and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, I(KACh) densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased I(KACh) densities. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.

  20. EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

    PubMed Central

    Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

    2013-01-01

    We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

  1. Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

    PubMed

    Hu, Jianpeng; Xuan, Xujun; Han, Conghui; Hao, Lin; Zhang, Peiying; Chen, Meng; He, Houguang; Fan, Tao; Dong, Binzheng

    2012-03-01

    To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

  2. 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). © 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.

  3. Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells.

    PubMed

    Marshall, G S; Fenger, D P; Stout, G G; Knights, M E; Hunt, L A

    1996-07-01

    Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

  4. Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo.

    PubMed

    Sapet, Cédric; Pellegrino, Christophe; Laurent, Nicolas; Sicard, Flavie; Zelphati, Olivier

    2012-05-01

    Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

  5. Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

    PubMed

    Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

    2017-12-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

  6. A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever.

    PubMed

    Sun, Yuan; Li, Hong-Yu; Tian, Da-Yong; Han, Qiu-Ying; Zhang, Xin; Li, Na; Qiu, Hua-Ji

    2011-10-26

    Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2014-08-01

    The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Respiratory adenovirus-like infection in a rose-ringed parakeet (Psittacula krameri).

    PubMed

    Desmidt, M; Ducatelle, R; Uyttebroek, E; Charlier, G; Hoorens, J

    1991-01-01

    Intranuclear inclusions were observed under light microscopy in the bronchial epithelial cells of a recently purchased female rose-ringed parakeet that died of chlamydiosis. Transmission electron microscopy revealed the presence of numerous particles of adenovirus morphology. A latent adenovirus infection may have become more severe following chlamydiosis and the stress of handling.

  9. 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. © 2016 Japanese Dermatological Association.

  10. Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

    PubMed Central

    Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

    2011-01-01

    Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. Materials and Methods In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Results Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. Conclusion This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics. PMID:23493491

  11. Cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony.

    PubMed

    Chen, Eunice C; Yagi, Shigeo; Kelly, Kristi R; Mendoza, Sally P; Tarara, Ross P; Canfield, Don R; Maninger, Nicole; Rosenthal, Ann; Spinner, Abigail; Bales, Karen L; Schnurr, David P; Lerche, Nicholas W; Chiu, Charles Y

    2011-07-01

    Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing <57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals (2/81, or 2.5%). These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses

  12. [Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

    PubMed

    Cao, Yi-zhan; Hao, Chun-qiu; Feng, Zhi-hua; Zhou, Yong-xing; Li, Jin-ge; Jia, Zhan-sheng; Wang, Ping-zhong

    2003-02-01

    To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

  13. 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.

  14. Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin.

    PubMed

    Skog, Johan; Mei, Ya-Fang; Wadell, Göran

    2002-06-01

    Most currently used adenovirus vectors are based upon adenovirus serotypes 2 and 5 (Ad2 and Ad5), which have limited efficiencies for gene transfer to human neural cells. Both serotypes bind to the known adenovirus receptor, CAR (coxsackievirus and adenovirus receptor), and have restricted cell tropism. The purpose of this study was to find vector candidates that are superior to Ad5 in infecting human neural tumours. Using flow cytometry, the vector candidates Ad4p, Ad11p and Ad17p were compared to the commonly used adenovirus vector Ad5v for their binding capacity to neural cell lines derived from glioblastoma, medulloblastoma and neuroblastoma cell lines. The production of viral structural proteins and the CAR-binding properties of the different serotypes were also assessed in these cells. Computer-based models of the fibre knobs of Ad4p and Ad17 were created based upon the crystallized fibre knob structure of adenoviruses and analysed for putative receptor-interacting regions that differed from the fibre knob of Ad5. The non CAR-binding vector candidate Ad11p showed clearly the best binding capacity to all of the neural cell lines, binding more than 90% of cells of all of the neural cell lines tested, in contrast to 20% or less for the commonly used vector Ad5v. Ad4p and Ad11p were also internalized and produced viral proteins more successfully than Ad5. Ad4p showed a low binding ability but a very efficient capacity for infection in cell culture. Ad17p virions neither bound or efficiently infected any of the neural cell lines studied.

  15. Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes.

    PubMed

    Peng, Zhi; Luo, Hong-Wei; Yuan, Ying; Shi, Jing; Huang, Shi-Feng; Li, Chun-Li; Cao, Wei-Xi; Huang, Zong-Gan; Feng, Wen-Li

    2011-05-01

    The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

  16. Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

    PubMed

    Maler, Mareike D; Nielsen, Peter J; Stichling, Nicole; Cohen, Idan; Ruzsics, Zsolt; Wood, Connor; Engelhard, Peggy; Suomalainen, Maarit; Gyory, Ildiko; Huber, Michael; Müller-Quernheim, Joachim; Schamel, Wolfgang W A; Gordon, Siamon; Jakob, Thilo; Martin, Stefan F; Jahnen-Dechent, Willi; Greber, Urs F; Freudenberg, Marina A; Fejer, György

    2017-08-01

    The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages

  17. Serotype chimeric oncolytic adenovirus coding for GM-CSF for treatment of sarcoma in rodents and humans.

    PubMed

    Bramante, Simona; Koski, Anniina; Kipar, Anja; Diaconu, Iulia; Liikanen, Ilkka; Hemminki, Otto; Vassilev, Lotta; Parviainen, Suvi; Cerullo, Vincenzo; Pesonen, Saila K; Oksanen, Minna; Heiskanen, Raita; Rouvinen-Lagerström, Noora; Merisalo-Soikkeli, Maiju; Hakonen, Tiina; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Hemminki, Akseli

    2014-08-01

    Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing. © 2013 UICC.

  18. Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

    PubMed

    Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

    2005-01-01

    We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

  19. Killing effect of TNF-mediated by conditionally replicating adenovirus on esophageal cancer and lung cancer cell lines.

    PubMed

    Jiang, Yue-Quan; Zhang, Zhi; Cai, Hua-Rong; Zhou, Hong

    2015-01-01

    The killing effect of TNF mediated by conditionally replicating adenovirus SG502 on human cancer cell lines was assessed by in vivo and in vitro experiments. The recombinant adenovirus SG502-TNF was used to infect human lung cancer cell line A549 and human esophageal cancer cell line TE-1. The expression of the exogenous gene and its inhibitory effect on the tumor cell lines were thus detected. Tumor transplantation experiment was performed in mice with the purpose of assessing the inhibitory effect of the adenovirus on tumor cells and tumor formation. The targeting of the adenovirus and the mechanism of tumor inhibition were discussed by in vivo imaging technology, HE staining and TUNEL assay. Recombinant adenovirus SG502-TNF targeted the tumor cells specifically with stable expression of TNF, which produced a killing effect on tumor cells by regulating the apoptotic signaling pathway. Recombinant adenovirus SG502-TNF possessed significant killing effect on TE-1 cells either in vivo or in vitro. This finding demonstrated the potential clinical application of adenovirus SG502.

  20. Structure, function and dynamics in adenovirus maturation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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

  1. Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

    PubMed Central

    Brady, J; Radonovich, M; Thoren, M; Das, G; Salzman, N P

    1984-01-01

    We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294. Images PMID:6321950

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

    PubMed Central

    Everett, R D; Dunlop, M

    1984-01-01

    This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA isolated after infection. The results showed that Herpes simplex viruses 1 and 2, Pseudorabies virus, Variella Zoster virus, Human Cytomegalovirus, Equine herpes virus-1 and Adenovirus-2 activate transcription from both promoters tested. In contrast, SV40 did not activate transcription in trans in this assay. The possible mechanisms of this activation are discussed. Images PMID:6089105

  3. 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

  4. The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating*

    PubMed Central

    Pérez-Berná, Ana J.; Ortega-Esteban, Alvaro; Menéndez-Conejero, Rosa; Winkler, Dennis C.; Menéndez, Margarita; Steven, Alasdair C.; Flint, S. Jane; de Pablo, Pedro J.; San Martín, Carmen

    2012-01-01

    Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus. PMID:22791715

  5. An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector

    PubMed Central

    Lichtenstein, DL; Spencer, JF; Doronin, K; Patra, D; Meyer, JM; Shashkova, EV; Kuppuswamy, M; Dhar, D; Thomas, MA; Tollefson, AE; Zumstein, LA; Wold, WSM; Toth, K

    2012-01-01

    Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 × 1010 viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007. PMID:19197324

  6. An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.

    PubMed

    Lichtenstein, D L; Spencer, J F; Doronin, K; Patra, D; Meyer, J M; Shashkova, E V; Kuppuswamy, M; Dhar, D; Thomas, M A; Tollefson, A E; Zumstein, L A; Wold, W S M; Toth, K

    2009-08-01

    Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

  7. E1A promoter of bovine adenovirus type 3.

    PubMed

    Xing, Li; Tikoo, Suresh Kumar

    2006-12-01

    Conserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR. As such, the promoter activity of ITR was tested in the context of a BAdV-3 vector or a plasmid-based system. Different regions of the left end of the BAdV-3 genome initiated transcription of the red fluorescent protein gene in a plasmid-based system. Moreover, BAdV-3 mutants in which the open reading frame of E1A was placed immediately downstream of the ITR produced E1A transcript and could be propagated in non-E1A-complementing Madin-Darby bovine kidney cells. These results suggest that the left ITR contains the sole BAdV-3 E1A promoter.

  8. 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

  9. Crystal structure of the human adenovirus proteinase with its 11 amino acid cofactor.

    PubMed Central

    Ding, J; McGrath, W J; Sweet, R M; Mangel, W F

    1996-01-01

    The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X-ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy. Images PMID:8617222

  10. Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials.

    PubMed

    Kallel, Héla; Kamen, Amine A

    2015-05-01

    Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines. This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing. Additionally, advancements in vector characterization will greatly facilitate the development of novel vectored vaccine candidates. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The anatomy of a major late-stage thrust and implications for models of late-stage collisional orogenesis in the Caledonian crust of northern Scandinavia

    NASA Astrophysics Data System (ADS)

    Anderson, Mark; Hames, Willis; Stokes, Alison

    2010-05-01

    Within the stack of Caledonian crystalline thrust sheets of northern Scandinavia, a single amphibolite facies lithotectonic unit, the Småtinden nappe, is identified as a major, basement-coupled ("stretching") shear zone. This dominantly pelitic unit achieved peak metamorphic conditions of 535-550°C and 8-9kbars, and the stretching geometry suggests that this most likely occurred in response to overthrusting of a hot, pre-assembled Caledonian thrust stack. Along-strike variations in microstructural geometries and patterns of mineral zoning in widely developed porphyroblast phases suggest, however, subsequent strain partitioning within the zone during late-stage decoupling of the thrust stack from the basement along major out-of-sequence thrusts. Large parts of the nappe are characterised by relatively late, static growth preserving concordant Si-Se relationships, and typically symmetrical external fabrics consistent with formation under dominantly pure shear conditions. In the Salangen area, however, the nappe is characterised by early garnet growth, with discordant Si-Se relationships and asymmetric external fabric geometries consistent with formation during ESE-directed simple shear. Remarkably consistent thermometric estimates from chlorites in both regimes (post- and syn-shearing) suggest that out-of-sequence ramping occurred at temperatures in the range 370-400 ̊C, within the typical range of blocking temperatures for argon retention in muscovite. 40Ar-39Ar dating of muscovites from S-C fabrics in the out-of-sequence shear zone suggest that late-stage thrusting occurred during the middle-late Devonian (ca. 395-375 Ma). Hanging-wall and footwall geometries coupled with these radiometric dates indicate that the development of these late thrusts closely relates to reactivation of pre-Caledonian Baltic basement during the Devonian (400-370 Ma). East-west contraction during the upper end of this time frame is peculiar considering that this was the period of large

  12. Characterization of an avian adenovirus associated with inclusion body hepatitis in day-old turkeys.

    PubMed

    Guy, J S; Barnes, H J

    1997-01-01

    A group I avian adenovirus isolated from day-old turkeys with inclusion body hepatitis (IBH) was identified as turkey adenovirus serotype 2 (TAV2) based on cross-neutralization assays and DNA restriction endonuclease analyses. Yolk sac inoculation of embryonated turkey eggs resulted in embryo mortality and significantly (P < 0.01) decreased hatchability compared with sham-inoculated controls. Embryo mortality occurred primarily between day 24 of incubation and the time embryos hatched. Focal necrosis was detected in livers of 11/52 virus-inoculated embryos that died postinoculation and 1/27 hatchlings; in three embryos, areas of necrosis contained intranuclear inclusion bodies. These findings identify the IBH isolate as TAV2, incriminate the virus as a potential cause of suboptimal hatchability in turkeys, and provide additional evidence for causal involvement in IBH.

  13. Conditionally replicative adenovirus for gastrointestinal cancers.

    PubMed

    Yamamoto, Masato

    2004-08-01

    The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic and oesophageal cancers) is dismal, despite the advance of conventional therapeutic strategies. Cancer gene therapy is a category of new therapeutics, among which conditionally replicative adenovirus (CRAd) is one promising strategy to overcome existing obstacles of cancer gene therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of the replication biology of adenovirus. Some CRAds have already been tested in clinical trials, but have fallen short of initial expectations. Concerns for clinical applicability include therapeutic potency, replication selectivity and interval end points in clinical trials. In addition, improvement of experimental animal models is needed for a deeper understanding of CRAd biology. Despite these obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical utility. Further virological and oncological research will eventually lead to full realisation of the therapeutic potential of CRAds in the field of GI cancers.

  14. 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).

  15. Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

    PubMed

    Hama, S; Kimura, G

    1980-01-01

    Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

  16. Major depressive disorder in late life: a multifocus perspective on care needs.

    PubMed

    Houtjes, W; van Meijel, B; Deeg, D J H; Beekman, A T F

    2010-09-01

    The effectiveness of late-life depression treatment can be improved by tailoring interventions to patients' needs. Unmet needs perceived by patients suffering from a severe mental illness, e.g. depression, may have a negative impact on their recovery. The aim of this study is to gain insight into the needs of outpatients with late-life depression. Ninety-nine outpatients (aged 58-92) receiving treatment for major depressive disorder were recruited from six specialized mental health care facilities in the Netherlands. They were interviewed using the Dutch version of the Camberwell Assessment of Needs for the Elderly (CANE-NL) to identify met and unmet needs. The Montgomery-Asberg Depression Rating Scale was administered to measure depression severity. Depression severity levels varied from remission (23%), mild (31%), moderate (31%) to severe depression (15%). The average number of needs reported was 8.86, comprising 6.5 met needs and 2.3 unmet needs. Most of the unique variance in depression severity was explained by psychological unmet needs, more in particular by needs representing psychological distress. The environmental, social or physical unmet needs, respectively, showed less or no meaningful predictive value for variance in depression severity. The psychological needs category of the CANE appeared to be the strongest predictor of depression severity. Systematic needs assessment may be considered as a necessary complement to medical examination and a prerequisite for the development of tailored treatment plans for older people with depression.

  17. Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

    PubMed Central

    Jakubczak, John L.; Rollence, Michele L.; Stewart, David A.; Jafari, Jonathon D.; Von Seggern, Dan J.; Nemerow, Glen R.; Stevenson, Susan C.; Hallenbeck, Paul L.

    2001-01-01

    A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector. PMID:11222722

  18. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

    PubMed Central

    Kirshenbaum, L A; MacLellan, W R; Mazur, W; French, B A; Schneider, M D

    1993-01-01

    Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. Images PMID:8326005

  19. The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

    PubMed Central

    Suomalainen, Maarit; Zheng, Yueting; Boucke, Karin

    2017-01-01

    The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection. PMID:28628648

  20. Effect of Relaxin Expressing Adenovirus on Scar Remodeling: A Preliminary Study

    PubMed Central

    Jung, Bok Ki; Lee, Won Jai; Kang, Eunhye; Ahn, Hyo Min; Kim, Yong Oock; Rah, Dong Kyun; Yun, Chae-Ok

    2017-01-01

    Background Relaxin is a transforming growth factor β1 antagonist. To determine the effects of relaxin on scar reduction, we investigated the scar remodeling process by injecting relaxin-expressing adenoviruses using a pig scar model. Methods Scars with full thickness were generated on the backs of Yorkshire pigs. Scars were divided into two groups (relaxin [RLX] and Control). Adenoviruses were injected into the RLX (expressing relaxin) and Control (not expressing relaxin) groups. Changes in the surface areas, color index and pliability of scars were compared. Results Fifty days after treatment, the surface areas of scars decreased, the color of scars was normalized, and the pliability of scars increased in RLX group. Conclusion Relaxin-expressing adenoviruses improved the surface area, color, and pliability of scars. The mechanism of therapeutic effects on scar formation should be further investigated. PMID:28913296

  1. 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

  2. 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.

  3. 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).

  4. Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

    PubMed

    Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

    2006-12-01

    Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

  5. A recombinant adenovirus bicistronically expressing porcine interferon-α and interferon-γ enhances antiviral effects against foot-and-mouth disease virus.

    PubMed

    Kim, Su-Mi; Kim, Se-Kyung; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Ko, Young-Joon; Lee, Hyang-Sim; Seo, Min-Goo; Shin, Yeun-Kyung; Kim, Byounghan

    2014-04-01

    Foot-and-mouth disease (FMD) is a virulent and economically costly disease in domestic livestock. Since the current vaccine available against FMD provides no protection until 7days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is by the application of anti-viral agents. The combination of recombinant adenovirus expressing type I interferon (IFN-α) and adenovirus expressing type II IFN (IFN-γ) has been reported to be an effective anti-viral treatment strategy against FMDV. Nevertheless, the recombinant adenovirus mixture may be inefficient because of the low anti-viral efficiency of IFN-γ compared to that of IFN-α. In this study, we generated a recombinant adenovirus co-expressing porcine IFN-α and IFN-γ in tandem using an FMDV 2A sequence to mediate effective cleavage of the two proteins (referred to as Ad-porcine IFN-αγ). We demonstrated that both recombinant porcine IFN-α and IFN-γ were expressed and interferon stimulated gene (ISG)s related with IFN-α and IFN-γ were induced in porcine kidney (IBRS-2) cells infected with Ad-porcine IFN-αγ. Additionally, the anti-viral effects of Ad-porcine IFN-αγ against FMDV were enhanced both in IBRS-2 cells and in CD-1 (ICR) suckling mice compared to that of adenovirus expressing only a single protein. We propose that Ad-porcine IFN-αγ could be a rapid, highly efficient, convenient anti-viral agent against FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

    PubMed Central

    Cotten, M; Wagner, E; Zatloukal, K; Birnstiel, M L

    1993-01-01

    Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus. Images PMID:8099627

  7. 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

  8. The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

    PubMed

    Hale, T K; Braithwaite, A W

    1999-08-20

    Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

  9. Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

    PubMed Central

    Madara, Jonathan; Krewet, James A; Shah, Maulik

    2005-01-01

    In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

  10. 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.

  11. ☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

    PubMed

    Zou, Xiao-Hui; Bi, Zhi-Xiang; Guo, Xiao-Juan; Zhang, Zun; Zhao, Yang; Wang, Min; Zhu, Ya-Lu; Jie, Hong-Ying; Yu, Yang; Hung, Tao; Lu, Zhuo-Zhuang

    2018-07-01

    Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Complete protection of cats against feline panleukopenia virus challenge by a recombinant canine adenovirus type 2 expressing VP2 from FPV.

    PubMed

    Yang, Songtao; Xia, Xianzhu; Qiao, Jun; Liu, Quan; Chang, Shuang; Xie, Zhijing; Ju, Huiyan; Zou, Xiaohuan; Gao, Yuwei

    2008-03-10

    Feline panleukopenia virus (FPV) is an important infectious pathogen of all members of the family Felidae. Here, we describe construction of a replication-competent recombinant canine adenovirus type 2 (CAV-2) expressing the VP2 protein of FPV (CAV-2-VP2) by transfection of MDCK cells with recombinant CAV-2 genome carrying a VP2 expression cassette. Ten 3-month-old cats were vaccinated with the recombinant virus with two boosters at 15-day intervals. All cats developed neutralizing antibodies of titers 1:16-1:32 by day 15 post-primary vaccination, increasing to 1:64-1:128 by day 45. Examination for clinical signs and viral presence, and total white blood cell counts in peripheral blood following FPV challenge, showed that all were completely protected. This recombinant virus appears to provide an effective alternative to attenuated and inactivated vaccines in immunizing cats against feline panleukopenia.

  13. Prospects for Oral Replicating Adenovirus-Vectored Vaccines

    PubMed Central

    Deal, Cailin; Pekosz, Andrew; Ketner, Gary

    2013-01-01

    Orally delivered replicating adenovirus (Ad) vaccines have been used for decades to prevent adenovirus serotype 4 and 7 respiratory illness in military recruits, demonstrating exemplary safety and high efficacy. That experience suggests that oral administration of live recombinant Ads (rAds) holds promise for immunization against other infectious diseases, including those that have been refractory to traditional vaccination methods. Live rAds can express intact antigens from free-standing transgenes during replication in infected cells. Alternatively, antigenic epitopes can be displayed on the rAd capsid itself, allowing presentation of the epitope to the immune system both prior to and during replication of the virus. Such capsid-display rAds offer a novel vaccine approach that could be used either independently of or in combination with transgene expression strategies to provide a new tool in the search for protection from infectious disease. PMID:23707160

  14. Neuropsychological predictors of dementia in late-life major depressive disorder.

    PubMed

    Potter, Guy G; Wagner, H Ryan; Burke, James R; Plassman, Brenda L; Welsh-Bohmer, Kathleen A; Steffens, David C

    2013-03-01

    Major depressive disorder is a likely risk factor for dementia, but some cases of major depressive disorder in older adults may actually represent a prodrome of this condition. The purpose of this study was to use neuropsychological test scores to predict conversion to dementia in a sample of depressed older adults diagnosed as nondemented at the time of neuropsychological testing. Longitudinal, with mean follow-up of 5.45 years. Outpatient depression treatment study at Duke University. Thirty nondemented individuals depressed at the time of neuropsychological testing and later diagnosed with incident dementia; 149 nondemented individuals depressed at the time of neuropsychological testing and a diagnosis of cognitively normal. All participants received clinical assessment of depression, were assessed to rule out prevalent dementia at the time of study enrollment, completed neuropsychological testing at the time of study enrollment, and were diagnosed for cognitive disorders on an annual basis. Nondemented, acutely depressed older adults who converted to dementia during the study period exhibited broadly lower cognitive performances at baseline than acutely depressed individuals who remained cognitively normal. Discriminant function analysis indicated that 2 neuropsychological tests, Recognition Memory (from the Consortium to Establish a Registry for Alzheimer's Disease neuropsychological battery) and Trail Making B, best predicted dementia conversion. Depressed older adults with cognitive deficits in the domains of memory and executive functions during acute depression are at higher risk for developing dementia. Some cases of late-life depression may reflect a prodrome of dementia in which clinical manifestation of mood changes may co-occur with emerging cognitive deficits. Copyright © 2013 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.

  15. 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

  16. Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

    PubMed

    Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

    2015-09-01

    Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Interferon-α Silencing by Small Interference RNA Increases Adenovirus Transduction and Transgene Expression in Huh7 Cells.

    PubMed

    Sobrevilla-Navarro, Ana Alondra; Sandoval-Rodríguez, Ana; García-Bañuelos, Jesús Javier; Armendariz-Borunda, Juan; Salazar-Montes, Adriana María

    2018-04-01

    Adenoviruses are the most common vectors used in clinical trials of gene therapy. In 2017, 21.2% of clinical trials used rAds as vectors. Systemic administration of rAds results in high tropism in the liver. Interferon types α and β are the major antiviral cytokines which orchestrate the host's immune response against rAd, limiting therapeutic gene expression and preventing subsequent vector administration. siRNA is small double-strand RNAs that temporally inhibit the expression of a specific gene. The aim is to evaluate the effect of IFN-α blocking by a specific siRNA on Ad-GFP transduction and on transgene expression in Huh7 cells in culture. Huh7 cells were cultured in DMEM and transfected with 70 nM of siRNA-IFN-α. Six hours later, the cells were exposed to 1 × 10 9  vp/ml of rAd-GFP for 24 h. Expression of IFN-α, TNF-α and the PKR gene was determined by RT-qPCR. Percentage of transduction was analyzed by flow cytometry and by qPCR. GFP expression was determined by western blot. 70 nM of siRNA-IFN-α inhibited 96% of IFN-α and 65% of TNF-α gene expression compared to an irrelevant siRNA. Percentage of transduction and transgene expression increased in these cells compared to an irrelevant siRNA. Inhibition of IFN-α expression by siRNA-IFN-α enabled a higher level of transduction and transgene expression GFP, highlighting the role of IFN-α in the elimination of adenovirus in transduced cells and thus suggesting that its inhibition could be an important strategy for gene therapy in clinical trials using adenovirus as a vector directed to liver diseases.

  18. Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells.

    PubMed

    Prill, Jan-Michael; Espenlaub, Sigrid; Samen, Ulrike; Engler, Tatjana; Schmidt, Erika; Vetrini, Francesco; Rosewell, Amanda; Grove, Nathan; Palmer, Donna; Ng, Philip; Kochanek, Stefan; Kreppel, Florian

    2011-01-01

    In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

  19. The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells.

    PubMed

    Patzke, Christopher; Max, Klaas E A; Behlke, Joachim; Schreiber, Jadwiga; Schmidt, Hannes; Dorner, Armin A; Kröger, Stephan; Henning, Mechthild; Otto, Albrecht; Heinemann, Udo; Rathjen, Fritz G

    2010-02-24

    The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

  20. Thin-section computed tomography findings in 104 immunocompetent patients with adenovirus pneumonia.

    PubMed

    Park, Chan Kue; Kwon, Hoon; Park, Ji Young

    2017-08-01

    Background To date, there has been no computed tomography (CT) evaluation of adenovirus pneumonia in a large number of immunocompetent patients. Purpose To describe the thin-section CT findings of immunocompetent patients with adenovirus pneumonia. Material and Methods We prospectively enrolled 104 patients with adenovirus pneumonia from a military hospital. CT scans of each patient were retrospectively and independently assessed by two radiologists for the presence of abnormalities, laterality and zonal predominance of the parenchymal abnormalities, and dominant imaging patterns and their anatomic distributions. Results CT findings included consolidation (n = 92), ground-glass opacity (GGO; n = 82), septal thickening (n = 34), nodules (n = 46), bronchial wall thickening (n = 32), pleural effusion (n = 16), and lymphadenopathy (n = 3). Eighty-four patients (81%) exhibited unilateral parenchymal abnormalities and 57 (57%) exhibited lower lung zone abnormalities. The most frequently dominant CT pattern was consolidation with surrounding GGO (n = 50), with subpleural (70%) and peribronchovascular (94%) distributions. Consolidation-the second-most common pattern (n = 33)-also exhibited subpleural (79%) and peribronchovascular (97%) distributions. The dominant nodule pattern (n = 14) exhibited mixed (64%) and peribronchovascular (100%) distributions. A dominant GGO pattern was only observed in four patients; none had central distribution. Conclusion Although the manifestations of adenovirus pneumonia on CT are varied, we found the most frequent pattern was consolidation with or without surrounding GGO, with subpleural and peribronchovascular distributions. Parenchymal abnormalities were predominantly unilateral and located in the lower lung zone. If dominant consolidation findings are present in immunocompetent patients during the early stages, adenovirus pneumonia should be considered.

  1. Pulmonary vasculature directed adenovirus increases epithelial lining fluid alpha-1 antitrypsin levels.

    PubMed

    Buggio, Maurizio; Towe, Christopher; Annan, Anand; Kaliberov, Sergey; Lu, Zhi Hong; Stephens, Calvin; Arbeit, Jeffrey M; Curiel, David T

    2016-01-01

    Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities. Copyright © 2016 John Wiley & Sons, Ltd.

  2. 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

  3. [Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities].

    PubMed

    Grinenko, N F; Savitskaia, N V; Pashvykina, G V; Al'tshteĭn, A D

    2003-06-01

    A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

  4. Experimental oral immunization of ferret badgers (Melogale moschata) with a recombinant canine adenovirus vaccine CAV-2-E3Δ-RGP and an attenuated rabies virus SRV9.

    PubMed

    Zhao, Jinghui; Liu, Ye; Zhang, Shoufeng; Fang, Lijun; Zhang, Fei; Hu, Rongliang

    2014-04-01

    Ferret badgers (Melogale moschata) are a major reservoir of rabies virus in southeastern China. Oral immunization has been shown to be a practical method for wildlife rabies management in Europe and North America. Two groups of 20 ferret badgers were given a single oral dose of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3Δ-RGP, or an experimental attenuated rabies virus vaccine, SRV9. At 21 days, all ferret badgers had seroconverted, with serum virus-neutralizing antibodies ranging from 0.1 to 4.5 IU/mL. Titers were >0.50 IU/mL (an acceptable level) in 17/20 and 16/20 animals receiving CAV-2-E3Δ-RGP or SRV9, respectively. The serologic results indicate that the recombinant CAV-2-E3Δ-RGP is at least as effective as the attenuated rabies virus vaccine. Both may be considered for additional research as oral rabies vaccine candidates for ferret badgers.

  5. Protection of Nonhuman Primates Against Two Species of Ebola Virus Infection With a Single Complex Adenovirus Vector

    DTIC Science & Technology

    2010-04-01

    glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus -based vector (CAdVax). We evaluated our vaccine ...recombinant complex adenovirus vaccine (CAdVax) system, which provides multivalent protection of NHPs against multiple species of filoviruses (33). The...CAdVax vaccine platform is based on a complex, replication-defective adenovirus 5 (Ad5) vector (28–30, 37, 38) that allows for the incorporation of

  6. Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

    PubMed

    Stachelek, S J; Song, C; Alferiev, I; Defelice, S; Cui, X; Connolly, J M; Bianco, R W; Levy, R J

    2004-01-01

    The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen

  7. 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.

  8. Serological and Molecular Biological Studies of Parvovirus B19, Coxsackie B Viruses, and Adenoviruses as Potential Cardiotropic Viruses in Bulgaria.

    PubMed

    Ivanova, Stefka Kr; Angelova, Svetla G; Stoyanova, Asya P; Georgieva, Irina L; Nikolaeva-Glomb, Lubomira K; Mihneva, Zafira G; Korsun, Neli St

    2016-12-01

    Inflammatory diseases of the heart (myocarditis, pericarditis) are commonly caused by viruses. Among the human cardiotropic viruses, parvovirus B19, Coxsackie B viruses, and adenoviruses play a leading role. The aim of the present study was to determine the presumptive causative role of parvovirus B19, Coxsackie B viruses, and adenoviruses in the development of myocarditis, pericarditis and dilated cardiomyopathy by demonstrating the presence of specific antiviral antibodies or viral DNA in patients' serum samples. We tested serum samples collected between 2010 and 2014 from 235 patients with myocarditis (n=108), pericarditis (n=79), myopericarditis (n=19), dilated cardiomyopathy (n=7), and fever of unknown origin accompanied by cardiac complaints (n=22). The mean age of patients with the standard deviation was 33 ± 18 years. Serological and molecular methods (ELISA for specific IgM/IgG antibodies to parvovirus B19 and IgM antibodies to Coxsackie B viruses and adenoviruses, and PCR for detection of parvovirus B19 in serum samples, respectively) were used in the study. Of all tested 235 serum samples, in 60 (25.5%) positive results for at least one of the three tested viruses were detected. Forty out of these 235 serum samples (17%) were Coxsackie B virus IgM positive. They were found in 17% (18/108) of the patients with myocarditis, in 15% (12/79) of those with pericarditis, in 16% (3/19) of those with myopericarditis and in 32% (7/22) in those with fever of unknown origin. The 63 Coxsackie B virus IgM negative patient's serum samples were tested by ELISA for presence of adenovirus IgM antibodies. Such were found in 4 patients with pericarditis and in 2 patients with fever of unknown origin. Every IgM negative sample (n=189) for Coxsackie B and adenovirus was further tested by ELISA for parvovirus B19 IgM/IgG antibodies. B19-IgM antibodies were detected in 14 patients (7.4%). The percentages for B19-IgM antibodies was 8% (7/90), 5% (3/63) and 31% (4/13) in the

  9. Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs.

    PubMed

    Taguchi, Masayuki; Namikawa, Kazuhiko; Maruo, Takuya; Orito, Kensuke; Lynch, Jonathan; Sahara, Hiroeki

    2011-09-01

    Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.

  10. Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs

    PubMed Central

    Taguchi, Masayuki; Namikawa, Kazuhiko; Maruo, Takuya; Orito, Kensuke; Lynch, Jonathan; Sahara, Hiroeki

    2011-01-01

    Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age. PMID:22379198

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

    PubMed

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

    2008-12-01

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

  12. Antiviral T Cells for Adenovirus in the Pretransplant Period: A Bridge Therapy for Severe Combined Immunodeficiency.

    PubMed

    Miller, Holly K; Hanley, Patrick J; Lang, Haili; Lazarski, Christopher A; Chorvinsky, Elizabeth A; McCormack, Sarah; Roesch, Lauren; Albihani, Shuroug; Dean, Marcus; Hoq, Fahmida; Adams, Roberta H; Bollard, Catherine M; Keller, Michael D

    2018-05-09

    Viral infections can be life threatening in patients with severe combined immunodeficiency (SCID) and other forms of profound primary immunodeficiency disorders both before and after hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with virus-specific T cells (VSTs) has been utilized in many patients in the setting of HSCT, but has very rarely been attempted for treatment of viral infections before HSCT. Here we describe the use of VSTs in an infant with RAG1 SCID who had developed disseminated adenovirus which failed to improve on cidofovir. Adenovirus cleared following 2 doses of VSTs and marrow infusion from a matched unrelated donor, without incidence of graft versus host disease. T cell receptor-b sequencing demonstrated expansion of adenovirus-specific T cell fraction of the VSTs, suggesting that infusion facilitated viral clearance. This report suggests that VSTs are likely safe in the pre-HSCT period, and may be a useful bridge therapy for infants with SCID and persistent viral infections. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  13. A One Year Study on the Concentrations of Norovirus and Enteric Adenoviruses in Wastewater and A Surface Drinking Water Source in Norway.

    PubMed

    Grøndahl-Rosado, Ricardo C; Yarovitsyna, Ekaterina; Trettenes, Elin; Myrmel, Mette; Robertson, Lucy J

    2014-12-01

    Enteric viruses transmitted via the faecal-oral route occur in high concentrations in wastewater and may contaminate drinking water sources and cause disease. In order to quantify enteric adenovirus and norovirus genotypes I and II (GI and GII) impacting a drinking source in Norway, samples of surface water (52), wastewater inlet (64) and outlet (59) were collected between January 2011 and April 2012. Samples were concentrated in two steps, using an electropositive disc filter and polyethylene glycol precipitation, followed by nucleic acid extraction and analysis by quantitative polymerase chain reaction. Virus was detected in 47/52 (90.4%) of surface water, 59/64 (92%) of wastewater inlet and 55/59 (93%) of wastewater outlet samples. Norovirus GI occurred in the highest concentrations in surface water (2.51e + 04) and adenovirus in wastewater (2.15e + 07). While adenovirus was the most frequently detected in all matrices, norovirus GI was more frequently detected in surface water and norovirus GII in wastewater. This study is the first in Norway to monitor both sewage and a drinking water source in parallel, and confirms the year-round presence of norovirus and adenovirus in a Norwegian drinking water source.

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

    PubMed Central

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

    2004-01-01

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

  15. 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.

  16. Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

    PubMed

    Carinhas, Nuno; Pais, Daniel A M; Koshkin, Alexey; Fernandes, Paulo; Coroadinha, Ana S; Carrondo, Manuel J T; Alves, Paula M; Teixeira, Ana P

    2016-03-23

    Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

  17. Disrupted adenovirus-based vaccines against small addictive molecules circumvent anti-adenovirus immunity.

    PubMed

    De, Bishnu P; Pagovich, Odelya E; Hicks, Martin J; Rosenberg, Jonathan B; Moreno, Amira Y; Janda, Kim D; Koob, George F; Worgall, Stefan; Kaminsky, Stephen M; Sondhi, Dolan; Crystal, Ronald G

    2013-01-01

    Adenovirus (Ad) vaccine vectors have been used for many applications due to the capacity of the Ad capsid proteins to evoke potent immune responses, but these vectors are often ineffective in the context of pre-existing anti-Ad immunity. Leveraging the knowledge that E1(-)E3(-) Ad gene transfer vectors are potent immunogens, we have developed a vaccine platform against small molecules by covalently coupling analogs of small molecules to the capsid proteins of disrupted Ad (dAd5). We hypothesized that the dAd5 platform would maintain immunopotency even in the context of anti-Ad neutralizing antibodies. To test this hypothesis, we coupled cocaine and nicotine analogs, GNE and AM1, to dAd5 capsid proteins to generate dAd5GNE and dAd5AM1, respectively. Mice were pre-immunized with Ad5Null, resulting in high titer anti-Ad5 neutralizing antibodies comparable to those observed in the human population. The dAd5GNE and dAd5AM1 vaccines elicited high anti-cocaine and anti-nicotine antibody titers, respectively, in both naive and Ad5-immune mice, and both functioned to prevent cocaine or nicotine from reaching the brain of anti-Ad immune mice. Thus, disrupted Ad5 evokes potent humoral immunity that is effective in the context of pre-existing neutralizing anti-Ad immunity, overcoming a major limitation for current Ad-based vaccines.

  18. Disrupted Adenovirus-Based Vaccines Against Small Addictive Molecules Circumvent Anti-Adenovirus Immunity

    PubMed Central

    De, Bishnu P.; Pagovich, Odelya E.; Hicks, Martin J.; Rosenberg, Jonathan B.; Moreno, Amira Y.; Janda, Kim D.; Koob, George F.; Worgall, Stefan; Kaminsky, Stephen M.; Sondhi, Dolan

    2013-01-01

    Abstract Adenovirus (Ad) vaccine vectors have been used for many applications due to the capacity of the Ad capsid proteins to evoke potent immune responses, but these vectors are often ineffective in the context of pre-existing anti-Ad immunity. Leveraging the knowledge that E1−E3− Ad gene transfer vectors are potent immunogens, we have developed a vaccine platform against small molecules by covalently coupling analogs of small molecules to the capsid proteins of disrupted Ad (dAd5). We hypothesized that the dAd5 platform would maintain immunopotency even in the context of anti-Ad neutralizing antibodies. To test this hypothesis, we coupled cocaine and nicotine analogs, GNE and AM1, to dAd5 capsid proteins to generate dAd5GNE and dAd5AM1, respectively. Mice were pre-immunized with Ad5Null, resulting in high titer anti-Ad5 neutralizing antibodies comparable to those observed in the human population. The dAd5GNE and dAd5AM1 vaccines elicited high anti-cocaine and anti-nicotine antibody titers, respectively, in both naive and Ad5-immune mice, and both functioned to prevent cocaine or nicotine from reaching the brain of anti-Ad immune mice. Thus, disrupted Ad5 evokes potent humoral immunity that is effective in the context of pre-existing neutralizing anti-Ad immunity, overcoming a major limitation for current Ad-based vaccines. PMID:23140508

  19. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

    PubMed Central

    Stevenson, S C; Rollence, M; Marshall-Neff, J; McClelland, A

    1997-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange

  20. 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

    2017-08-18

    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.

  1. Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses.

    PubMed

    Hackenbrack, Nicole; Rogers, Matthew B; Ashley, Robert E; Keel, M Kevin; Kubiski, Steven V; Bryan, John A; Ghedin, Elodie; Holmes, Edward C; Hafenstein, Susan L; Allison, Andrew B

    2017-01-15

    Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV. Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged

  2. Three-year serologic immunity against canine parvovirus type 2 and canine adenovirus type 2 in dogs vaccinated with a canine combination vaccine.

    PubMed

    Larson, L J; Schultz, R D

    2007-01-01

    A group of client-owned dogs and a group of dogs at a commercial kennel were evaluated for duration of antibody responses against canine parvovirus type 2 (CPV-2) and canine adenovirus type 1 (CAV-1) after receiving a combination vaccine containing recombinant canarypox-vectored canine distemper virus (CDV) and modified-live CPV-2, CAV-2, and canine parainfluenza virus, with (C6) or without (C4) two serovars of Leptospira (Recombitek C4 or C6, Merial). Duration of antibody, which correlates with protective immunity, was found to be at least 36 months in both groups. Recombitek combination vaccines can confidently be given every 3 years with assurance of protection in immunocompetent dogs against CPV-2 and CAV-1 as well as CDV. This allows this combination vaccine, like other, similar modified- live virus combination products containing CDV, CAV-2, and CPV-2, to be administered in accordance with the recommendations of the American Animal Hospital Association Canine Vaccine Task Force.

  3. Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica.

    PubMed

    Park, Yon Mi; Kim, Jeong-Hoon; Gu, Se Hun; Lee, Sook Young; Lee, Min-Goo; Kang, Yoon Kyoo; Kang, Sung-Ho; Kim, Hak Jun; Song, Jin-Won

    2012-01-05

    Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Therapeutic effect of targeted Fas-expressing adenoviruses method combining γδ T cells in a mouse model of human ovarian carcinoma.

    PubMed

    Zeng, Dingyuan; Lin, Jiajing; He, Hongying; Tan, Guangping; Lan, Ying; Jiang, Fuyan; Sheng, Shuting

    2018-02-01

    The present study aimed to investigate the therapeutic effect and safety of targeted use of Fas-expressing adenoviruses combined with γδ T cell-mediated killing to treat human ovarian cancer xenografts in BALB/c mice. Shuttle plasmids containing control elements of human telomerase reverse transcriptase promoter and two-step transcriptional amplification system were constructed and packaged into adenovirus-5 vectors to generate expression of an exogenous Fas gene. A mouse xenograft model of human ovarian carcinoma was constructed. A total of 35 BALB/c mice were randomly divided into five groups, which were injected with PBS, γδ T cells, Fas-expressing adenoviruses, taxol, or Fas-expressing adenovirus and γδ T cells. The weight and volume of tumors in mice in each group was monitored. Tissue sections of the various tissues of mice in the Fas-expressing adenovirus and γδ T cells group was compared with those in the PBS group to evaluate the safety of Fas-expressing adenovirus and γδ T cells in the treatment of human ovarian cancer xenograft tumors. The results of the present study indicated that mice in all treatment groups were alive at the end of the treatment course. Tumor weight and volume was the highest in the PBS group, followed successively by the adenovirus group, the γδ T cell group, the adenovirus and γδ T cell group, and the taxol group. The weight and volume inhibition rate in adenovirus and γδ T cell group were significantly higher compared with in the PBS group (P<0.05). Pathological observation of tissue samples revealed that none of vital organs in the adenovirus and γδ T cell group developed any evident morphological changes during treatment, when compared with healthy controls. In conclusion, the combined therapy with Fas-expressing adenoviruses and γδ T cells is efficient and safe for the treatment of mouse human ovarian carcinoma xenografts.

  5. Co-expression of Erns and E2 genes of classical swine fever virus by replication-defective recombinant adenovirus completely protects pigs against virulent challenge with classical swine fever virus.

    PubMed

    Sun, Yongke; Yang, Yuai; Zheng, Huanli; Xi, Dongmei; Lin, Mingxing; Zhang, Xiaomin; Yang, Linfu; Yan, Yulin; Chu, Xiaohui; Bi, Baoliang

    2013-04-01

    The objective of this study was to construct a recombinant adenovirus for future CSFV vaccines used in the pig industry for the reduction of losses involved in CSF outbreaks. The Erns and E2 genes of classical swine fever virus (CSFV), which encode the two main protective glycoproteins from the "Shimen" strain of CSFV, were combined and inserted into the replication-defective human adenovirus type-5 and named the rAd-Erns-E2. Nine pigs were randomly assigned to three treatment groups (three pigs in each group) including the rAd-Erns-E2, hAd-CMV control and DMEM control. Intramuscular vaccination with 2×10(6) TCID(50) of the rAd-Erns-E2 was administered two times with an interval of 21 days. At 42 days post inoculation, pigs in all groups were challenged with a lethal dose of 1×10(3) TCID(50) CSFV "Shimen" strain. Observation of clinical signs was made and the existence of CSFV RNA was detected. Animals in the hAd-CMV and DMEM groups showed severe clinical CSF symptoms and were euthanized from 7 to 10 days after the challenge. However, no adverse clinical CSF signs were observed in vaccinated pigs after the administration of rAd-Erns-E2 and even after CSFV challenge. Neither CSFV RNA nor pathological changes were detected in the tissues of interest of the above vaccinated pigs. These results implied that the recombination adenovirus carrying the Erns-E2 genes could be used to prevent swine from classical swine fever. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens.

    PubMed

    Zhang, Dongchao; Long, Yuqing; Li, Meng; Gong, Jianfang; Li, Xiaohui; Lin, Jing; Meng, Jiali; Gao, Keke; Zhao, Ruili; Jin, Tianming

    2018-04-01

    Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P < 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

  7. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

    PubMed Central

    Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

    1993-01-01

    To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

  8. 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

  9. Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine.

    PubMed

    Lokhandwala, Shehnaz; Waghela, Suryakant D; Bray, Jocelyn; Sangewar, Neha; Charendoff, Chloe; Martin, Cameron L; Hassan, Wisam S; Koynarski, Tsvetoslav; Gabbert, Lindsay; Burrage, Thomas G; Brake, David; Neilan, John; Mwangi, Waithaka

    2017-01-01

    African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.

  10. Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine

    PubMed Central

    Waghela, Suryakant D.; Bray, Jocelyn; Sangewar, Neha; Charendoff, Chloe; Martin, Cameron L.; Hassan, Wisam S.; Koynarski, Tsvetoslav; Gabbert, Lindsay; Burrage, Thomas G.; Brake, David; Neilan, John; Mwangi, Waithaka

    2017-01-01

    African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy. PMID:28481911

  11. The Insignificance of Major Mergers in Driving Star Formation at z approximately equal to 2

    NASA Technical Reports Server (NTRS)

    Kaviraj, S.; Cohen, S.; Windhorst, R. A.; Silk, J.; O'Connell, R. W.; Dopita, M. A.; Dekel, A.; Hathi, N. P.; Straughn, A.; Rutkowski, M.

    2012-01-01

    We study the significance of major mergers in driving star formation in the early Universe, by quantifying the contribution of this process to the total star formation budget in 80 massive (M(*) > 10(exp 10) Solar M) galaxies at z approx = 2. Employing visually-classified morphologies from rest-frame V-band HST imaging, we find that 55(exp +/-14)% of the star formation budget is hosted by non-interacting late-types, with 27(exp +/-18% in major mergers and 18(exp +/- 6)% in spheroids. Given that a system undergoing a major merger continues to experience star formation driven by other processes at this epoch (e.g. cold accretion, minor mergers), approx 27% is a likely upper limit for the major-merger contribution to star formation activity at this epoch. The ratio of the average specific star formation rate in major mergers to that in the non-interacting late-types is approx 2.2:1, suggesting that the typical enhancement of star formation due to major merging is modest and that just under half the star formation in systems experiencing major mergers is unrelated to the merger itself. Taking this into account, we estimate that the actual major-merger contribution to the star formation budget may be as low as approx 15%. While our study does not preclude a major-merger-dominated. era in the very early Universe, if the major-merger contribution to star formation does not evolve significantly into larger look-back times, then this process has a relatively insignificant role in driving stellar mass assembly over cosmic time.

  12. Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom.

    PubMed

    Walker, David; Fee, Seán A; Hartley, Gill; Learmount, Jane; O'Hagan, Maria J H; Meredith, Anna L; de C Bronsvoort, Barend M; Porphyre, Thibaud; Sharp, Colin P; Philbey, Adrian W

    2016-10-31

    Canine adenovirus type 1 (CAV-1) causes infectious canine hepatitis (ICH), a frequently fatal disease which primarily affects canids. In this study, serology (ELISA) and molecular techniques (PCR/qPCR) were utilised to investigate the exposure of free-ranging red foxes (Vulpes vulpes) to CAV-1 in the United Kingdom (UK) and to examine their role as a wildlife reservoir of infection for susceptible species. The role of canine adenovirus type 2 (CAV-2), primarily a respiratory pathogen, was also explored. In foxes with no evidence of ICH on post-mortem examination, 29 of 154 (18.8%) red foxes had inapparent infections with CAV-1, as detected by a nested PCR, in a range of samples, including liver, kidney, spleen, brain, and lung. CAV-1 was detected in the urine of three red foxes with inapparent infections. It was estimated that 302 of 469 (64.4%) red foxes were seropositive for canine adenovirus (CAV) by ELISA. CAV-2 was not detected by PCR in any red foxes examined. Additional sequence data were obtained from CAV-1 positive samples, revealing regional variations in CAV-1 sequences. It is concluded that CAV-1 is endemic in free-ranging red foxes in the UK and that many foxes have inapparent infections in a range of tissues.

  13. "Engage" therapy: Prediction of change of late-life major depression.

    PubMed

    Alexopoulos, George S; O'Neil, Robert; Banerjee, Samprit; Raue, Patrick J; Victoria, Lindsay W; Bress, Jennifer N; Pollari, Cristina; Arean, Patricia A

    2017-10-15

    Engage grew out of the need for streamlined psychotherapies that can be accurately used by community therapists in late-life depression. Engage was based on the view that dysfunction of reward networks is the principal mechanism mediating depressive symptoms. Accordingly, Engage uses "reward exposure" (exposure to meaningful activities) and assumes that repeated activation of reward networks will normalize these systems. This study examined whether change in a behavioral activation scale, an index of reward system function, predicts change in depressive symptomatology. The participants (N = 48) were older adults with major depression treated with 9 weekly sessions of Engage and assessed 27 weeks after treatment. Depression was assessed with the 24-item Hamilton Depression Rating Scale (HAM-D) and behavioral activation with the four subscales of Behavioral Activation for Depression Scale (activation, avoidance/rumination, work impairment, social impairment) at baseline, 6 weeks (mid-treatment), 9 weeks (end of treatment), and 36 weeks. Change only in the Activation subscale during successive periods of assessment predicted depression severity (HAM-D) at the end of each period (F 1, 47 = 21.05, p<0.0001). An increase of one standard deviation in the Activation score resulted in a 2.04 (95% CI: 1.17-2.92) point decrease in HAM-D. For every one point increase in the Activation score, HAM-D was decreased by 0.22 points (95% CI: 0.12-0.31). No comparison group. Partial overlap of Activation Subscale with HAM-D, lack of detailed neurocognitive assessment and social support. Change in behavioral activation predicts improvement of depressive symptoms and signs in depressed older adults treated with Engage. Copyright © 2017. Published by Elsevier B.V.

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

    PubMed

    Richardson, Jason S; Yao, Michel K; Tran, Kaylie N; Croyle, Maria A; Strong, James E; Feldmann, Heinz; Kobinger, Gary P

    2009-01-01

    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. 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. 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 adenovirus-based Ebola vaccine. Understanding and improving the

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

    PubMed

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

    2013-12-01

    Abstract :  The persistence of Giardia, Cryptosporidium, Rotavirus, and Adenovirus in samples of raw and treated sewage collected monthly in 2010 at the Biological Wastewater Treatment Plant of Ribeirão Preto, SP, Brazil, was analyzed. The USEPA Method 1623 was used to detect and quantify Giardia and Cryptosporidium. An enzyme immunoassay was carried out to test Rotavirus and Adenovirus antigen optical density (Rotascreen® and Adenoscreen®). The results show a significant decrease in the concentrations of Giardia, Rotavirus and Adenovirus (P < 0.05) and a trend of decreasing Cryptosporidium densities, without statistical significance. Giardia concentrations ranged from 120 to 2,200 cysts/L in raw sewage and from 0.45 to 3.5 cysts/L in treated sewage. Cryptosporidium concentration ranged from undetectable to 28.9 oocysts/L in raw sewage and undetectable to 1.05 oocysts/L in treated sewage. Rotavirus presented absorbance values that ranged from 1.17 ± 0.81 in raw sewage to 0.46 ± 0.32 in treated sewage. Adenovirus, in turn, presented absorbance values of 0.64 ± 0.20 in raw sewage and of 0.45 ± 0.04 in treated sewage. There was no significant seasonal tendency observed in the distribution of protozoa (oo)cysts and in the viral antigen density in the monthly sewage samples during 2010 (P > 0.05). Even though these pathogenic agents decreased after treatment, the remaining loads observed in treated sewage can reach the watercourses receiving it. Giardia, Cryptosporidium, Rotavirus, and Adenovirus are pathogens with very low infectious doses, representing a public health risk especially for vulnerable groups, such as children living near these watercourses and homeless people using this water for various purposes. Studies addressing the environmental persistence of opportunistic pathogens in watercourses are hugely important in the public health sphere, especially in developing countries, where economic, social, cultural, and environmental factors still persist

  16. uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors.

    PubMed

    Sobrevals, Luciano; Mato-Berciano, Ana; Urtasun, Nerea; Mazo, Adela; Fillat, Cristina

    2014-01-01

    Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133(+) population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells. © 2013.

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

    PubMed Central

    Gao, Peng

    2016-01-01

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

  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. Impact of CO2 and continental configuration on Late Cretaceous ocean dynamics

    NASA Astrophysics Data System (ADS)

    Puceat, Emmanuelle; Donnadieu, Yannick; Moiroud, Mathieu; Guillocheau, François; Deconinck, Jean-François

    2014-05-01

    The Late Cretaceous period is characterized by a long-term climatic cooling (Huber et al., 1995; Pucéat et al., 2003; Friedrich et al., 2012) and by major changes in continental configuration with the widening of the Atlantic Ocean, the initiation of the Tethyan ocean closure, and the deepening of the Central Atlantic Gateway. The Late Cretaceous also marks the end of the occurrence of Oceanic Anoxic Events (OAEs), that are associated to enhanced organic carbon burial, to major crises of calcifying organisms, and to possible ocean acidification (Jenkyns, 2010). It has been suggested that the evolution in continental configuration and climate occurring during the Late Cretaceous could have induced a reorganization in the oceanic circulation, that may have impacted the oxygenation state of the oceanic basins and contributed to the disappearance of OAEs (Robinson et al., 2010; Robinson and Vance, 2012). Yet there is no consensus existing on the oceanic circulation modes and on their possible evolution during the Late Cretaceous, despite recent improvement of the spatial and temporal coverage of neodymium isotopic data (ɛNd), a proxy of oceanic circulation (MacLeod et al., 2008; Robinson et al., 2010; Murphy and Thomas, 2012; Robinson and Vance, 2012; Martin et al., 2012; Moiroud et al., 2012). Using the fully coupled ocean-atmosphere General Circulation Model FOAM, we explore in this work the impact on oceanic circulation of changes in continental configuration between the mid- and latest Cretaceous. Two paleogeography published by Sewall et al. (2007) were used, for the Cenomanian/Turonian boundary and for the Maastrichtian. For each paleogeography, 3 simulations have been realized, at 2x, 4x, and 8x the pre-industrial atmospheric CO2 level, in order to test the sensitivity of the modelled circulation to CO2. Our results show for both continental configurations a bipolar mode for the oceanic circulation displayed by FOAM. Using the Cenomanian/Turonian land-sea mask

  20. Molecular Epidemiology of Emerging Adenovirus 14 Associated Respiratory Disease in the United States

    DTIC Science & Technology

    2010-01-01

    nucleotides and 99.6% amino acids), including pos- session of a single 3-nucleotide GTG insertion corresponding to amino acid 148 (Ser) that was present in all...trainees: the Adenovirus Surveillance Program, 1966–1971. Am J Epidemiol 1973; 97:187–98. 4. Gray GC, Goswami PR, Malasig MD, et al. Adult adenovirus...facility and tertiary-care hospital. Clin Infect Dis 2001; 32:694–700. 43. Gray GC, McCarthy T, Lebeck MG, et al. Genotype prevalence and risk factors

  1. A cost effective real-time PCR for the detection of adenovirus from viral swabs

    PubMed Central

    2013-01-01

    Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture. PMID:23758993

  2. Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

    PubMed

    Kano, M; Matsushita, K; Rahmutulla, B; Yamada, S; Shimada, H; Kubo, S; Hiwasa, T; Matsubara, H; Nomura, F

    2016-01-01

    Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late

  3. In vitro inactivation of Chlamydia trachomatis and of a panel of DNA (HSV-2, CMV, adenovirus, BK virus) and RNA (RSV, enterovirus) viruses by the spermicide benzalkonium chloride.

    PubMed

    Bélec, L; Tevi-Benissan, C; Bianchi, A; Cotigny, S; Beumont-Mauviel, M; Si-Mohamed, A; Malkin, J E

    2000-11-01

    Kinetics of inactivation by the detergent spermicide benzalkonium chloride (BZK) of Chlamydia trachomatis and of a panel of DNA viruses [herpes simplex virus hominis type 2 (HSV-2), cytomegalovirus (CMV), adenovirus (ADV) and BK virus (BKV)] and RNA [respiratory syncytial virus (RSV) and enterovirus (ENV)] were established in accordance with a standardized in vitro protocol. After a 5 min incubation, inactivation of >95% of HSV-2 and CMV was obtained at a concentration of 0.0025% (w/v) (25 Ig/L); concentrations as low as 0.0005%, 0.0050% and 0.0125%, induced a 3.0 log10 reduction in infectivity of HSV-2 and CMV, RSV and ADV, respectively. After a 60 min incubation, concentrations of 0.0125% and 0.050% provided a 3.0 log10 reduction in infectivity of ENV and BKV, respectively. These features indicate that sensitivity to BZK was very high (HSV-2 and CMV) or high (RSV) for enveloped viruses, intermediate (ADV) or low (ENV and BKV) for non-enveloped viruses. Furthermore, BZK had marked antichlamydial activity, showing >99% killing after only a 1 min incubation at a concentration of 0.00125%. BZK demonstrates potent in vitro activity against the majority of microorganisms causing sexually transmitted infectious diseases, including those acting as major genital cofactors of human immunodeficiency virus transmission. These attributes qualify BZK as a particularly attractive candidate for microbicide development.

  4. 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

  5. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

    PubMed Central

    Yang, Y; Nunes, F A; Berencsi, K; Furth, E E; Gönczöl, E; Wilson, J M

    1994-01-01

    An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes. Images PMID:8183921

  6. 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.

  7. Mapping of Ppd-B1, a Major Candidate Gene for Late Heading on Wild Emmer Chromosome Arm 2BS and Assessment of Its Interactions with Early Heading QTLs on 3AL.

    PubMed

    Zhou, Wei; Wu, Shasha; Ding, Mingquan; Li, Jingjuan; Shi, Zhaobin; Wei, Wei; Guo, Jialian; Zhang, Hua; Jiang, Yurong; Rong, Junkang

    2016-01-01

    Wheat heading date is an important agronomic trait determining maturation time and yield. A set of common wheat (Triticum aestivum var. Chinese Spring; CS)-wild emmer (T. turgidum L. subsp. dicoccoides (TDIC)) chromosome arm substitution lines (CASLs) was used to identify and allocate QTLs conferring late or early spike emergence by examining heading date. Genetic loci accelerating heading were found on TDIC chromosome arms 3AL and 7BS, while loci delaying heading were located on 4AL and 2BS. To map QTLs conferring late heading on 2BS, F2 populations derived from two cross combinations of CASL2BS × CS and CASL3AL × CASL2BS were developed and each planted at two times, constituting four F2 mapping populations. Heading date varied continuously among individuals of these four populations, suggesting quantitative characteristics. A genetic map of 2BS, consisting of 23 SSR and one single-stranded conformation polymorphism (SSCP) marker(s), was constructed using these F2 populations. This map spanned a genetic length of 53.2 cM with average marker density of 2.3 cM. The photoperiod-sensitivity gene Ppd-B1 was mapped to chromosome arm 2BS as a SSCP molecular marker, and was validated as tightly linked to a major QTL governing late heading of CASL2BS in all mapping populations. A significant dominance by additive effect of Ppd-B1 with the LUX gene located on 3AL was also detected. CS had more copies of Ppd-B1 than CASL2BS, implying that increased copy number could elevate the expression of Ppd-1 in CS, also increasing expression of LUX and FT genes and causing CS to have an earlier heading date than CASL2BS in long days.

  8. Mapping of Ppd-B1, a Major Candidate Gene for Late Heading on Wild Emmer Chromosome Arm 2BS and Assessment of Its Interactions with Early Heading QTLs on 3AL

    PubMed Central

    Ding, Mingquan; Li, Jingjuan; Shi, Zhaobin; Wei, Wei; Guo, Jialian; Zhang, Hua; Jiang, Yurong; Rong, Junkang

    2016-01-01

    Wheat heading date is an important agronomic trait determining maturation time and yield. A set of common wheat (Triticum aestivum var. Chinese Spring; CS)-wild emmer (T. turgidum L. subsp. dicoccoides (TDIC)) chromosome arm substitution lines (CASLs) was used to identify and allocate QTLs conferring late or early spike emergence by examining heading date. Genetic loci accelerating heading were found on TDIC chromosome arms 3AL and 7BS, while loci delaying heading were located on 4AL and 2BS. To map QTLs conferring late heading on 2BS, F2 populations derived from two cross combinations of CASL2BS × CS and CASL3AL × CASL2BS were developed and each planted at two times, constituting four F2 mapping populations. Heading date varied continuously among individuals of these four populations, suggesting quantitative characteristics. A genetic map of 2BS, consisting of 23 SSR and one single-stranded conformation polymorphism (SSCP) marker(s), was constructed using these F2 populations. This map spanned a genetic length of 53.2 cM with average marker density of 2.3 cM. The photoperiod-sensitivity gene Ppd-B1 was mapped to chromosome arm 2BS as a SSCP molecular marker, and was validated as tightly linked to a major QTL governing late heading of CASL2BS in all mapping populations. A significant dominance by additive effect of Ppd-B1 with the LUX gene located on 3AL was also detected. CS had more copies of Ppd-B1 than CASL2BS, implying that increased copy number could elevate the expression of Ppd-1 in CS, also increasing expression of LUX and FT genes and causing CS to have an earlier heading date than CASL2BS in long days. PMID:26848576

  9. Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

    PubMed

    Westerberg, Sonja; Hagbom, Marie; Rajan, Anandi; Loitto, Vesa; Persson, B David; Allard, Annika; Nordgren, Johan; Sharma, Sumit; Magnusson, Karl-Eric; Arnberg, Niklas; Svensson, Lennart

    2018-04-01

    Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41

  10. Pancreatic cancer therapy with combined mesothelin-redirected chimeric antigen receptor T cells and cytokine-armed oncolytic adenoviruses

    PubMed Central

    Watanabe, Keisuke; Luo, Yanping; Da, Tong; Scholler, John; Keith, Brian; Young, Regina M.; Sorsa, Suvi; Siurala, Mikko; Havunen, Riikka; Tähtinen, Siri; Hemminki, Akseli

    2018-01-01

    Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA. PMID:29618658

  11. 2D CFT partition functions at late times

    NASA Astrophysics Data System (ADS)

    Dyer, Ethan; Gur-Ari, Guy

    2017-08-01

    We consider the late time behavior of the analytically continued partition function Z( β + it) Z( β - it) in holographic 2 d CFTs. This is a probe of information loss in such theories and in their holographic duals. We show that each Virasoro character decays in time, and so information is not restored at the level of individual characters. We identify a universal decaying contribution at late times, and conjecture that it describes the behavior of generic chaotic 2 d CFTs out to times that are exponentially large in the central charge. It was recently suggested that at sufficiently late times one expects a crossover to random matrix behavior. We estimate an upper bound on the crossover time, which suggests that the decay is followed by a parametrically long period of late time growth. Finally, we discuss gravitationally-motivated integrable theories and show how information is restored at late times by a series of characters. This hints at a possible bulk mechanism, where information is restored by an infinite sum over non-perturbative saddles.

  12. Cost-effectiveness analysis of reacquiring and using adenovirus types 4 and 7 vaccines in naval recruits.

    PubMed

    Hyer, R N; Howell, M R; Ryan, M A; Gaydos, J C

    2000-05-01

    Adenovirus vaccines have controlled acute respiratory disease (ARD) in military recruits since 1971. Vaccine production, however, ceased and new facilities are required. We assessed whether reacquiring and using vaccines in naval recruits is cost-effective. Three policy options were evaluated: no vaccination, seasonal vaccination, and year-round vaccination. Morbidity (outpatient and inpatient), illness costs (medical and lost training), and vaccine program costs (start-up, acquisition, and distribution) were modeled using a decision-analytic method. Results were based on a cohort of 49,079 annual trainees, a winter vaccine-preventable ARD rate of 2.6 cases per 100 person-weeks, a summer incidence rate at 10% of the winter rate, a hospitalization rate of 7.6%, and a production facility costing US$12 million. Compared to no vaccination, seasonal vaccination prevented 4,015 cases and saved $2.8 million per year. Year-round vaccination prevented 4,555 cases and saved $2.6 million. Reacquiring and using adenovirus vaccines seasonally or year-round saves money and averts suffering.

  13. Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

    PubMed

    Wang, Xiaoyan; Wang, Jian-Ping; Rao, Xiao-Mei; Price, Janet E; Zhou, Heshan S; Lachman, Lawrence B

    2005-01-01

    Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was

  14. Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum.

    PubMed

    Cianciola, Nicholas L; Chung, Stacey; Manor, Danny; Carlin, Cathleen R

    2017-03-15

    Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich "lipid rafts." Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune

  15. Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa.

    PubMed

    van Heerden, J; Ehlers, M M; Heim, A; Grabow, W O K

    2005-01-01

    Human adenoviruses (HAds), of which there are 51 serotypes, are associated with gastrointestinal, respiratory, urinary tract and eye infections. The importance of water in the transmission of HAds and the potential health risks constituted by HAds in these environments are widely recognized. Adenoviruses have not previously been quantified in river and treated drinking water samples. In this study, HAds in river water and treated drinking water sources in South Africa were detected, quantified and typed. Adenoviruses were recovered from the water samples using a glass wool adsorption-elution method followed by polyethylene glycol/NaCl precipitation for secondary concentration. The sensitivity and specificity of two nested PCR methods were compared for detection of HAds in the water samples. Over a 1-year period (June 2002 to July 2003), HAds were detected in 5.32% (10/188) of the treated drinking water and 22.22% (10/45) of river water samples using the conventional nested PCR method. The HAds detected in the water samples were quantified using a real-time PCR method. The original treated drinking water and river water samples had an estimate of less than one copy per litre of HAd DNA present. The hexon-PCR products used for typing HAds were directly sequenced or cloned into plasmids before sequencing. In treated drinking water samples, species D HAds predominated. In addition, adenovirus serotypes 2, 40 and 41 were each detected in three different treated drinking water samples. Most (70%) of the HAds detected in river water samples analysed were enteric HAds (serotypes 40 and 41). One HAd serotype 2 and two species D HAds were detected in the river water. Adenoviruses detected in river and treated drinking water samples were successfully quantified and typed. The detection of HAds in drinking water supplies treated and disinfected by internationally recommended methods, and which conform to quality limits for indicator bacteria, warrants an investigation of the

  16. Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts.

    PubMed

    Huang, Jing Li; LaRocca, Christopher J; Yamamoto, Masato

    2016-09-19

    Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

  17. Adenovirus-vectored Ebola vaccines.

    PubMed

    Gilbert, Sarah C

    2015-01-01

    The 2014 outbreak of Ebola virus disease in West Africa has highlighted the need for the availability of effective vaccines against outbreak pathogens that are suitable for use in frontline workers who risk their own health in the course of caring for those with the disease, and also for members of the community in the affected area. Along with effective contact tracing and quarantine, use of a vaccine as soon as an outbreak is identified could greatly facilitate rapid control and prevent the outbreak from spreading. This review describes the progress that has been made in producing and testing adenovirus-based Ebola vaccines in both pre-clinical and clinical studies, and considers the likely future use of these vaccines.

  18. Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

    PubMed

    Biasiotto, Roberta; Akusjärvi, Göran

    2015-01-28

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Xinheng; Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642; South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642

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

  20. Neuropsychological Predictors of Dementia in Late-Life Major Depressive Disorder

    PubMed Central

    Potter, Guy G.; Wagner, H. Ryan; Burke, James R.; Plassman, Brenda L.; Welsh-Bohmer, Kathleen A.; Steffens, David C.

    2012-01-01

    Objective Major Depressive Disorder (MDD) is a likely risk factor for dementia, but some cases of MDD in older adults may actually represent a prodrome of this condition. The purpose of this study was to use neuropsychological test scores to predict conversion to dementia in a sample of depressed older adults diagnosed as nondemented at time of neuropsychological testing. Design Longitudinal, with mean follow-up of 5.45 years. Setting Outpatient depression treatment study at Duke University Participants 30 nondemented individuals depressed at time of neuropsychological testing and later diagnosed with incident dementia; 149 nondemented individuals depressed at time of neuropsychological testing and a diagnosis of cognitively normal. Methodology All participants received clinical assessment of depression, were assessed to rule out prevalent dementia at time of study enrollment, completed neuropsychological testing at time of study enrollment, and were diagnosed for cognitive disorders on an annual basis. Results Non-demented, acutely depressed older adults who converted to dementia during the study period exhibited broadly lower cognitive performances at baseline than acutely depressed individuals who remained cognitively normal. Discriminant function analysis indicated that 2 neuropsychological tests, CERAD Recognition Memory and Trail Making B, best predicted dementia conversion. Conclusions Depressed older adults with cognitive deficits in the domains of memory and executive functions during acute depression are at higher risk for developing dementia. Some cases of late-life depression may reflect a prodrome of dementia in which clinical manifestation of mood changes may co-occur with emerging cognitive deficits. PMID:23395197

  1. Comparison of intracerebral inoculation and osmotic blood-brain barrier disruption for delivery of adenovirus, herpesvirus, and iron oxide particles to normal rat brain.

    PubMed Central

    Muldoon, L. L.; Nilaver, G.; Kroll, R. A.; Pagel, M. A.; Breakefield, X. O.; Chiocca, E. A.; Davidson, B. L.; Weissleder, R.; Neuwelt, E. A.

    1995-01-01

    Delivery of adenovirus, herpes simplex virus (HSV), and paramagnetic monocrystalline iron oxide nanoparticles (MION) to rat brain (n = 64) was assessed after intracerebral inoculation or osmotic disruption of the blood-brain barrier (BBB). After intracerebral inoculation, the area of distribution was 7.93 +/- 0.43 mm2 (n = 9) for MION and 9.17 +/- 1.27 mm2 (n = 9) for replication-defective adenovirus. The replication-compromised HSV RH105 spread to 14.00 +/- 0.87 mm2 (n = 8), but also had a large necrotic center (3.54 +/- 0.47 mm2). No infection was detected when virus was administered intra-arterially without hyperosmotic mannitol. After osmotic BBB disruption, delivery of the viruses and MIONs was detected throughout the disrupted cerebral cortex. Positive staining was found in 4 to 845 cells/100 microns thick coronal brain section (n = 7) after adenovirus administration, and in 13 to 197 cells/section (n = 8) after HSV administration. Cells of glial morphology were more frequently stained after administration of adenovirus, whereas neuronal cells were preferentially stained after delivery of both HSV vectors and MION. In a preliminary test of vector delivery in the feline, MION was detected throughout the white matter tracts after inoculation into normal cat brain. Thus MION may be a tool for use in vivo, to monitor the delivery of virus to the central nervous system. Additionally, BBB disruption may be an effective method to globally deliver recombinant viruses to the CNS. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7495307

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

    PubMed Central

    2011-01-01

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

  3. Adenovirus is a key pathogen in hemorrhagic cystitis associated with bone marrow transplantation.

    PubMed

    Akiyama, H; Kurosu, T; Sakashita, C; Inoue, T; Mori Si; Ohashi, K; Tanikawa, S; Sakamaki, H; Onozawa, Y; Chen, Q; Zheng, H; Kitamura, T

    2001-05-01

    Late-onset hemorrhagic cystitis (HC) is a well-known complication of bone marrow transplantation (BMT) that is mainly attributed to infection with BK virus (BKV) and adenovirus (AdV). From 1986 through 1998, 282 patients underwent BMT, and 45 of them developed HC. Urine samples tested positive for AdV in 26 patients, of which 22 showed virus type 11. Among patients who underwent allogeneic BMT, logistic regression analysis revealed acute graft-versus-host disease (grade, > or = 2) to be the most significant predictive factor for HC (P < .0001). In addition, a total of 193 urine samples regularly obtained from 26 consecutive patients who underwent allogeneic BMT were examined for BKV, JC virus (JCV), and AdV by means of polymerase chain reaction. Of patients without HC, approximately 30% of the specimens tested positive for BKV (58 samples) and JCV (55 samples), whereas 5 (3%) tested positive for AdV. Of the 3 samples obtained from patients with HC, the numbers of positive results for BKV, JCV, and AdV were 3, 1, and 1, respectively; the numbers of positive results increased to 14 of 17, 9 of 17, and 10 of 17, respectively, when we added another 14 samples obtained from 14 patients with HC (P < .0001, P = .026, and P < .0001, respectively). In conclusion, there was significant correlation between AdV and HC in the patients we studied.

  4. A dynamical systems model for combinatorial cancer therapy enhances oncolytic adenovirus efficacy by MEK-inhibition.

    PubMed

    Bagheri, Neda; Shiina, Marisa; Lauffenburger, Douglas A; Korn, W Michael

    2011-02-01

    Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced

  5. Oncolytic Adenovirus Complexes Coated with Lipids and Calcium Phosphate for Cancer Gene Therapy.

    PubMed

    Chen, Jianhua; Gao, Pei; Yuan, Sujing; Li, Rongxin; Ni, Aimin; Chu, Liang; Ding, Li; Sun, Ying; Liu, Xin-Yuan; Duan, Yourong

    2016-12-27

    Oncolytic adenovirus (Onco Ad ) is a promising therapeutic agent for treating cancer. However, the therapeutic potential of Onco Ad is hindered by hepatic sequestration and the host immune response in vivo. Here, we constructed a PEG/Lipids/calcium phosphate (CaP)-Onco Ad (PLC-Onco Ad ) delivery system for ZD55-IL-24, an oncolytic adenovirus that carries the IL-24 gene. The negatively charged PLC-ZD55-IL-24 were disperse and resisted serum-induced aggregation. Compared to naked ZD55-IL-24, the systemic administration of PLC-ZD55-IL-24 in BALB/c mice resulted in reduced liver sequestration and systemic toxicity and evaded the innate immune response. In addition, masking the surface of Onco Ad protected it from neutralization by pre-existing neutralizing antibody. PLC-Onco Ad achieved efficient targeted delivery in Huh-7-bearing nude mice, and intravenous administration of a high dose of PLC-ZD55-IL-24 increased therapeutic efficacy without inducing toxicity. The developed PLC-Onco Ad delivery system represents a promising improvement for oncolytic adenovirus-based cancer gene therapy in vivo.

  6. Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ugai, Hideyo; Yamasaki, Takahito; Hirose, Megumi

    2005-06-17

    Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 deg C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-formingmore » units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10{sup 10} pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.« less

  7. Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

    PubMed Central

    Eichholz, Karsten; Bru, Thierry; Tran, Thi Thu Phuong; Fernandes, Paulo; Mennechet, Franck J. D.; Manel, Nicolas; Alves, Paula; Perreau, Matthieu

    2016-01-01

    Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs. PMID:27636895

  8. Characterization of a major late herpes simplex virus type 1 mRNA.

    PubMed

    Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K

    1981-05-01

    A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

  9. “Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

    PubMed Central

    Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

    2001-01-01

    Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

  10. Prospective inter-relationships between late adolescent personality and major depressive disorder in early adulthood.

    PubMed

    Wilson, S; DiRago, A C; Iacono, W G

    2014-02-01

    A well-established body of literature demonstrates concurrent associations between personality traits and major depressive disorder (MDD), but there have been relatively few investigations of their dynamic interplay over time. Prospective inter-relationships between late-adolescent personality and MDD in early adulthood were examined in a community sample of male and female twins from the Minnesota Twin Family Study (MTFS; n = 1252). Participants were classified into naturally occurring MDD groups based on the timing (adolescent versus adult onset) and course (chronic/recurrent versus remitting) of MDD. MDD diagnoses were assessed at ages 17, 20, 24 and 29 years, and personality traits [negative emotionality (NEM), positive emotionality (PEM) and constraint (CON)] were assessed at ages 17, 24 and 29 years. Multilevel modeling (MLM) analyses indicated that higher age-17 NEM was associated with the subsequent development of MDD, and any MDD, regardless of onset or course, was associated with higher NEM up to age 29. Moreover, the chronic/recurrent MDD groups failed to show the normative decrease in NEM from late adolescence to early adulthood. Lower age-17 PEM was also associated with the subsequent development of MDD but only among the chronic/recurrent MDD groups. Finally, the adolescent-onset MDD groups reported lower age-17 CON relative to the never-depressed and adult-onset MDD groups. Taken together, the results speak to the role of personality traits for conferring risk for the onset of MDD in late adolescence and early adulthood, in addition to the pernicious implications of chronic/recurrent MDD, particularly when it onsets during adolescence, for adaptive personality development.

  11. Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

    PubMed

    Warimwe, George M; Lorenzo, Gema; Lopez-Gil, Elena; Reyes-Sandoval, Arturo; Cottingham, Matthew G; Spencer, Alexandra J; Collins, Katharine A; Dicks, Matthew D J; Milicic, Anita; Lall, Amar; Furze, Julie; Turner, Alison V; Hill, Adrian V S; Brun, Alejandro; Gilbert, Sarah C

    2013-12-05

    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. 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. 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. 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.

  12. [Effect of topical application of a recombinant adenovirus carrying promyelocytic leukemia gene in a psoriasis-like mouse model].

    PubMed

    Wang, Qiongyu; Zhang, Aijun; Ma, Huiqun; Wang, Shijie; Ma, Yunyun; Zou, Xingwei; Li, Ruilian

    2013-03-01

    To investigate the effects of topical treatment with adenovirus-mediated promyelocytic leukemia gene (PML) gene in a psoriasis-like mouse model. The effect of adenovirus-mediated PML gene on the granular layer of mouse tail scale epidermis and epithelial mitosis were observed on longitudinal histological sections prepared from the tail skin and vaginal epithelium of the mice. Adenovirus-mediated PML gene significantly inhibited mitosis of mouse vaginal epithelial cells and promoted the formation of granular layer in mouse tail scale epidermis. The therapeutic effect of PML gene in the psoriasis-like mouse model may be associated with increased granular cells and suppressed epidemic cell proliferation.

  13. Climate, pCO2 and terrestrial carbon cycle linkages during late Palaeozoic glacial-interglacial cycles

    NASA Astrophysics Data System (ADS)

    Montañez, Isabel P.; McElwain, Jennifer C.; Poulsen, Christopher J.; White, Joseph D.; Dimichele, William A.; Wilson, Jonathan P.; Griggs, Galen; Hren, Michael T.

    2016-11-01

    Earth's last icehouse, 300 million years ago, is considered the longest-lived and most acute of the past half-billion years, characterized by expansive continental ice sheets and possibly tropical low-elevation glaciation. This atypical climate has long been attributed to anomalous radiative forcing promoted by a 3% lower incident solar luminosity and sustained low atmospheric pCO2 (<=300 ppm). Climate models, however, indicate a CO2 sensitivity of ice-sheet distribution and sea-level response that questions this long-standing climate paradigm by revealing major discrepancy between hypothesized ice distribution, pCO2, and geologic records of glacioeustasy. Here we present a high-resolution record of atmospheric pCO2 for 16 million years of the late Palaeozoic, developed using soil carbonate-based and fossil leaf-based proxies, that resolves the climate conundrum. Palaeo-fluctuations on the 105-yr scale occur within the CO2 range predicted for anthropogenic change and co-vary with substantial change in sea level and ice volume. We further document coincidence between pCO2 changes and repeated restructuring of Euramerican tropical forests that, in conjunction with modelled vegetation shifts, indicate a more dynamic carbon sequestration history than previously considered and a major role for terrestrial vegetation-CO2 feedbacks in driving eccentricity-scale climate cycles of the late Palaeozoic icehouse.

  14. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

    PubMed

    Stanley, Daphne A; Honko, Anna N; Asiedu, Clement; Trefry, John C; Lau-Kilby, Annie W; Johnson, Joshua C; Hensley, Lisa; Ammendola, Virginia; Abbate, Adele; Grazioli, Fabiana; Foulds, Kathryn E; Cheng, Cheng; Wang, Lingshu; Donaldson, Mitzi M; Colloca, Stefano; Folgori, Antonella; Roederer, Mario; Nabel, Gary J; Mascola, John; Nicosia, Alfredo; Cortese, Riccardo; Koup, Richard A; Sullivan, Nancy J

    2014-10-01

    Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

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

    PubMed Central

    2010-01-01

    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. PMID:21054831

  16. 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.

  17. 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

  18. 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...correlation with illness. Among the samples tested, only pathogens associated with FRI, such as adenovirus 4 and enterovirus 68, revealed strong temporal...In this study, RPM technology was used to explore the distribution of, and associations between, HAdV, picorna- viruses (HRV and human enterovirus [HEV

  19. Amplified and persistent immune responses generated by single-cycle replicating adenovirus vaccines.

    PubMed

    Crosby, Catherine M; Nehete, Pramod; Sastry, K Jagannadha; Barry, Michael A

    2015-01-01

    Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered "single-cycle" adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not

  20. Epizootiology and pathologic findings associated with a newly described adenovirus in the red squirrel, Sciurus vulgaris.

    PubMed

    Martínez-Jiménez, David; Graham, David; Couper, David; Benkö, Maria; Schöniger, Sandra; Gurnell, John; Sainsbury, Anthony W

    2011-04-01

    An infectious disease caused by Squirrelpox virus has contributed to the decline of red squirrels, Sciurus vulgaris, in the British Isles. Because of the heightened disease surveillance activity in red squirrels, adenovirus infection with associated mortality has been detected. Adenoviral disease is described in other rodent species usually associated with stressors. Here we 1) describe the pathologic findings in red squirrels found dead with adenoviral infection and gastrointestinal disease, and 2) investigate the epizootiology of the disease through pathologic investigation, scanning surveillance, and virologic studies. Ten red squirrels involved in conservation studies were diagnosed with adenoviral infection by electron microscopy or PCR. All squirrels exhibited diarrhea and small intestinal inflammation or hemorrhage was evident in seven cases. Lesions indicative of splenic lymphocytolysis were observed in one squirrel and leukocytic hepatitis in another. No adenovirus was detected in grey squirrels, Sciurus carolinensis, inhabiting the same forest area, but previous serologic studies showed that grey squirrels cannot be discounted as a reservoir of the virus. Scanning surveillance showed that 12% of 493 red squirrels had diarrheal disease and two of 13 free-living red squirrels with diarrheal disease had adenovirus infection. Adenoviral disease in declining free-living wild red squirrel populations in the British Isles occurs at a detectable frequency and its impact on the conservation of this species deserves further attention.

  1. Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses and male-specific coliphages from seawater.

    PubMed

    Gibbons, C D; Rodríguez, R A; Tallon, L; Sobsey, M D

    2010-08-01

    To evaluate the electropositive, alumina nanofibre (NanoCeram) cartridge filter as a primary concentration method for recovering adenovirus, norovirus and male-specific coliphages from natural seawater. Viruses were concentrated from 40 l of natural seawater using a NanoCeram cartridge filter and eluted from the filter either by soaking the filter in eluent or by recirculating the eluent continuously through the filter using a peristaltic pump. The elution solution consisted of 3% beef extract and 0.1 mol l(-1) of glycine. The method using a peristaltic pump was more effective in removing the viruses from the filter. High recoveries of norovirus and male-specific coliphages (>96%) but not adenovirus (<3%) were observed from seawater. High adsorption to the filter was observed for adenovirus and male-specific coliphages (>98%). The adsorption and recovery of adenovirus and male-specific coliphages were also determined for fresh finished water and source water. The NanoCeram cartridge filter was an effective primary concentration method for the concentration of norovirus and male-specific coliphages from natural seawater, but not for adenovirus, in spite of the high adsorption of adenovirus to the filter. This study demonstrates that NanoCeram cartridge filter is an effective primary method for concentrating noroviruses and male-specific coliphages from seawater, thereby simplifying collection and processing of water samples for virus recovery.

  2. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

    EPA Science Inventory

    Human adenovirus is relatively resistant to UV radiation and has been used as a conservative testing microbe for evaluations of UV disinfection systems as components of water treatment processes. In this study, we attempted to validate the applicability of integrated cell culture...

  3. Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues.

    PubMed

    Koski, Anniina; Bramante, Simona; Kipar, Anja; Oksanen, Minna; Juhila, Juuso; Vassilev, Lotta; Joensuu, Timo; Kanerva, Anna; Hemminki, Akseli

    2015-10-01

    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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se; Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se; Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se

    2012-11-25

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

  5. Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens.

    PubMed

    Dar, Arshud; Gomis, Susantha; Shirley, Ian; Mutwiri, George; Brownlie, Robert; Potter, Andrew; Gerdts, Volker; Tikoo, Suresh K

    2012-03-01

    Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

  6. 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.

  7. Inference of pCO2 Levels during the Late Cretaceous Using Fossil Lauraceae

    NASA Astrophysics Data System (ADS)

    Richey, J. D.; Upchurch, G. R.

    2011-12-01

    Botanical estimates of pCO2 for the Late Cretaceous have most commonly used Stomatal Index (SI) in fossil Ginkgo. Recently, SI in fossil Lauraceae has been used to infer changes in pCO2 across the Cenomanian-Turonian boundary, based on the relation between SI and pCO2 in extant Laurus and Hypodaphnis. To provide a broad-scale picture of pCO2 based on fossil Lauraceae, we examined dispersed cuticle of the leaf macrofossil genus Pandemophyllum from: 1) the early to middle Cenomanian of the Potomac Group of Maryland (Mauldin Mountain locality, lower Zone III) and 2) the Maastrichtian of southern Colorado (Raton Basin, Starkville South and Berwind Canyon localities). These samples fall within the Late Cretaceous decline in pCO2 inferred from geochemical modeling and other proxies. SI was calculated from fossil cuticle fragments using ImageJ and counts of up to 56,000 cells per sample, a far greater number of cells than are counted in most studies. CO2 levels were estimated using the relation between SI and CO2 published for Laurus nobilis and Hypodaphnis zenkeri. Early to middle Cenomanian atmospheric pCO2 is estimated at 362-536 parts per million (ppm). This represents the absolute minimum and maximum estimated CO2 levels from the ±95% confidence intervals (CI) of the relation between SI and CO2 for the modern equivalents, and SI ± 1 Standard Deviation (SD) in the fossil genus Pandemophyllum. Late Maastrichtian atmospheric pCO2 is estimated at 358-534 ppm. The Maastrichtian estimates falls within the range of published estimates from other proxies. The Cenomanian estimate, in contrast, is low relative to most other estimates. The 95% confidence intervals of our pCO2 estimates overlap each other and many of the assemblages published by Barclay et al. (2010) for Lauraceae across the Cenomanian-Turonian boundary. This could indicate that 1) pCO2 did not undergo a major long-term decline during the Late Cretaceous, 2) Lauraceae show low sensitivity to high pCO2, or 3

  8. Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes.

    PubMed

    Lee, Tim W R; Blair, G Eric; Matthews, David A

    2003-12-01

    During adenovirus infection, following capsid dissociation, core protein VII enters the host cell nucleus complexed with adenovirus DNA. In order to determine whether protein VII may have an active role in this nuclear import, regions of the preVII gene were amplified by PCR, and further oligonucleotide mutants were designed with site-directed mutation of codons for the basic amino acids arginine and lysine. Fragments were cloned into a mammalian expression plasmid to express the peptides as N-terminal fusions to enhanced green fluorescent protein. Results demonstrate that preVII protein contains both nuclear and nucleolar targeting sequences. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, the data suggest that protein VII may bind to human chromosomes by means of two distinct domains, one sharing homology with the N-terminal regulatory tail of histone H3.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-01-01

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

  11. Adenovirus type 5 intrinsic adsorption rates measured by surface plasmon resonance.

    PubMed

    Roper, D Keith; Nakra, Shamit

    2006-01-01

    Intrinsic adsorption rates of whole adenovirus type 5 (Ad5) onto a diethylaminoethyl (DEAE) anion exchange surface are measured for the first time by surface plasmon resonance (SPR). Fitting SPR sensorgrams to a two-compartment mass transport reaction model distinguishes intrinsic adsorption rates from slow diffusive Ad5 mass transport. Ad5 is a widely used viral vector for gene therapy that binds electrostatically to surfaces of cells and synthetics such as membranes, chromatographic resins, and glass. Increasing NaCl concentration from 4.8 to 14.4mM shifts binding of whole Ad5 from diffusion control to a regime where both sorption and diffusion affect binding. Intrinsic adsorption rates for Ad5-DEAE interaction are 16 times faster than intrinsic adsorption rates for Ad5 fiber knob interacting with soluble extracellular domain of coxsackievirus adenovirus receptors (s-CAR).

  12. Mechanisms of pathogenesis of emerging adenoviruses.

    PubMed

    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.

  13. Evaluation of sampling technique and transport media for the diagnostics of adenoviral eye infections. Adenovirus sampling and transport.

    PubMed

    Wölfel, Roman; Pfeffer, Martin; Essbauer, Sandra; Nerkelun, Sylke; Dobler, Gerhard

    2006-11-01

    Human adenoviruses (HAdV) may cause pharyngoconjunctival fever, follicular conjunctivitis or epidemic keratoconjunctivitis (EKC). Especially, outbreaks of the latter may lead to severe economic losses when preventive measures are implemented too late. Thus, a safe sampling method, proper specimen transport conditions and a fast and sensitive diagnostic technique is mandatory. Two commercially available virus transport systems (VTS) were compared with two NaCl-moisturised sampling devices, one of which comprises Dacron-tipped plastic-shafted swabs and the other a cotton-tipped wood-shafted swab, available in most ophthalmologists' offices. Downstream methods for specific detection of HAdV included direct immunofluorescence assay (IFA) of conjunctival swabs, virus isolation by cell culture and quantitative real-time polymerase chain reaction (qPCR). Furthermore, the influence of application of local anaesthetics prior to swabbing on subsequent detection of HAdV was investigated. Application of local anaesthetics had a positive influence on the amount of swabbed cells, thus increasing the chance of obtaining positive results by IFA. Neither isolation of HAdV by cell culture nor by qPCR was negatively influenced by this pretreatment. Surprisingly, both commercially available VTS performed significantly worse than the NaCl-moisturised swabs. This was shown with regard to virus recovery rates in cell culture as well as viral genome copy numbers in the qPCR. Based on our results, the following recommendations are provided to improve sampling, transport and diagnostic techniques regarding conjunctival swabs for diagnosis of human adenovirus infection: (1) application of local anaesthetics, (2) NaCl-moisturised VTS for shipment of specimens, and (3) detection of HAdV by qPCR. The latter method proved to be superior to virus isolation by cell culture, including subsequent identification by IFA, because it is faster, more sensitive and allows simultaneous handling of a number

  14. The late Miocene 'paradox' of the CO2 climate sensitivity (Invited)

    NASA Astrophysics Data System (ADS)

    Zhang, Y.; Pagani, M.

    2013-12-01

    Ancient climates provide opportunities for studying the impact of CO2 change on global temperatures. While advances in CO2-reconstruction techniques are yielding a clearer picture of the Cenozoic history of CO2 (Beerling and Royer, 2011), the late Miocene (~12-5 Ma) remains enigmatic. For example, recent sea-surface temperature reconstructions from 12-5 Ma have shown that mid-latitude and equatorial regions of the Pacific cooled 6°C (LaRiviere et al., 2012) and 2°C (Zhang et al., 2013), respectively. This cooling trend was probably initiated at the mid-Miocene climate transition (14 Ma), and continued into the Plio-Pleistocene. However, existing compilation of late Miocene - Pliocene CO2 records show little variability, with some indicating a rise in CO2 concurrent with global cooling. Here we present four continuous alkenone-based CO2 records using Pacific sediment samples (ODP Sites 769, 806, 850 and 1143), from late Miocene to Pliocene. Compound-specific carbon isotope measurements show a broad decrease in alkenone δ13C values in all four sites, suggesting increasing pCO2 levels in the late Miocene. Decreasing ocean temperature and increasing pCO2 in the late Miocene appears to challenge a leading climatic role for CO2 during this time. Alternatively, alkenone-CO2 estimates are flawed in the late Miocene because factors other than CO2, such as algal growth rate, cell geometry, and carbon-fixation pathways, can influence carbon isotopic fractionation during algae growth. We explore the uncertainty of the alkenone-CO2 methodology and assess the potential influence that non-CO2 variables have in producing spurious CO2 estimates and trends. Beerling, D.J., Royer, D.L., 2011. Convergent Cenozoic CO2 history. Nat. Geosci. 4, 418-420. LaRiviere, J.P., Ravelo, A.C., Crimmins, A., Dekens, P.S., Ford, H.L., Lyle, M., Wara, M.W., 2012. Late Miocene decoupling of oceanic warmth and atmospheric carbon dioxide forcing. Nature 486, 97-100. Zhang, Y.G., Pagani, M., Liu, Z

  15. 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.

  16. 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. © 2015 Wiley Periodicals, Inc.

  17. The Kinase Activity of Ataxia-Telangiectasia Mutated Interferes with Adenovirus E4 Mutant DNA Replication

    PubMed Central

    Gautam, Dipendra

    2013-01-01

    Adenovirus (Ad) mutants that lack early region 4 (E4) are unable to produce the early regulatory proteins that normally inactivate the Mre11/Rad50/Nbs1 (MRN) sensor complex, which is a critical component for the ability of cells to respond to DNA damage. E4 mutant infection therefore activates a DNA damage response, which in turn interferes with a productive viral infection. MRN complex proteins localize to viral DNA replication centers in E4 mutant-infected cells, and this complex is critical for activating the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which phosphorylate numerous substrates important for DNA repair, cell cycle checkpoint activation, and apoptosis. E4 mutant growth defects are substantially rescued in cells lacking an intact MRN complex. We have assessed the role of the downstream ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. We did not identify a role for either ATM or ATR in “repair” of E4 mutant genomes to form concatemers. ATR was also not observed to contribute to E4 mutant defects in late protein production. In contrast, the kinase activity of ATM was important for preventing efficient E4 mutant DNA replication and late gene expression. Our results suggest that the MRN complex interferes with E4 mutant DNA replication at least in part through its ability to activate ATM. PMID:23740981

  18. Immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying B and T cell epitopes of foot-and-mouth disease.

    PubMed

    Pan, Qunxing; Wang, Hui; Ouyang, Wei; Wang, Xiaoli; Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; He, Kongwang

    2016-01-20

    Virus-like particles (VLPs) vaccines combine many of the advantages of whole-virus vaccines and recombinant subunit vaccines, integrating key features that underlay their immunogenicity, safety and protective potential. We have hypothesized here the effective insertion of the VP1 epitopes (three amino acid residues 21-40, 141-160 and 200-213 in VP1, designated VPe) of foot-and-mouth disease (FMDV) within the external loops of PPV VP2 could be carried out without altering assembly based on structural and antigenic data. To investigate the possibility, development of two recombinant adenovirus rAd-PPV:VP2-FMDV:VPe a or rAd-PPV:VP2-FMDV:VPe b were expressed in HEK-293 cells. Out of the two insertion strategies tested, one of them tolerated an insert of 57 amino acids in one of the four external loops without disrupting the VLPs assembly. Mice were inoculated with the two recombinant adenoviruses, and an immunogenicity study showed that the highest levels of FMDV-specific humoral responses and T cell proliferation could be induced by rAd-PPV:VP2-FMDV:VPe b expressing hybrid PPV:VLPs (FMDV) in the absence of an adjuvant. Then, the protective efficacy of inoculating swine with rAd-PPV:VP2-FMDV:VPe b was tested. All pigs inoculated with rAd-PPV:VP2-FMDV:VPe b were protected from viral challenge, meanwhile the neutralizing antibody titers were significantly higher than those in the group inoculated with swine FMD type O synthetic peptide vaccine. Our results clearly demonstrate the potential usefulness of adenovirus-derived PPV VLPs as a vaccine strategy in prevention of FMDV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.

    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. Whenmore » 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.« less

  20. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

    PubMed Central

    Mittereder, N; March, K L; Trapnell, B C

    1996-01-01

    Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. [Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

    PubMed

    Ruan, Jia; Ren, Dong-xia; Yang, Dan-ni; Long, Pin-pin; Zhao, Hong-yue; Wang, Yi-qi; Li, Yong-xin

    2015-07-01

    To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.

  3. Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor.

    PubMed Central

    Zamanian, M; La Thangue, N B

    1992-01-01

    The retinoblastoma (Rb) gene product forms a complex with the cellular transcription factor DRTF1, a property assumed to be important for mediating negative growth control because certain viral oncogenes, such as adenovirus E1a, prevent this interaction and mutant Rb alleles, which have lost the capacity to regulate growth, encode proteins that fail to associate with DRTF1. In this study, we show that the wild-type Rb protein can specifically repress transcription from promoters driven by DRTF1 whereas a naturally occurring mutant Rb protein cannot. Furthermore, Rb-mediated transcriptional repression can be overridden by adenovirus E1a; this requires regions in E1a necessary for cellular transformation. The Rb protein therefore acts in trans to repress the transcriptional activity of DRTF1 whereas adenovirus E1a prevents this interaction and thus maintains DRTF1 in a constitutively active state. The Rb protein and adenovirus E1a therefore have opposite effects on the activity of a common molecular target. Transcriptional repression mediated by the Rb protein and inactivation of repression by the E1a protein are likely to play an important role in mediating their biological effects. Images PMID:1385776

  4. Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

    PubMed Central

    Liikanen, Ilkka; Ahtiainen, Laura; Hirvinen, Mari LM; Bramante, Simona; Cerullo, Vincenzo; Nokisalmi, Petri; Hemminki, Otto; Diaconu, Iulia; Pesonen, Sari; Koski, Anniina; Kangasniemi, Lotta; Pesonen, Saila K; Oksanen, Minna; Laasonen, Leena; Partanen, Kaarina; Joensuu, Timo; Zhao, Fang; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum—a possible indicator of immune response—increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy. PMID:23546299

  5. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. An adenovirus-vectored nasal vaccine confers rapid and sustained protection against anthrax in a single-dose regimen.

    PubMed

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

    2013-01-01

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

  7. Avian Influenza Vaccination in Chickens and Pigs with Replication-Competent Adenovirus–Free Human Recombinant Adenovirus 5

    PubMed Central

    Toro, Haroldo; van Ginkel, Frederik W.; Tang, De-chu C.; Schemera, Bettina; Rodning, Soren; Newton, Joseph

    2010-01-01

    SUMMARY Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular vaccination with replication-competent adenovirus (RCA)-free human recombinant adenovirus serotype 5 (Ad5) encoding AI virus H5 (AdTW68.H5) or H7 (AdCN94.H7) hemagglutinins. We evaluated bivalent in ovo vaccination with AdTW68.H5 and AdCN94.H7 and determined that vaccinated chickens developed robust hemagglutination inhibition (HI) antibody levels to both H5 and H7 AI strains. Additionally, we evaluated immune responses of 1-day-old chickens vaccinated via spray with AdCN94.H7. These birds showed increased immunoglobulin A responses in lachrymal fluids and increased interleukin-6 expression in Harderian gland–derived lymphocytes. However, specific HI antibodies were not detected in the sera of these birds. Because pigs might play a role as a “mixing vessel” for the generation of pandemic influenza viruses we explored the use of RCA-free adenovirus technology to immunize pigs against AI virus. Weanling piglets vaccinated intramuscularly with a single dose of RCA-free AdTW68.H5 developed strong systemic antibody responses 3 wk postvaccination. Intranasal application of AdTW68.H5 in piglets resulted in reduced vaccine coverage, i.e., 33% of pigs (2/6) developed an antibody response, but serum antibody levels in those successfully immunized animals were similar to intramuscularly vaccinated animals. PMID:20521636

  8. Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

    PubMed

    Doronin, K; Kuppuswamy, M; Toth, K; Tollefson, A E; Krajcsi, P; Krougliak, V; Wold, W S

    2001-04-01

    We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a

  9. The ACE Gene Is Associated with Late-Life Major Depression and Age at Dementia Onset in a Population-Based Cohort.

    PubMed

    Zettergren, Anna; Kern, Silke; Gustafson, Deborah; Gudmundsson, Pia; Sigström, Robert; Östling, Svante; Eriksson, Elias; Zetterberg, Henrik; Blennow, Kaj; Skoog, Ingmar

    2017-02-01

    Depression and dementia in the elderly have been suggested to share similar risk factors and pathogenetic background, and recently the authors reported that the APOEɛ4 allele is a risk factor for both disorders in the general population. The aim of the present study was to examine the influence of the well-known polymorphisms rs1799752 in the angiotensin-converting enzyme (ACE) and rs5186 in the angiotensin receptor II type 1 (AGTR1) on late-life depression and dementia in a population-based Swedish cohort of older individuals followed over 12 years. In 2000-2001, 900 individuals underwent neuropsychiatric and neuropsychological examinations. Follow-up evaluations were performed in 2005-2006 and 2009-2010, and register data on dementia to 2012 were included. Cross-sectional associations between genotypes/alleles and depression and dementia at baseline and between genotypes/alleles and depression on at least one occasion during the study period and dementia onset to 2012 were investigated. As previously found for rs1799752 in ACE, rs5186 in AGTR1 was associated with dementia at baseline (OR: 3.25 [CI: 1.42-7.06], z = 2.90, p = 0.004). These associations became substantially weaker, or disappeared, when dementia onset to 2012 was included. For rs1799752 this could be explained by a significant association with age at onset (mean: 79.5 [SD: 6.45] years for risk-genotype carriers and 81.7 [SD: 7.12] years for carriers of other genotypes, b = -2.43, t = -2.38, df = 192, p = 0.02). When individuals with major depression on at least one occasion were analyzed, a significant association (OR: 2.14 [95% CI: 1.13-4.20], z = 2.28, p = 0.02), remaining after exclusion of dementia, with rs1799752 in ACE was found. In this population-based sample of older individuals, genetic variations in ACE seem to be important both for late-life major depression and dementia. Copyright © 2017 American Association for Geriatric Psychiatry. Published by

  10. Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.

    PubMed

    Rakoczy, P E; Lai, M C; Baines, M G; Spilsbury, K; Constable, I J

    1998-10-01

    To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity

  11. Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

    PubMed

    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

    2016-08-16

    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.

  12. 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

  13. Acanthamoeba castellanii is not be an adequate model to study human adenovirus interactions with macrophagic cells

    PubMed Central

    Cateau, Estelle; Leveque, Nicolas; Kaaki, Sihem; Beby-Defaux, Agnès; Rodier, Marie-Hélène

    2017-01-01

    Free living amoebae (FLA) including Acanthamoeba castellanii, are protozoa that feed on different microorganisms including viruses. These microorganisms show remarkable similarities with macrophages in cellular structures, physiology or ability to phagocyte preys, and some authors have therefore wondered whether Acanthamoeba and macrophages are evolutionary related. It has been considered that this amoeba may be an in vitro model to investigate relationships between pathogens and macrophagic cells. So, we intended in this study to compare the interactions between a human adenovirus strain and A. castellanii or THP-1 macrophagic cells. The results of molecular and microscopy techniques following co-cultures experiments have shown that the presence of the adenovirus decreased the viability of macrophages, while it has no effect on amoebic viability. On another hand, the viral replication occurred only in macrophages. These results showed that this amoebal model is not relevant to explore the relationships between adenoviruses and macrophages in in vitro experiments. PMID:28591183

  14. 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

    2017-12-01

    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. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

  15. Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

    PubMed

    Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

    1998-05-01

    The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

  16. Development of parallel line analysis criteria for recombinant adenovirus potency assay and definition of a unit of potency.

    PubMed

    Ogawa, Yasushi; Fawaz, Farah; Reyes, Candice; Lai, Julie; Pungor, Erno

    2007-01-01

    Parameter settings of a parallel line analysis procedure were defined by applying statistical analysis procedures to the absorbance data from a cell-based potency bioassay for a recombinant adenovirus, Adenovirus 5 Fibroblast Growth Factor-4 (Ad5FGF-4). The parallel line analysis was performed with a commercially available software, PLA 1.2. The software performs Dixon outlier test on replicates of the absorbance data, performs linear regression analysis to define linear region of the absorbance data, and tests parallelism between the linear regions of standard and sample. Width of Fiducial limit, expressed as a percent of the measured potency, was developed as a criterion for rejection of the assay data and to significantly improve the reliability of the assay results. With the linear range-finding criteria of the software set to a minimum of 5 consecutive dilutions and best statistical outcome, and in combination with the Fiducial limit width acceptance criterion of <135%, 13% of the assay results were rejected. With these criteria applied, the assay was found to be linear over the range of 0.25 to 4 relative potency units, defined as the potency of the sample normalized to the potency of Ad5FGF-4 standard containing 6 x 10(6) adenovirus particles/mL. The overall precision of the assay was estimated to be 52%. Without the application of Fiducial limit width criterion, the assay results were not linear over the range, and an overall precision of 76% was calculated from the data. An absolute unit of potency for the assay was defined by using the parallel line analysis procedure as the amount of Ad5FGF-4 that results in an absorbance value that is 121% of the average absorbance readings of the wells containing cells not infected with the adenovirus.

  17. Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model.

    PubMed

    Badie, B; Hunt, K; Economou, J S; Black, K L

    1994-11-01

    The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease. Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses. Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy. We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model. Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity. Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively. Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo. Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells. Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells. We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo. Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.

  18. 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

  19. Contrasting Effects of Human, Canine, and Hybrid Adenovirus Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Clinical Efficacy▿

    PubMed Central

    Perreau, Matthieu; Mennechet, Franck; Serratrice, Nicolas; Glasgow, Joel N.; Curiel, David T.; Wodrich, Harald; Kremer, Eric J.

    2007-01-01

    Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis of Adenoviridae immunogenicity and that each may have specific clinical advantages. PMID:17229706

  20. Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

    PubMed Central

    Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

    1995-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber

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

    PubMed Central

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

    2013-01-01

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

  2. Adenovirus-mediated human paraoxonase1 gene transfer to provide protection against the toxicity of the organophosphorus pesticide toxicant diazoxon.

    PubMed

    Duysen, E G; Parikh, K; Aleti, V; Manne, V; Lockridge, O; Chilukuri, N

    2011-03-01

    Human paraoxonase1 (hPON1) is a potential therapeutic against the toxicity of organophosphorus (OP) pesticides and chemical warfare nerve agents. We tested whether PON1 gene transfer using adenovirus provides protection against the toxicity of the OP diazoxon. Using an adenovirus construct containing hPON1 gene, we showed elevated levels of recombinant hPON1 in vitro in 293A cells and in vivo in mice. The recombinant enzyme was secreted by 293A cells into culture medium and into the systemic circulation of mice. Western blotting revealed that the virally expressed hPON1 had the expected molecular weight of 45 kDa. Recombinant hPON1 in mice was in complex with mouse high-density lipoprotein (HDL) and migrated more slowly than endogenous hPON1 in the human HDL complex. Mice injected with adenovirus expressed PON1 at 600-3480 U ml(-1) on day 5 post-treatment, which is 8-50-fold above endogenous. Six mice expressing hPON1 survived 2LD(50) doses of diazoxon. Four of the six mice survived a second dose of diazoxon (for a total of 4LD(50)) administered 24 h later. In contrast, none of the three mice in the control group survived one 2LD(50) dose. These results show that hPON1 in mice functions as a prophylactic and offers significant protection against lethal doses of diazoxon.

  3. Two distinct cellular proteins interact with the EIa-responsive element of an adenovirus early promoter.

    PubMed Central

    Jansen-Durr, P; Wintzerith, M; Reimund, B; Hauss, C; Kédinger, C

    1990-01-01

    EIa-dependent transactivation of the adenovirus EIIa early (EIIaE) promoter is correlated with the activation of the cellular transcription factor E2F. In this study we identified a cellular protein, C alpha, that is distinct from E2F and that binds two sites in the EIIaE promoter, one of which overlaps with the proximal E2F binding site of the EIIaE promoter. The possible involvement of C alpha in the EIa responsiveness of this promoter is discussed. Images PMID:2139142

  4. Distribution and Molecular Characterization of Human Adenovirus and Epstein-Barr Virus Infections in Tonsillar Lymphocytes Isolated from Patients Diagnosed with Tonsillar Diseases.

    PubMed

    Assadian, Farzaneh; Sandström, Karl; Bondeson, Kåre; Laurell, Göran; Lidian, Adnan; Svensson, Catharina; Akusjärvi, Göran; Bergqvist, Anders; Punga, Tanel

    2016-01-01

    Surgically removed palatine tonsils provide a conveniently accessible source of T and B lymphocytes to study the interplay between foreign pathogens and the host immune system. In this study we have characterised the distribution of human adenovirus (HAdV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in purified tonsillar T and B cell-enriched fractions isolated from three patient age groups diagnosed with tonsillar hypertrophy and chronic/recurrent tonsillitis. HAdV DNA was detected in 93 out of 111 patients (84%), while EBV DNA was detected in 58 patients (52%). The most abundant adenovirus type was HAdV-5 (68%). None of the patients were positive for HCMV. Furthermore, 43 patients (39%) showed a co-infection of HAdV and EBV. The majority of young patients diagnosed with tonsillar hypertrophy were positive for HAdV, whereas all adult patients diagnosed with chronic/recurrent tonsillitis were positive for either HAdV or EBV. Most of the tonsils from patients diagnosed with either tonsillar hypertrophy or chronic/recurrent tonsillitis showed a higher HAdV DNA copy number in T compared to B cell-enriched fraction. Interestingly, in the majority of the tonsils from patients with chronic/recurrent tonsillitis HAdV DNA was detected in T cells only, whereas hypertrophic tonsils demonstrated HAdV DNA in both T and B cell-enriched fractions. In contrast, the majority of EBV positive tonsils revealed a preference for EBV DNA accumulation in the B cell-enriched fraction compared to T cell fraction irrespective of the patients' age.

  5. A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.

    PubMed

    Johnson, Michael A; Pooley, Catherine; Ignjatovic, Jagoda; Tyack, Scott G

    2003-06-20

    The spike peplomer S1 subunit sequence from avian infectious bronchitis virus (IBV) Vic S strain was expressed in a plasmid under the control of the fowl adenovirus (FAV) major late promoter (MLP). Two recombinants were constructed in FAV serotype 8 (FAV 8) by inserting the expression cassette between the SnaBI and XbaI restriction enzyme sites (clone DA3) or between the SpeI sites (clone CA6-20). Expression of the S1 gene in the recombinants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by 20h post-infection. Commercial broiler chickens were orally vaccinated at day 0 or day 6 post-hatch and challenged at day 35 post-hatch. FAV antibody ELISA confirmed that maternal antibody directed against inclusion body hepatitis (serotype 8) had decayed in control birds and that FAV specific serum IgG responses were produced in vaccinated birds at the time of challenge. Further, an S1 specific antibody response was detected prior to challenge. Birds were challenged with either Vic S (serotype B) or N1/62 (serotype C) strains of IBV. The tracheas of challenged birds were analyzed by RT-PCR and re-isolation of virus. In birds vaccinated at day 6, 90-100% protection at the trachea was induced against either homologous or heterologous challenge. The construction of a recombinant FAV expressing S1 of IBV demonstrates the potential of an alternative vaccination strategy against IBV.

  6. Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

    PubMed Central

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

    2015-01-01

    Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection. PMID:25768646

  7. Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

    PubMed

    Meng, Fanfeng; Dong, Guiwei; Zhang, Yubiao; Tian, Sibao; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2018-05-01

    In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

  8. LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

    PubMed

    Shepelev, Mikhail V; Korobko, Elena V; Vinogradova, Tatiana V; Kopantsev, Eugene P; Korobko, Igor V

    2013-03-04

    Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

  9. 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

  10. Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication

    PubMed Central

    Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K.; McCormick, Frank; Graeber, Thomas G.; Christofk, Heather R.

    2014-01-01

    SUMMARY Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. While recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. PMID:24703700

  11. Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene tissue fixative and formalin.

    PubMed

    Kap, Marcel; Arron, Georgina I; Loibner, M; Hausleitner, Anja; Siaulyte, Gintare; Zatloukal, Kurt; Murk, Jean-Luc; Riegman, Peter

    2013-08-01

    Formalin fixation is known to inactivate most viruses in a vaccine production context, but nothing is published about virus activity in tissues treated with alternative, non-crosslinking fixatives. We used a model assay based on cell culture to test formalin and PAXgene Tissue fixative for their virus-inactivating abilities. MDCK, A549, and MRC-5 cells were infected with Influenza A virus, Adenovirus, and Cytomegalovirus, respectively. When 75% of the cells showed a cytopathic effect (CPE), the cells were harvested and incubated for 15 min, or 1, 3, 6, or 24 hours, with PBS (positive control), 4% formalin, or PAXgene Tissue Fix. The cells were disrupted and the released virus was used to infect fresh MDCK, A549, and MRC-5 cells cultured on cover slips in 24-well plates. The viral cultures were monitored for CPE and by immunocytochemistry (ICC) to record viral replication and infectivity. Inactivation of Adenovirus by formalin occurred after 3 h, while Influenza A virus as well as Cytomegalovirus were inactivated by formalin after 15 min. All three virus strains were inactivated by PAXgene Tissue fixative after 15 min. We conclude that PAXgene Tissue fixative is at least as effective as formalin in inactivating infectivity of Influenza A virus, Adenovirus, and Cytomegalovirus.

  12. A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

    PubMed

    Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

    2015-08-01

    We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and

  13. A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens

    PubMed Central

    Mahairas, Gregory G.; Shaw, Carolyn E.; Huang, Meei-Li; Koelle, David M.; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M.

    2015-01-01

    ABSTRACT We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4+ and CD8+ T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of

  14. Seroprevalence of Neutralizing Antibodies against Human Adenovirus Type-5 and Chimpanzee Adenovirus Type-68 in Cancer Patients.

    PubMed

    Zhao, Hua; Xu, Can; Luo, Xiaoli; Wei, Feng; Wang, Ning; Shi, Huiying; Ren, Xiubao

    2018-01-01

    Since the preclinical results about chimpanzee adenovirus serotype-68 (AdC68)-based vaccine showed an encouraging results, it reminded us that AdC68 may be a suitable cancer vaccine vector. Previous study indicated that the seroprevalence of neutralizing antibodies (NAbs) against adenovirus was different between cancer patients and healthy volunteers. Knowledge regarding the prevalence rates of AdC68 NAbs for cancer patients is lacking. Therefore, assessing the preexistence of NAbs against AdC68 in cancer patients could provide useful insights for developing future AdC68-based cancer vaccines. In this study, 440 patients with different pathological types of tumors and 204 healthy adult volunteers were enrolled to evaluate the NAbs against AdC68 and human adenovirus serotype-5 (AdHu5). The seroprevalence of NAbs against AdC68 was much lower than that against AdHu5 in cancer subjects (43.64 vs. 67.05%, P  < 0.01). The seroprevalence rates of NAbs to AdC68 in the cancer subjects were statistically higher than those detected in the healthy adult volunteers (43.64 vs. 23.53%, P  = 0.000). The seroprevalence rates of AdC68 NAbs were much lower in lung, laryngeal, esophageal, and cervical cancer patients compared with oropharyngeal, colon, and rectal cancer patients. Furthermore, the seroprevalence rates of AdC68 NAbs were much lower in lung adenocarcinoma patients than in lung squamous cell carcinoma patients (35.00 vs. 70.00%, P  < 0.05). No significant difference in the AdC68 NAbs among patients with different clinical stages of cancer was detected. The percentage of NAbs against AdC68 was significantly lower than that against AdHu5 ( P  < 0.05) in stage-I, -II, and -III cancer patients. No significant difference between the percentage of NAbs against AdC68 and AdHu5 in the subjects with stage-IV cancer was detected. The study also demonstrated the distribution of AdHu5 and AdC68 NAb titers for the positive samples. It showed that very low NAb titers

  15. Seroprevalence of Neutralizing Antibodies against Human Adenovirus Type-5 and Chimpanzee Adenovirus Type-68 in Cancer Patients

    PubMed Central

    Zhao, Hua; Xu, Can; Luo, Xiaoli; Wei, Feng; Wang, Ning; Shi, Huiying; Ren, Xiubao

    2018-01-01

    Since the preclinical results about chimpanzee adenovirus serotype-68 (AdC68)-based vaccine showed an encouraging results, it reminded us that AdC68 may be a suitable cancer vaccine vector. Previous study indicated that the seroprevalence of neutralizing antibodies (NAbs) against adenovirus was different between cancer patients and healthy volunteers. Knowledge regarding the prevalence rates of AdC68 NAbs for cancer patients is lacking. Therefore, assessing the preexistence of NAbs against AdC68 in cancer patients could provide useful insights for developing future AdC68-based cancer vaccines. In this study, 440 patients with different pathological types of tumors and 204 healthy adult volunteers were enrolled to evaluate the NAbs against AdC68 and human adenovirus serotype-5 (AdHu5). The seroprevalence of NAbs against AdC68 was much lower than that against AdHu5 in cancer subjects (43.64 vs. 67.05%, P < 0.01). The seroprevalence rates of NAbs to AdC68 in the cancer subjects were statistically higher than those detected in the healthy adult volunteers (43.64 vs. 23.53%, P = 0.000). The seroprevalence rates of AdC68 NAbs were much lower in lung, laryngeal, esophageal, and cervical cancer patients compared with oropharyngeal, colon, and rectal cancer patients. Furthermore, the seroprevalence rates of AdC68 NAbs were much lower in lung adenocarcinoma patients than in lung squamous cell carcinoma patients (35.00 vs. 70.00%, P < 0.05). No significant difference in the AdC68 NAbs among patients with different clinical stages of cancer was detected. The percentage of NAbs against AdC68 was significantly lower than that against AdHu5 (P < 0.05) in stage-I, -II, and -III cancer patients. No significant difference between the percentage of NAbs against AdC68 and AdHu5 in the subjects with stage-IV cancer was detected. The study also demonstrated the distribution of AdHu5 and AdC68 NAb titers for the positive samples. It showed that very low NAb titers against

  16. Correlations between Stroop task performance and white matter lesion measures in late-onset major depression.

    PubMed

    Dalby, Rikke B; Frandsen, Jesper; Chakravarty, M Mallar; Ahdidan, Jamila; Sørensen, Leif; Rosenberg, Raben; Østergaard, Leif; Videbech, Poul

    2012-05-31

    Cerebral white matter lesions (WMLs) are believed to play an important role in a subset of patients with late-onset depression by affecting the white matter connectivity in circuitries essential for mood and cognition. In this study we used diffusion tensor imaging-based (DTI-based) tractography to assess white matter fiber tracts affected by deep WMLs (DWMLs) in patients with late-onset major depression and age- and gender-matched controls. Tractography outcome, illustrated as pathways affected by DWMLs, was analyzed for associations with cognitive performance on the Stroop Test (ST). The patients (n=17) performed significantly worse on the ST than the controls (n=22). Poor performance on the ST correlated with higher lesion load. Regression analysis showed a significant correlation between poor performance on the ST and tracts affected by DWMLs in multiple brain areas in the control group, but very sparse correlation in the patient group. Our results suggest that DWMLs play an important role in the cognitive performance of controls,whereas their influence in depressed patients is overruled by additional, state-dependent factors. Future focus on the tract-specific localization of WMLs using DTI tractography may reveal important associations between neuroconnectivity and clinical measures. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

    PubMed

    Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Archer, Linda L; Waltzek, Thomas B; Gray, Gregory C; Wellehan, James F X

    2017-03-01

    California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

  18. A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein.

    PubMed

    Fingerut, E; Gutter, B; Gallili, G; Michael, A; Pitcovski, J

    2003-06-20

    In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

  19. 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

  20. Fowl adenoviruses isolated from chickens with inclusion body hepatitis in Japan, 2009-2010.

    PubMed

    Mase, Masaji; Nakamura, Kikuyasu; Minami, Fujiko

    2012-08-01

    Nine fowl adenoviruses (FAdVs) isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2009 to 2010 were characterized serologically and genetically. These isolates were all neutralized by antisera against the SR-48 strain (FAdV-2). Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all isolates were almost identical except one isolate in 2009. This suggests a common ancestor for the FAdVs obtained from chickens with IBH in Japan in 2010.

  1. Late-Glacial to Late-holocene Shifts in Global Precipitation Delta(sup 18)O

    NASA Technical Reports Server (NTRS)

    Jasechko, S.; Lechler, A.; Pausata, F.S.R.; Fawcett, P.J.; Gleeson, T.; Cendon, D.I.; Galewsky, J.; LeGrande, A. N.; Risi, C.; Sharp, Z. D.; hide

    2015-01-01

    Reconstructions of Quaternary climate are often based on the isotopic content of paleo-precipitation preserved in proxy records. While many paleo-precipitation isotope records are available, few studies have synthesized these dispersed records to explore spatial patterns of late-glacial precipitation delta(sup 18)O. Here we present a synthesis of 86 globally distributed groundwater (n 59), cave calcite (n 15) and ice core (n 12) isotope records spanning the late-glacial (defined as 50,000 to 20,000 years ago) to the late-Holocene (within the past 5000 years). We show that precipitation delta(sup 18)O changes from the late-glacial to the late-Holocene range from -7.1% (delta(sup 18)O(late-Holocene) > delta(sup 18)O(late-glacial) to +1.7% (delta(sup 18)O(late-glacial) > delta(sup 18)O(late-Holocene), with the majority (77) of records having lower late-glacial delta(sup 18)O than late-Holocene delta(sup 18)O values. High-magnitude, negative precipitation delta(sup 18)O shifts are common at high latitudes, high altitudes and continental interiors.

  2. Single-cycle adenovirus vectors in the current vaccine landscape.

    PubMed

    Barry, Michael

    2018-02-01

    Traditional inactivated and protein vaccines generate strong antibodies, but struggle to generate T cell responses. Attenuated pathogen vaccines generate both, but risk causing the disease they aim to prevent. Newer gene-based vaccines drive both responses and avoid the risk of infection. While these replication-defective (RD) vaccines work well in small animals, they can be weak in humans because they do not replicate antigen genes like more potent replication-competent (RC) vaccines. RC vaccines generate substantially stronger immune responses, but also risk causing their own infections. To circumvent these problems, we developed single-cycle adenovirus (SC-Ad) vectors that amplify vaccine genes, but that avoid the risk of infection. This review will discuss these vectors and their prospects for use as vaccines. Areas covered: This review provides a background of different types of vaccines. The benefits of gene-based vaccines and their ability to replicate antigen genes are described. Adenovirus vectors are discussed and compared to other vaccine types. Replication-defective, single-cycle, and replication-competent Ad vaccines are compared. Expert commentary: The potential utility of these vaccines are discussed when used against infectious diseases and as cancer vaccines. We propose a move away from replication-defective vaccines towards more robust replication-competent or single-cycle vaccines.

  3. Adenovirus-mediated E2-EPF UCP gene transfer prevents autoamputation in a mouse model of hindlimb ischemia.

    PubMed

    Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

    2012-04-01

    E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

  4. Pandemic Influenza Virus 2009 H1N1 and Adenovirus in a High Risk Population of Young Adults: Epidemiology, Comparison of Clinical Presentations, and Coinfection

    DTIC Science & Technology

    2014-01-08

    Pandemic Influenza Virus 2009 H1N1 and Adenovirus in a High Risk Population of Young Adults: Epidemiology, Comparison of Clinical Presentations, and... H1N1 influenza virus (2009 H1N1 ) emerged worldwide, causing morbidity and mortality that disproportionately affected young adults. Upper respiratory...adenovirus and 2009 H1N1 were prospectively collected. Results: 375 trainees with URI enrolled and were tested for both adenovirus and 2009 H1N1 by

  5. The Late Ordovician Extinction: How it became the best understood of the five major extinctions.

    NASA Astrophysics Data System (ADS)

    Sheehan, P.

    2003-04-01

    The end Ordovician extinction has become arguably the best-understood major extinction event in Earth History. A plethora of workers have established the pattern of faunal change and causes of the extinction with remarkably little disagreement. The first indication of increased extinction at the end of the Ordovician was a graph of global diversity patterns by Norman Newell in 1967, although he did not recognize it as a major event. The presence of a major extinction event became clear as William Berry and Art Boucot assembled data for Silurian correlation charts in the late 1960s. The first reports of North African glaciation in the late 1960s provided a cause for the extinction and study of the event snowballed. It was no accident that recognition of the extinction began in North America, because it was there that the extinction completely overturned faunas in the epicontinental seas. Glacio-eustatic regression of shallow seaway coincided with the disappearance of endemic Laurentian faunas and replacement by a highly cosmopolitan fauna in the Silurian. Once the event was established in North America, paleontologists soon found evidence of the event around the globe. The well-documented Hirnantia Fauna was found to correspond to the glacial interval, and Pat Brenchley soon recognized that there were two pulses of extinction, at the beginning and end of the glaciation. At the same time that the faunal changes were being documented geologic studies of the glaciation provided information on the environmental changes associated with the extinction. The timing of the glacial maximum was established in Africa and by the presence of dropstones in high latitude marine rocks. The 1990s saw geochemical techniques employed that allowed examination of atmospheric CO2 and temperature changes. In many places carbonate deposition declined. Glacio-eustatic regression was obvious in many areas, and a sea-level decline in the range of 50-100 m was established. Shallow

  6. 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...

  7. 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 ...

  8. Immunogenicity and protective efficacy of a replication-defective infectious bronchitis virus vaccine using an adenovirus vector and administered in ovo.

    PubMed

    Zeshan, Basit; Zhang, Lili; Bai, Juan; Wang, Xinglong; Xu, Jiarong; Jiang, Ping

    2010-06-01

    In ovo vaccination remains an attractive option for a cost effective, uniform and mass application of vaccines for commercial poultry. However, the vaccines which can be delivered safely by this method are limited and there is no currently licensed embryo-safe vaccine against infectious bronchitis virus (IBV). In this study, a recombinant adenovirus expressing the S1 gene of nephropathogenic IBV (rAd-S1) was constructed and the immune responses and protective efficacy against homologous challenge were evaluated after in ovo vaccination. The results showed that the rAd-S1 led to dramatic augmentation of humoral and cellular responses in birds vaccinated in ovo followed by an intramuscular inoculation. Both IFN-gamma and IL-4 in chicken's lymphocytes were produced by this strategy. Following challenge with IBV, the chickens vaccinated with recombinant adenovirus showed fewer nephropathic lesions and less severe clinical signs as compared to those receiving wild-type adenovirus or PBS. The construction of non-replicating human adenovirus vector encoding S1 gene of IBV and its in ovo delivery demonstrated the potential of an alternative vaccination strategy against IBV. Copyright 2010 Elsevier B.V. All rights reserved.

  9. Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

    PubMed

    Kimball, Kristopher J; Rivera, Angel A; Zinn, Kurt R; Icyuz, Mert; Saini, Vaibhav; Li, Jing; Zhu, Zeng B; Siegal, Gene P; Douglas, Joanne T; Curiel, David T; Alvarez, Ronald D; Borovjagin, Anton V

    2009-01-01

    We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

  10. Adenovirus and Herpesvirus Diversity in Free-Ranging Great Apes in the Sangha Region of the Republic of Congo

    PubMed Central

    Seimon, Tracie A.; Olson, Sarah H.; Lee, Kerry Jo; Rosen, Gail; Ondzie, Alain; Cameron, Kenneth; Reed, Patricia; Anthony, Simon J.; Joly, Damien O.; 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. PMID:25781992

  11. 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.

  12. Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle.

    PubMed

    Dulal, Pawan; Wright, Daniel; Ashfield, Rebecca; Hill, Adrian V S; Charleston, Bryan; Warimwe, George M

    2016-04-29

    Development of safe and efficacious vaccines whose potency is unaffected by long-term storage at ambient temperature would obviate major vaccine deployment hurdles and limit wastage associated with breaks in the vaccine cold chain. Here, we evaluated the immunogenicity of a novel chimpanzee adenovirus vectored Rift Valley Fever vaccine (ChAdOx1-GnGc) in cattle, following its thermostabilisation by slow desiccation on glass fiber membranes in the non-reducing sugars trehalose and sucrose. Thermostabilised ChAdOx1-GnGc vaccine stored for 6 months at 25, 37 or 45 ° C elicited comparable Rift Valley Fever virus neutralising antibody titres to those elicited by the 'cold chain' vaccine (stored at -80 ° C throughout) at the same dose, and these were within the range associated with protection against Rift Valley Fever in cattle. The results support the use of sugar-membrane thermostabilised vaccines in target livestock species. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector

    PubMed Central

    Serratrice, Nicolas; Cubizolle, Aurelie; Ibanes, Sandy; Mestre-Francés, Nadine; Bayo-Puxan, Neus; Creyssels, Sophie; Gennetier, Aurelie; Bernex, Florence; Verdier, Jean-Michel; Haskins, Mark E.; Couderc, Guilhem; Malecaze, Francois; Kalatzis, Vasiliki; Kremer, Eric J.

    2015-01-01

    Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes. PMID:24607662

  14. 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

  15. Three-year duration of immunity in dogs following vaccination against canine adenovirus type-1, canine parvovirus, and canine distemper virus.

    PubMed

    Gore, Thomas C; Lakshmanan, Nallakannu; Duncan, Karen L; Coyne, Michael J; Lum, Melissa A; Sterner, Frank J

    2005-01-01

    A challenge-of-immunity study was conducted to demonstrate immunity in dogs 3 years after their second vaccination with a new multivalent, modified-live vaccine containing canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), and canine distemper virus (CDV). Twenty-three seronegative pups were vaccinated at 7 and 11 weeks of age. Eighteen seronegative pups, randomized into groups of six dogs, served as challenge controls. Dogs were kept in strict isolation for 3 years following the vaccination and then challenged sequentially with virulent canine adenovirus type 1 (CAV-1), CPV, and CDV. For each viral challenge, a separate group of six control dogs was also challenged. Clinical signs of CAV-1, CPV, and CDV infections were prevented in 100% of vaccinated dogs, demonstrating that the multivalent, modified-live test vaccine provided protection against virulent CAV-1, CPV, and CDV challenge in dogs 7 weeks of age or older for a minimum of 3 years following second vaccination.

  16. A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

    PubMed

    Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

    2018-05-02

    Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

  17. Protection of Chickens against Avian Influenza with Non-Replicating Adenovirus-Vectored Vaccine

    PubMed Central

    Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Shi, Z.

    2009-01-01

    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 an H7 AI hemagglutinin (AdChNY94.H7). Chickens vaccinated in ovo with an Ad vector encoding an AI H5 (AdTW68.H5) previously described, which were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Antibody responses to Ad vector in ovo vaccination follow a dose-response kinetic. The use of a synthetic AI H5 gene codon optimized to match the chicken cell tRNA pool was more potent than the cognate H5 gene. The use of Ad-vectored vaccines to increase resistance of chicken populations against multiple AI strains could reduce the risk of an avian-originating influenza pandemic in humans. PMID:18384919

  18. Characterization of a Novel Bat Adenovirus Isolated from Straw-Colored Fruit Bat (Eidolon helvum).

    PubMed

    Ogawa, Hirohito; Kajihara, Masahiro; Nao, Naganori; Shigeno, Asako; Fujikura, Daisuke; Hang'ombe, Bernard M; Mweene, Aaron S; Mutemwa, Alisheke; Squarre, David; Yamada, Masao; Higashi, Hideaki; Sawa, Hirofumi; Takada, Ayato

    2017-12-04

    Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats ( Eidolon helvum ) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus . The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E , F and G , from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name " Bat mastadenovirus H ". Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission.

  19. Characterization of a Novel Bat Adenovirus Isolated from Straw-Colored Fruit Bat (Eidolon helvum)

    PubMed Central

    Kajihara, Masahiro; Nao, Naganori; Shigeno, Asako; Fujikura, Daisuke; Hang’ombe, Bernard M.; Mweene, Aaron S.; Mutemwa, Alisheke; Yamada, Masao; Higashi, Hideaki; Sawa, Hirofumi; Takada, Ayato

    2017-01-01

    Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name “Bat mastadenovirus H”. Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission. PMID:29207524

  20. Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro.

    PubMed

    Gao, Hong; Liu, Lei; Qu, Zhang-Yi; Wei, Feng-Xiang; Wang, Shu-Qiu; Chen, Guang; Qin, Le; Jiang, Fu-Yang; Wang, Ying-Chen; Shang, Lei; Gao, Chun-Yan

    2011-01-01

    Radix Lithosperm eyrthrorhizon is a common prescription compound in traditional Chinese medicine. Shikonin is a major component of Radix Lithospermi and has various biological activities. We have investigated the inhibitory effect of shikonin on the growth of adenovirus type 3 (AdV3) in vitro. The antiviral function of shikonin against AdV3 and its virus inhibition ratio were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT). The expression of hexon protein in AdV3 was determined by immunofluorescence assay using laser scanning confocal microscopy (LSCM) and Western blot analysis. In addition, the rate of apoptosis in cells infected by AdV3 was determined by flow cytometry. Shikonin (0.0156-1 µM) inhibited growth of AdV3 in a concentration-dependent manner with a virus inhibition rate of 23.8-69.1%. Expression of hexon protein in AdV3 was higher in the virus control group than in the shikonin-treated groups as determined by immunofluorescence assay and Western blotting (p<0.05). The rate of shikonin-treated HeLa cell apoptosis had a statistically significant decrease with increasing concentration of drug (p<0.05). Our data demonstrate that shikonin possesses anti-AdV3 capabilities and that the potential antiviral mechanism might involve inhibiting the degree of apoptosis and hexon protein expression of AdV.

  1. Two Adenovirus Serotype 3 Outbreaks Associated with Febrile Respiratory Disease and Pharyngoconjunctival Fever in Children under 15 Years of Age in Hangzhou, China, during 2011

    PubMed Central

    Xie, Li; Yu, Xin-Fen; Sun, Zhou; Yang, Xu-Hui; Huang, Ren-Jie; Wang, Jing; Yu, Apeng; Zheng, Lin; Yu, Man-Chu; Hu, Xiao-Wei; Wang, Ban-Ma; Chen, Jin; Pan, Jing-Cao

    2012-01-01

    Adenovirus serotype 3 and 7 outbreaks have occurred periodically in northern, eastern, and southern China since 1955, but there has been no report since the adenovirus serotype 7 outbreak first occurred in Hangzhou, China, in 1991. Here we explored the epidemiology and etiology of two adenovirus serotype 3 outbreaks in Hangzhou in 2011. One acute respiratory outbreak was found in Chun'an County, where a total of 371 cases were confirmed in 5 of 23 towns from 4 to 31 May 2011. The outbreak affected 18.57% (13/70) of schools and 14.49% (90/621) of classes. The incidence was 5.18% (371/7,163). The population was distributed among individuals ages 7 to 15 years. No parents or teachers were infected. Another pharyngoconjunctival fever outbreak was discovered in the Chenjinglun Swimming Center located in the Xihu District between 1 and 15 July 2011. A total of 134 cases were confirmed in 900 amateur swimmers, with an incidence of 14.89% (134/900). The ages ranged from 4 to 9 years. The two outbreaks had no severe complications or death. The viruses in 66.67% (10/15) of throat swabs from children with acute respiratory infections and 100% (10/10) of the swabs from children with pharyngoconjunctival fever were confirmed to be adenovirus serotype 3 with 100% homology by PCR. Of these samples, 60.0% (12/20) had a classical characteristic cytopathic effect, presented as grape-like clusters at 72 h after infection in HEp-2 cells. In conclusion, the acute respiratory infection and pharyngoconjunctival fever outbreak in Hangzhou were caused by the completely homologous type 3 adenovirus in subgenus B. Moreover, these outbreaks demonstrated rapid transmission rates, possibly due to close contact and droplet transmission. PMID:22442311

  2. Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

    PubMed

    Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

    2014-04-01

    Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Novel Human Adenovirus Causing Nosocomial Epidemic Keratoconjunctivitis▿

    PubMed Central

    Ishiko, Hiroaki; Shimada, Yasushi; Konno, Tsunetada; Hayashi, Akio; Ohguchi, Takeshi; Tagawa, Yoshitsugu; Aoki, Koki; Ohno, Shigeaki; Yamazaki, Shudo

    2008-01-01

    In 2000, we encountered cases of nosocomial infections with epidemic keratoconjunctivitis (EKC) at a university hospital in Kobe, in the western part of Japan. Two human adenovirus (HAdV) strains, Kobe-H and Kobe-S, were isolated from patients with nosocomial EKC infection. They were untypeable by existing neutralizing antisera; however, the isolate was neutralized with homologous antisera. We then encountered several cases of EKC due to nosocomial infections in eye clinics in different parts of Japan. A total of 80 HAdVs were isolated from patients with EKC at eight different hospitals. The partial hexon gene sequences of the isolates were determined and compared to those of the prototype strains of 51 serotypes. All isolates had identical partial hexon nucleotide sequences. Phylogenetic analysis classified these isolates into species of HAdV-D. The isolates showed 93.9 to 96.7% nucleotide identity with HAdV-D prototype strains, while all 32 HAdV-D prototype strains ranged from 93.2 to 99.2% identity. The sequences of the loop 2 and fiber knob regions from the representative strain, Kobe-H, were dissimilar in all prototype strains of 51 serotypes. We believe that this virus is a novel serotype of HAdV that causes EKC. PMID:18385435

  4. Enhanced growth inhibition of prostate cancer in vitro and in vivo by a recombinant adenovirus-mediated dual-aptamer modified drug delivery system.

    PubMed

    Jing, Pei; Cao, Shousong; Xiao, Shuangli; Zhang, Xiaoqin; Ke, Siyun; Ke, Famin; Yu, Xin; Wang, Li; Wang, Shurong; Luo, Yuling; Zhong, Zhirong

    2016-12-28

    The peptide aptamer DUP-1 targets prostate-specific membrane antigen (PSMA)-negative cells, while the RNA aptamer A10-3.2 targets PSMA-positive prostate cancer cells. Moreover, the tumor-suppressor gene phosphatase and tensin homolog (PTEN) and the chemotherapeutic agent doxorubicin (DOX) effectively inhibit prostate cancer, and a recombinant adenovirus (Ad5) mediates high gene transfer efficiency. Here, we design a dual-aptamer modified tumor targeting gene and DOX delivery system mediated by recombinant adenovirus (A10-3.2(DOX)/DUP-1-PEG-Ad5, ADDP-Ad5). DUP-1 and A10-3.2 are connected to the adenovirus through polyethylene glycol (PEG), PTEN is integrated into Ad5, and DOX is embedded into the double chain of aptamer A10-3.2. The PEG-modification rate of Ad5 is 98.70 ± 2.43%. The DUP-1 and A10-3.2 modified products yield 80.40 ± 1.36% and 82.20 ± 2.14%, respectively. The uptake of ADDP-Ad5 and the expression of the reporter gene are enhanced by the system in PSMA-positive LNCaP and PSMA-negative PC3 human prostate cancer cells. ADDP-Ad5 significantly inhibits the cell growth of both LNCaP and PC3 cells. More importantly, ADDP-Ad5 is active in vivo against LNCaP and PC3 tumor xenografts and exhibits no significant toxicity to the mice. Therefore, ADDP-Ad5 may have clinical potential in prostate cancer therapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Akt/protein kinase B activation by adenovirus vectors contributes to NFkappaB-dependent CXCL10 expression.

    PubMed

    Liu, Qiang; White, Lindsay R; Clark, Sharon A; Heffner, Daniel J; Winston, Brent W; Tibbles, Lee Anne; Muruve, Daniel A

    2005-12-01

    In gene therapy, the innate immune system is a significant barrier to the effective application of adenovirus (Ad) vectors. In kidney epithelium-derived (REC) cells, serotype 5 Ad vectors induce the expression of the chemokine CXCL10 (IP-10), a response that is dependent on NFkappaB. Compared to the parental vector AdLuc, transduction with the RGD-deleted vector AdL.PB resulted in reduced CXCL10 activation despite increasing titers, implying that RGD-alpha(V) integrin interactions contribute to adenovirus induction of inflammatory genes. Akt, a downstream effector of integrin signaling, was activated within 10 min of transduction with Ad vectors in a dose-dependent manner. Akt activation was not present following transduction with AdL.PB, confirming the importance of capsid-alpha(V) integrin interactions in Ad vector Akt activation. Inhibition of the phosphoinositide-3-OH kinase/Akt pathway by Wortmannin or Ly294002 compounds decreased Ad vector induction of CXCL10 mRNA. Similarly, adenovirus-mediated overexpression of the dominant negative AktAAA decreased CXCL10 mRNA expression compared to the reporter vector AdLacZ alone. The effect of Akt on CXCL10 mRNA expression occurred via NFkappaB-dependent transcriptional activation, since AktAAA overexpression and Ly294002 both inhibited CXCL10 and NFkappaB promoter activation in luciferase reporter experiments. These results show that Akt plays a role in the Ad vector activation of NFkappaB and CXCL10 expression. Understanding the mechanism underlying the regulation of host immunomodulatory genes by adenovirus vectors will lead to strategies that will improve the efficacy and safety of these agents for clinical use.

  6. 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...

  7. Fowl adenovirus serotype 4: Epidemiology, pathogenesis, diagnostic detection, and vaccine strategies.

    PubMed

    Li, P H; Zheng, P P; Zhang, T F; Wen, G Y; Shao, H B; Luo, Q P

    2017-08-01

    Fowl adenovirus (FAdV) serotype-4 is highly pathogenic for chickens, especially for broilers aged 3 to 5 wk, and it has emerged as one of the foremost causes of economic losses to the poultry industry in the last 30 years. The liver is a major target organ of FAdV-4 infections, and virus-infected chickens usually show symptoms of hydropericardium syndrome. The virus is very contagious, and it is spread both vertically and horizontally. It can be isolated from infected liver homogenates and detected by several laboratory diagnostic methods (including an agar gel immunodiffusion test, indirect immunofluorescence assays, counterimmunoelectrophoresis, enzyme-linked immunosorbent assays, restriction endonuclease analyses, polymerase chain reaction (PCR), real-time PCR, and high-resolution melting-curve analyses). Although inactivated vaccines have been deployed widely to control the disease, attenuated live vaccines and subunit vaccines also have been developed, and they are more attractive vaccine candidates. This article provides a comprehensive review of FAdV-4, including its epidemiology, pathogenesis, diagnostic detection, and vaccine strategies. © 2017 Poultry Science Association Inc.

  8. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    PubMed

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  9. Concentration of Reovirus and Adenovirus from Sewage and Effluents by Protamine Sulfate (Salmine) Treatment 1

    PubMed Central

    England, Beatrice

    1972-01-01

    Protamine sulfate was employed to recover reoviruses, adenoviruses, and certain enteroviruses from sewage and treated effluents; 50- to 400-fold concentration of viral content was achieved. PMID:4342842

  10. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    PubMed

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Trajectories of postsurgical pain in children: risk factors and impact of late pain recovery on long-term health outcomes after major surgery.

    PubMed

    Rabbitts, Jennifer A; Zhou, Chuan; Groenewald, Cornelius B; Durkin, Lindsay; Palermo, Tonya M

    2015-11-01

    Over 1 million children undergo inpatient surgery annually in the United States. Emerging research indicates that many children have longer-term problems with pain. However, limited data exist on the course of pain over time and the impact of pain recovery on long-term health outcomes. We sought to prospectively characterize children's postsurgical pain trajectories using repeated assessments over 12 months. In addition, we identified presurgical child and parent psychological risk factors associated with persistent pain and examined relationships between pain trajectories and long-term health outcomes. Sixty children aged 10 to 18 years undergoing major surgery and their parent/guardian were enrolled. Participants completed assessments at 5 time points: presurgery, inhospital, 2 weeks, 4 months, and 1 year postsurgery. Child and parent pain catastrophizing was assessed during the week before surgery. Children completed daily monitoring with an electronic pain diary and reported on pain characteristics, health-related quality of life, and activity limitations. Group-based longitudinal modeling revealed 2 distinct trajectories of postsurgical pain: early recovery (n = 49, 82%) and late recovery (n = 11, 18%). In a logistic regression model controlling for age and sex, parental pain catastrophizing before surgery significantly predicted membership in the late recovery group (odds ratio = 1.11, P = 0.03), whereas child catastrophizing and baseline pain did not (Ps < 0.05). In a multivariate regression controlling for age and sex, late pain recovery was significantly associated with poorer health-related quality of life (β = -10.7, P = 0.02) and greater activity limitations (β = 3.6, P = 0.04) at 1 year. Our findings suggest that preoperative interventions that modify parent behaviors and cognitions might be beneficial in this population.

  12. Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

    PubMed Central

    Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

    2012-01-01

    E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis. PMID:22294149

  13. Fowl adenovirus serotype 9 vectored vaccine for protection of avian influenza virus

    USDA-ARS?s Scientific Manuscript database

    A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...

  14. Comparative trial of the canine parvovirus, canine distemper virus and canine adenovirus type 2 fractions of two commercially available modified live vaccines.

    PubMed

    Bergman, J G H E; Muniz, M; Sutton, D; Fensome, R; Ling, F; Paul, G

    2006-11-25

    The results of vaccinating two groups of puppies with commercial vaccines, both of which claimed to provide adequate protection with a final vaccination at 10 weeks of age, were compared. Groups of 19 and 20 puppies with similar titres of maternally derived antibodies against canine parvovirus (cpv), canine distemper virus (cdv) and canine adenovirus type 2 (cav-2) at four weeks of age were vaccinated at six and 10 weeks of age and their responses to each vaccination were measured by comparing the titres against cpv, cdv and cav-2 in the serum samples taken immediately before the vaccination and four weeks later. After the vaccination at six weeks of age, all 19 of the puppies in group 1 had responded to cpv and cdv, and 14 had responded to cav-2; in group 2, 17 of the 20 had responded to cpv, 19 to cdv and 15 to cav-2. In both groups the puppies that did not respond to the first vaccination had responded serologically to cpv, cdv and cav-2 at 10 weeks of age.

  15. Lytic and transforming functions of individual products of the adenovirus E1A gene.

    PubMed Central

    Moran, E; Grodzicker, T; Roberts, R J; Mathews, M B; Zerler, B

    1986-01-01

    To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus. Images PMID:2936898

  16. Comparative Inactivation of Murine Norovirus, Human Adenovirus, and Human JC Polyomavirus by Chlorine in Seawater

    PubMed Central

    de Abreu Corrêa, Adriana; Carratala, Anna; Barardi, Celia Regina Monte; Calvo, Miquel; Bofill-Mas, Sílvia

    2012-01-01

    Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log10 GC reductions and a 2.3- and 2.4-log10 PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log10 GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log10 GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration. PMID:22773637

  17. Adenovirus-vectored shRNAs targeted to the highly conserved regions of VP1 and 2B in tandem inhibits replication of foot-and-mouth disease virus both in vitro and in vivo.

    PubMed

    Xu, Yan-Fang; Shen, Hai-Yan; Zhao, Ming-Qiu; Chen, Li-Jun; Li, Yin-Guang; Liao, Ming; Jia, Jun-Tao; Lv, Ying-Ran; Yi, Lin; Chen, Jin-Ding

    2012-04-01

    Foot-and-mouth disease is a highly contagious and economically important disease of cloven-hoofed animals. RNA interference (RNAi) can be used as a rapid and specific antiviral approach. It was shown that treatment with recombinant adenovirus (Ad(VP1-2B)) carrying shRNAs targeted to the VP1 and 2B genes of FMDV expressed in tandem had marked antiviral effects against FMDV both in IBRS-2 cells and guinea pigs. Treatment with Ad(VP1-2B) both before and after FMDV infection was most effective in IBRS-2 cells, as the FMDV RNA transcripts could not be detected within 48 h post-challenge (hpc), and the viral RNA copy number at 72 hpc was only 0.02% of that in the positive control group. Delivery of Ad(VP1-2B) reduced significantly the susceptibility of guinea pigs to FMDV infection. All guinea pigs were protected within 3 days post challenge (dpc) when they were injected twice with the same dose of Ad(VP1-2B), and a third treatment with the same dose of Ad(VP1-2B) at 3 dpc was necessary to confer longer lasting protection (up to 6 dpc). In conclusion, application of such a adenovirus vector to inhibit more than one viral gene may be an advantageous method for prevention and therapy of FMDV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Zheng, Fei-qun; Xu, Yin; Yang, Ren-jie; Wu, Bin; Tan, Xiao-hua; Qin, Yi-de; Zhang, Qun-wei

    2009-01-01

    Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting. Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo. Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma. PMID:19363518

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin

    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 representsmore » 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.« less

  20. Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

    PubMed Central

    El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K.; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M.; Rommelaere, Jean

    2012-01-01

    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. PMID:22787235

  1. 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.

  2. Incidence of human adenoviruses and Hepatitis A virus in the final effluent of selected wastewater treatment plants in Eastern Cape Province, South Africa.

    PubMed

    Osuolale, Olayinka; Okoh, Anthony

    2015-06-24

    Municipal effluent constitutes a large reservoir of human enteric viruses and bacteria. Contemporary monitoring practices rely on indicator bacteria, and do not test for viruses. Different viruses, including Norwalk-like viruses, Hepatitis A virus (HAV), adenoviruses, and rotaviruses, are important agents of illnesses in humans. The burden of disease caused by adenoviruses manifests as pneumonia, bronchiolitis, otitis media, conjunctivitis, and tonsillitis, whereas HAV infection can manifest as acute inflammatory diseases of the liver, fever, anorexia, malaise, nausea, and abdominal discomfort, followed by jaundice and dark urine. The public health implications of these viruses depend upon the physiological status of the wastewater microbial community. The occurrence of human adenovirus (HAdV) and HAV was determined in the final effluents of five wastewater treatment plants (WWTPs) in the Eastern Cape, South Africa, over 12 months (September 2012-August 2013). The viruses were detected with real-time PCR, and conventional PCR was used for serotyping. Adenovirus was detected in effluent samples from all five WWTPs and in 64 % of the total samples, whereas HAV was not detected in any effluent sample. At WWPT-A, samples were collected from the final effluent tank (adenoviral concentrations ranged from 1.05 × 10(1) to 1.10 × 10(4) genome/L, with a 41.7 % detection rate) and the discharge point (adenoviral concentrations ranged between 1.2 × 10(1) and 2.8 × 10(4) genome/L, with a 54.5 % detection rate). At WWPT-B, HAdV was detected in 91.7 % of samples, with viral concentrations of 7.92 × 10(1)-2.37 × 10(5) genome/L. The HAdV concentrations at WWPT-C were 5.32 × 10(1)-2.20 × 10(5) genome/L, and the detection rate was 75 %. The adenoviral concentrations at WWPT-D were 1.23 × 10(3)-1.05 × 10(4) genome/L, and the detection rate was 66.7 %. At WWPT-E, the viral concentrations were 1.08 × 10(1)-5.16 × 10

  3. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice.

    PubMed

    Martín, V; Pascual, E; Avia, M; Rangel, G; de Molina, A; Alejo, A; Sevilla, N

    2016-01-06

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Inactivation of Adenoviruses, Enteroviruses, and Murine Norovirus in Water by Free Chlorine and Monochloramine▿

    PubMed Central

    Cromeans, Theresa L.; Kahler, Amy M.; Hill, Vincent R.

    2010-01-01

    Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5°C. CT values (disinfectant concentration × time) for 2- to 4-log10 (99 to 99.99%) reductions in virus titers were calculated by using the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg·min/liter (pH 7 and 8) for 4-log10 inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18 mg·min/liter for 2- and 3-log10 reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log10 reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. These data will be useful in drinking water risk assessment studies and disinfection system planning. PMID:20023080

  5. Encapsulation of adenovirus serotype 5 in anionic lecithin liposomes using a bead-based immunoprecipitation technique enhances transfection efficiency.

    PubMed

    Mendez, Natalie; Herrera, Vanessa; Zhang, Lingzhi; Hedjran, Farah; Feuer, Ralph; Blair, Sarah L; Trogler, William C; Reid, Tony R; Kummel, Andrew C

    2014-11-01

    Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140 to 180 nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4 × higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Encapsulation of Adenovirus Serotype 5 in Anionic Lecithin Liposomes using a Bead-Based Immunoprecipitation Technique Enhances Transfection Efficiency

    PubMed Central

    Mendez, N.; Herrera, V.; Zhang, L.; Hedjran, F.; Feuer, R.; Blair, S.; Trogler, W.; Reid, T.

    2014-01-01

    Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140–180nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4× higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations. PMID:25154663

  7. A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus.

    PubMed

    Lin, Donghai; Tang, Thompson; Jed Harrison, D; Lee, William E; Jemere, Abebaw B

    2015-06-15

    We report on the development of a regenerable sensitive immunosensor based on electrochemical impedance spectroscopy for the detection of type 5 adenovirus. The multi-layered immunosensor fabrication involved successive modification steps on gold electrodes: (i) modification with self-assembled layer of 1,6-hexanedithiol to which gold nanoparticles were attached via the distal thiol groups, (ii) formation of self-assembled monolayer of 11-mercaptoundecanoic acid onto the gold nanoparticles, (iii) covalent immobilization of monoclonal anti-adenovirus 5 antibody, with EDC/NHS coupling reaction on the nanoparticles, completing the immunosensor. The immunosensor displayed a very good detection limit of 30 virus particles/ml and a wide linear dynamic range of 10(5). An electrochemical reductive desorption technique was employed to completely desorb the components of the immunosensor surface, then re-assemble the sensing layer and reuse the sensor. On a single electrode, the multi-layered immunosensor could be assembled and disassembled at least 30 times with 87% of the original signal intact. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Persistence of transgene expression influences CD8+ T-cell expansion and maintenance following immunization with recombinant adenovirus.

    PubMed

    Finn, Jonathan D; Bassett, Jennifer; Millar, James B; Grinshtein, Natalie; Yang, Teng Chih; Parsons, Robin; Evelegh, Carole; Wan, Yonghong; Parks, Robin J; Bramson, Jonathan L

    2009-12-01

    Previous studies determined that the CD8(+) T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8(+) T-cell maintenance following immunization with a recombinant adenovirus.

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8+ T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors. PMID:27467954

  11. 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.

  12. Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

    PubMed

    Oki, Hiroshi; Yazawa, Takuya; Baba, Yasuko; Kanegae, Yumi; Sato, Hanako; Sakamoto, Seiko; Goto, Takahisa; Saito, Izumu; Kurahashi, Kiyoyasu

    2017-07-01

    Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 10 9 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  13. Immunogenicity and efficacy in mice of an adenovirus-based bicistronic rotavirus vaccine expressing NSP4 and VP7.

    PubMed

    Xie, Li; Yan, Min; Wang, Xiaonan; Ye, Jing; Mi, Kai; Yan, Shanshan; Niu, Xianglian; Li, Hongjun; Sun, Maosheng

    2015-12-02

    NSP4 and VP7 are important functional proteins of rotavirus. Proper combination of viral gene expression is favorable to improving the protection effect of subunit vaccine. In the present study, We evaluated the immunogenicity and efficacy of the bicistronic recombinant adenovirus (rAd-NSP4-VP7) and two single-gene expressing adenoviruses (rAd-NSP4, rAd-VP7). The three adenovirus vaccines were used to immunize mice by intramuscular or intranasal administration. The data showed significant increases in serum antibodies, T lymphocyte subpopulations proliferation, and cytokine secretions of splenocyte in all immunized groups. However, the serum IgA and neutralizing antibody levels of the rAd-NSP4-VP7 or rAd-VP7 groups were significantly higher than those of the rAd-NSP4, while the splenocyte numbers of IFN-γ secretion in the rAd-NSP4-VP7 or rAd-NSP4 groups was greater than that of the rAd-VP7. Furthermore, the efficacy evaluation in a suckling mice model indicated that only rAd-NSP4-VP7 conferred significant protection against rotavirus shedding challenge. These results suggest that the co-expression of NSP4 and VP7 in an adenovirus vector induce both humoral and cell-mediated immune responses efficiently, and provide potential efficacy for protection against rotavirus disease. It is possible to represent an efficacious subunits vaccine strategy for control of rotavirus infection and transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

    PubMed

    Carey, John B; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V S; Draper, Simon J; Moore, Anne C

    2014-08-21

    Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP1₄₂, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP1₄₂ also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP1₄₂ using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

  15. Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

    PubMed Central

    Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

    2014-01-01

    Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. PMID:25142082

  16. 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.

  17. 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 Central

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

    2014-01-01

    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. PMID:24307239

  18. Adenovirus E1A and E1B-19K Proteins Protect Human Hepatoma Cells from Transforming Growth Factor β1-induced Apoptosis

    PubMed Central

    Tarakanova, Vera L.; Wold, William S. M.

    2009-01-01

    Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor β1 (TGFβ). We report that infection with species C human adenovirus conferred resistance to TGFβ-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFβ-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFβ pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFβ-induced apoptosis. PMID:19854227

  19. 2 CFR 176.120 - Determinations on late requests.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 2 Grants and Agreements 1 2012-01-01 2012-01-01 false Determinations on late requests. 176.120 Section 176.120 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS NATIONAL POLICY...

  20. 2 CFR 176.120 - Determinations on late requests.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 2 Grants and Agreements 1 2010-01-01 2010-01-01 false Determinations on late requests. 176.120 Section 176.120 Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS Reserved AWARD TERMS FOR ASSISTANCE AGREEMENTS THAT INCLUDE FUNDS UNDER THE AMERICAN...

  1. 2 CFR 176.120 - Determinations on late requests.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 2 Grants and Agreements 1 2013-01-01 2013-01-01 false Determinations on late requests. 176.120 Section 176.120 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS NATIONAL POLICY...

  2. Triple infection with agamid adenovirus 1, Encephaliton cuniculi-like microsporidium and enteric coccidia in a bearded dragon (Pogona vitticeps).

    PubMed

    Schilliger, Lionel; Mentré, Véronique; Marschang, Rachel E; Nicolier, Alexandra; Richter, Barbara

    2016-10-12

    A 2-month-old juvenile central bearded dragon was presented for anorexia and cachexia. Another specimen from the same cage had died suddenly 2 weeks prior. Fecal analysis revealed a high quantity of Isospora amphiboluri and a few pinworm eggs. Other examinations were not performed and the animal died a few days later despite supportive care. A third individual from the same cage presented with anorexia and a distended cœlom and was euthanized. In this third dragon, histological examination revealed intestinal coccidiosis, basophilic intranuclear inclusions compatible with adenovirus infection, acute hepatic necrosis with intrahepatocytic and intraenteritic organisms typical of microsporidia and renal gout. A PCR confirmed the diagnosis of adenovirosis. Sequencing showed that the PCR product was 100% identical to the corresponding portion of the agamid adenovirus 1 genome. A PCR for the detection of Encephalitozoon (E.) cuniculi was positive. Partial sequencing revealed 100% identity to an E. cuniculi-like organism previously found in bearded dragons. In cases where environmental factors such as poor hygiene or stress can be excluded, the presence of opportunistic pathogens in high numbers can be due to a systemic (viral) infection with temporary immunosuppression.

  3. 2 CFR 176.120 - Determinations on late requests.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Determinations on late requests. 176.120 Section 176.120 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS Reserved AWARD TERMS...

  4. 2 CFR 176.120 - Determinations on late requests.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 2 Grants and Agreements 1 2011-01-01 2011-01-01 false Determinations on late requests. 176.120 Section 176.120 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS [Reserved] AWARD TERMS...

  5. Immune responses in pigs induced by recombinant canine adenovirus 2 expressing the glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

    PubMed

    Zhou, J-X; Xue, J-D; Yu, T; Zhang, J-B; Liu, Y; Jiang, N; Li, Y-L; Hu, R-L

    2010-04-01

    To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection.

  6. Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII

    PubMed Central

    Fischer, Thomas H.; Herting, Jonas; Mason, Fleur E.; Hartmann, Nico; Watanabe, Saera; Nikolaev, Viacheslav O.; Sprenger, Julia U.; Fan, Peidong; Yao, Lina; Popov, Aron-Frederik; Danner, Bernhard C.; Schöndube, Friedrich; Belardinelli, Luiz; Hasenfuss, Gerd; Maier, Lars S.; Sossalla, Samuel

    2015-01-01

    Aims Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca2+-leak in atrial cardiomyocytes (CMs). Methods and results In murine atrial CMs, SR-Ca2+-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca2+-leak. Conclusion Late INa exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late

  7. Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

    PubMed Central

    Condezo, Gabriela N.; Marabini, Roberto; Ayora, Silvia; Carazo, José M.; Alba, Raúl; Chillón, Miguel

    2015-01-01

    ABSTRACT Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in

  8. Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions.

    PubMed

    Xie, Liangzhi; Pilbrough, Warren; Metallo, Christian; Zhong, Tanya; Pikus, Lana; Leung, John; Auniņs, John G; Zhou, Weichang

    2002-12-05

    PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production. Copyright 2002 Wiley Periodicals, Inc.

  9. Latent class analysis of diagnostic tests for adenovirus, Bordetella pertussis and influenza virus infections in German adults with longer lasting coughs.

    PubMed

    Sobotzki, C; Riffelmann, M; Kennerknecht, N; Hülsse, C; Littmann, M; White, A; Von Kries, R; Wirsing VON König, C H

    2016-03-01

    Laboratory tests in adult outpatients with longer lasting coughs to identify a potential causal pathogen are rarely performed, and there is no gold standard for these diagnostic tests. While the diagnostic validity of serological tests for pertussis is well established their potential contribution for diagnosing adenovirus and influenza virus A and B infections is unclear. A sentinel study into the population-based incidence of longer lasting coughs in adults was done in Rostock (former East Germany) and Krefeld (former West Germany). A total of 971 outpatients who consulted general practitioners or internists were included. Inclusion criteria were coughing for ⩾1 week and no chronic respiratory diseases. We evaluated the performance of polymerase chain reaction (PCR) as well as IgG and IgA serology, applying a latent class model for diagnosing infections with adenovirus, B. pertussis, and influenza virus A and B. The adult outpatients first sought medical attention when they had been coughing for a median of 3 weeks. In this situation, direct detection of infectious agents by PCR had a low sensitivity. Modelling showed that additional serological tests equally improved sensitivity and specificity for diagnosis for adenovirus, B. pertussis and influenza virus A and B infections. The combination of serology and PCR may improve the overall performance of diagnostic tests for B. pertussis and also for adenovirus, and influenza virus A and B infections.

  10. The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway

    PubMed Central

    Mui, Melissa Z.; Zhou, Yiwang; Blanchette, Paola; Chughtai, Naila; Knight, Jennifer F.; Gruosso, Tina; Papadakis, Andreas I.; Huang, Sidong; Park, Morag; Gingras, Anne-Claude

    2015-01-01

    ABSTRACT When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2AB55 phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2AB55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2AB55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCE The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been

  11. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response

    USDA-ARS?s Scientific Manuscript database

    We previously demonstrated that an adenovirus-based FMDV serotype A24 subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1Campos (...

  12. ELECTRON MICROSCOPY OF HELA CELLS INFECTED WITH ADENOVIRUSES

    PubMed Central

    Harford, Carl G.; Hamlin, Alice; Parker, Esther; van Ravenswaay, Theodore

    1956-01-01

    HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies. PMID:13357696

  13. 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.

  14. 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)...

  15. Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

    PubMed

    Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

    2014-02-01

    Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

  16. The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

    PubMed Central

    Gabitzsch, Elizabeth S.; Tsang, Kwong Yok; Palena, Claudia; David, Justin M.; Fantini, Massimo; Kwilas, Anna; Rice, Adrian E.; Latchman, Yvette; Hodge, James W.; Gulley, James L.; Madan, Ravi A.; Heery, Christopher R.; Balint, Joseph P.

    2015-01-01

    Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies. PMID:26374823

  17. 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.

  18. 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

  19. Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice.

    PubMed

    Zinn, Kurt R; Chaudhuri, Tandra R; Krasnykh, Victor N; Buchsbaum, Donald J; Belousova, Natalya; Grizzle, William E; Curiel, David T; Rogers, Buck E

    2002-05-01

    To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. The hSSTr2 and TK

  20. Inhibitory effect of Survivin promoter-regulated oncolytic adenovirus carrying P53 gene against gallbladder cancer.

    PubMed

    Liu, Chen; Sun, Bin; An, Ni; Tan, Weifeng; Cao, Lu; Luo, Xiangji; Yu, Yong; Feng, Feiling; Li, Bin; Wu, Mengchao; Su, Changqing; Jiang, Xiaoqing

    2011-12-01

    Gene therapy has become an important strategy for treatment of malignancies, but problems remains concerning the low gene transferring efficiency, poor transgene expression and limited targeting specific tumors, which have greatly hampered the clinical application of tumor gene therapy. Gallbladder cancer is characterized by rapid progress, poor prognosis, and aberrantly high expression of Survivin. In the present study, we used a human tumor-specific Survivin promoter-regulated oncolytic adenovirus vector carrying P53 gene, whose anti-cancer effect has been widely confirmed, to construct a wide spectrum, specific, safe, effective gene-viral therapy system, AdSurp-P53. Examining expression of enhanced green fluorecent protein (EGFP), E1A and the target gene P53 in the oncolytic adenovirus system validated that Survivin promoter-regulated oncolytic adenovirus had high proliferation activity and high P53 expression in Survivin-positive gallbladder cancer cells. Our in vitro cytotoxicity experiment demonstrated that AdSurp-P53 possessed a stronger cytotoxic effect against gallbladder cancer cells and hepatic cancer cells. The survival rate of EH-GB1 cells was lower than 40% after infection of AdSurp-P53 at multiplicity of infection (MOI) = 1 pfu/cell, while the rate was higher than 90% after infection of Ad-P53 at the same MOI, demonstrating that AdSurp-P53 has a potent cytotoxicity against EH-GB1 cells. The tumor growth was greatly inhibited in nude mice bearing EH-GB1 xenografts when the total dose of AdSurp-P53 was 1 × 10(9) pfu, and terminal dUTP nick end-labeling (TUNEL) revealed that the apoptotic rate of cancer cells was (33.4 ± 8.4)%. This oncolytic adenovirus system overcomes the long-standing shortcomings of gene therapy: poor transgene expression and targeting of only specific tumors, with its therapeutic effect better than the traditional Ad-P53 therapy regimen already on market; our system might be used for patients with advanced gallbladder cancer and

  1. Characterizing Bacteriophage PR772 as a Potential Surrogate for Adenovirus in Water Disinfection: A Comparative Analysis of Inactivation Kinetics and Replication Cycle Inhibition by Free Chlorine.

    PubMed

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2016-03-01

    Elucidating mechanisms by which pathogenic waterborne viruses become inactivated by drinking water disinfectants would facilitate the development of sensors to detect infectious viruses and novel disinfection strategies to provide safe water. Using bacteriophages as surrogates for human pathogenic viruses could assist in elucidating these mechanisms; however, an appropriate viral surrogate for human adenovirus (HAdV), a medium-sized virus with a double-stranded DNA genome, needs to be identified. Here, we characterized the inactivation kinetics of bacteriophage PR772, a member of the Tectiviridae family with many similarities in structure and replication to HAdV. The inactivation of PR772 and HAdV by free chlorine had similar kinetics that could be represented with a model previously developed for HAdV type 2 (HAdV-2). We developed and tested a quantitative assay to analyze several steps in the PR772 replication cycle to determine if both viruses being inactivated at similar rates resulted from similar replication cycle events being inhibited. Like HAdV-2, we observed that PR772 inactivated by free chlorine still attached to host cells, and viral DNA synthesis and early and late gene transcription were inhibited. Consequently, free chlorine exposure inhibited a replication cycle event that was post-binding but took place prior to early gene synthesis for both PR772 and HAdV-2.

  2. Evidence for a Single-Stranded Adenovirus-Associated Virus Genome: Isolation and Separation of Complementary Single Strands

    PubMed Central

    Berns, K. I.; Rose, J. A.

    1970-01-01

    Single-stranded adenovirus-associated virus type 2 deoxyribonucleic acid (AAV-2 DNA) has been isolated from the virion after enzymatic pretreatment of the particles by heating at 53 C for 1 hr in 0.015 m NaCl plus 0.0015 m sodium citrate in the presence of 1% sodium dodecyl sulfate. Double-stranded AAV-2 DNA present as a marker is not denatured by this treatment. AAV-2 single-stranded DNA is composed of two complementary species which can be separated in neutral CsCl when 5-bromodeoxyuridine has been substituted for thymidine in the DNA. The present report is the first documented instance of the separation of complementary strands of an animal virus DNA. PMID:5429749

  3. Recombinant adenovirus of SEA and CD80 genes driven by MMRE and mouse TERT promoter induce effective antitumor immune responses against different types of tumor cells in vitro and in vivo.

    PubMed

    Si, Shao-Yan; Liu, Jun-Li; Liu, Jun-Lian; Xu, Bing-Xin; Li, Jian-Zhong; Qin, Ya-Ya; Song, Shu-Jun

    2017-05-01

    Staphylococcus enterotoxin A (SEA) is a powerful immunostimulant and can stimulate T cells bearing certain T-cell receptor β-chain variable regions when bound to major histocompatibility complex II molecules. SEA is widely used in research of antitumor therapy. The low affinity T-cell receptor (TCR) interaction with SEA in the absence of MHC class II antigens is sufficient for the induction of cytotoxicity but requires additional CD28/B7 signaling to result in proliferation of resting T cells. In this study, we constructed recombinant adenovirus (named as Ad-MMRE-mTERT-BIS) carrying membrane-expressing SEA (named as SEAtm) and CD80 driven by Myc-Max response elements (MMRE) and mouse telomerase reverse transcriptase (mTERT) promoter to reduce toxicity and to improve safety and efficiency. We demonstrated that Ad-MMRE-mTERT-BIS could make SEAtm and CD80 to co-express highly on the surface of Hepa1-6 and B16 cells, at low level on the surface of CT26 cells, but not in NIH3T3. Hepa1-6 and B16 cells infected by the recombinant adenovirus induced proliferation of CD4+ and CD8+ T cells and increased cytokine [interleukin (IL)-2, tumor necrosis factor (TNF)-α, interferon (IFN)-γ] production in vitro. Intratumoral injection of Ad-MMRE-mTERT-BIS in hepatoma and melanoma mouse models induced tumor-specific cytotoxic T cells in the spleen. Moreover, hepatoma and melanoma xenografts were suppressed by treatment with Ad-MMRE-mTERT-BIS and the survival time of treated mice was prolonged. These findings suggest that recombinant adenovirus of SEA and CD80 genes driven by mTERT promoter could induce effective antitumor immune responses against different kinds of tumor cells in vitro and in vivo.

  4. Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

    PubMed

    Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

    2010-06-22

    Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

  5. Detection of adenovirus and respiratory syncytial virus in patients with chronic obstructive pulmonary disease: Exacerbation versus stable condition.

    PubMed

    Kokturk, Nurdan; Bozdayi, Gulendam; Yilmaz, Senay; Doğan, Bora; Gulbahar, Ozlem; Rota, Seyyal; Tatlicioglu, Turkan

    2015-08-01

    Latent infection with adenovirus and respiratory syncytial virus (RSV) is associated with chronic obstructive pulmonary disease (COPD). The role of respiratory viral infections are emerging in COPD exacerbations. The present study aimed to investigate the prevalence of adenovirus and RSV serotypes A and B in individuals with acute exacerbations of COPD (COPD-AE) and stable COPD. Twenty seven patients with COPD-AE were evaluated using a prospective longitudinal study design. Induced sputum, sera and nasal smears were sampled from patients experiencing COPD-AE and those in a stable condition. Adenoplex® multiplex polymerase chain reaction (PCR) kits and Invitek RTP® DNA/RNA Virus Mini kits were used for PCR assays of adenovirus and RSV, respectively. Eighteen patients who experienced a COPD-AE were also evaluated while in a stable condition. The results showed that three sputum samples were positive for adenovirus in patients experiencing an exacerbation, while one was positive among the patients in a stable condition. RSV serotype A was detected in 17/27 (63%) patients with COPD-AE and 10/18 (55.6%) patients in a stable condition. RSV serotype B was not detected. Patients with COPD-AE, who were positive for RSV serotype A exhibited higher serum fibrinogen levels than those who were negative (438.60 ± 126.08 mg/dl compared with 287.60 ± 85.91 mg/dl; P=0.004). Eight/ten patients who were positive for RSV serotype A while in a stable condition, were also positive during COPD-AE. The results of the present study suggested that RSV infection may be prevalent in patients with COPD-AE and in those in a stable condition. Therefore, chronic RSV infection may occur in COPD. The detection and prevention of RSV may be useful in the management of COPD.

  6. Biodistribution and Safety Assessment of Bladder Cancer Specific Recombinant Oncolytic Adenovirus in Subcutaneous Xenografts Tumor Model in Nude Mice

    PubMed Central

    Wang, Fang; Wang, Zhiping; Tian, Hongwei; Qi, Meijiao; Zhai, Zhenxing; Li, Shuwen; Li, Renju; Zhang, Hongjuan; Wang, Wenyun; Fu, Shenjun; Lu, Jianzhong; Rodriguez, Ronald; Guo, Yinglu; Zhou, Liqun

    2012-01-01

    Background The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. Materials and Method Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific Uroplakin II (UP II) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. Results General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5×108 pfu or higher dose (5×109 pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5×109 pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. Conclusions Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5×107 pfu and 5×108 pfu intratumorally injection in mice, without any discernable effects on general health

  7. Biodistribution and safety assessment of bladder cancer specific recombinant oncolytic adenovirus in subcutaneous xenografts tumor model in nude mice.

    PubMed

    Wang, Fang; Wang, Zhiping; Tian, Hongwei; Qi, Meijiao; Zhai, Zhenxing; Li, Shuwen; Li, Renju; Zhang, Hongjuan; Wang, Wenyun; Fu, Shenjun; Lu, Jianzhong; Rodriguez, Ronald; Guo, Yinglu; Zhou, Liqun

    2012-04-01

    The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials. Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific UroplakinII(UPII) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay. General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5x10(8) pfu or higher dose (5x10(9) pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5x10(9) pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay. Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5x10(7) pfu and 5x10(8) pfu intratumorally injection in mice, without any discernable effects on general health and behavior.

  8. The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

    PubMed

    Reddy, Vijay S

    2017-09-01

    Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing continuous hexagonal network formed by the protein IX molecules is conserved among different human adenoviruses (HAdVs), but not in non-HAdVs. In this report, we propose a distinct role for the hexon protein as a "molecular mold" in enabling the formation of such hexagonal protein IX network that has been shown to preserve the stability and infectivity of HAdVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

    PubMed Central

    Dowdle, W. R.; Lambriex, M.; Hierholzer, J. C.

    1971-01-01

    A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO4 is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections. Images PMID:4325021

  10. Molecular Identification and Epidemiological Features of Human Adenoviruses Associated with Acute Respiratory Infections in Hospitalized Children in Southern China, 2012-2013.

    PubMed

    Chen, Yi; Liu, Fanghua; Wang, Changbing; Zhao, Mingqi; Deng, Li; Zhong, Jiayu; Zhang, Yingying; Ye, Jun; Jing, Shuping; Cheng, Zetao; Guan, Yongxin; Ma, Yi; Sun, Yuanyuan; Zhu, Bing; Zhang, Qiwei

    2016-01-01

    Acute respiratory infections (ARI) are the major worldwide health problem associated with high morbidity and mortality rates. Human adenovirus (HAdV) is one of the most common pathogens associated with viral ARI, and thus calls for specific diagnosis and better understanding of the epidemiology and clinical characteristics. Total 4,130 children with ARI requiring hospitalization from 2012 to 2013 were retrospectively studied. Throat swab specimens were collected from each patient. Fluorescence Quantitative PCR was performed to detect adenovirus as well as other common ARI-related pathogens. The seven HAdV hypervariable regions (HVRs) of the hexon gene from fifty-seven HAdVs-positive samples collected in the seasonal peaks were sequenced. Phylogenetic analysis of HVRs was also conducted to confirm the molecular types and genetic variation. In addition, epidemiological features and co-infection with other human respiratory pathogens were investigated and analyzed. Of 4,130 hospitalized pediatric patients tested, the positive rates of respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP), and HAdV were 13.7%, 13.2%, and 12.0%, respectively. The HAdV positive patients accounted for 7.9%, 17.2%, 17.5% and 10.7% in age groups <1, 1-3, 3-6 and 6-14 years, respectively. Eighty-four HAdV positive children were co-infected with other respiratory pathogens (84/495, 17.0%). The most common co-infection pathogens with HAdV were MP (57.1%) and Human Bocavirus (HBoV) (16.7%). The majority of HAdV infected patients were totally recovered (96.9%, 480/495); However, four (0.8%) patients, who were previously healthy and at the age of 2 years or younger died of pneumonia. Seasonal peaks of HAdV infection occurred in the summer season of 2012 and 2013; the predominant HAdV type was HAdV-3 (70%), followed by HAdV-7 (28%). These epidemiological features were different from those in Northern China. The HAdV-55 was identified and reported for the first time in Guangzhou

  11. MicroRNA-155 attenuates late sepsis-induced cardiac dysfunction through JNK and β-arrestin 2.

    PubMed

    Zhou, Yu; Song, Yan; Shaikh, Zahir; Li, Hui; Zhang, Haiju; Caudle, Yi; Zheng, Shouhua; Yan, Hui; Hu, Dan; Stuart, Charles; Yin, Deling

    2017-07-18

    Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Major transgression during Late Cretaceous constrained by basin sediments in northern Africa: implication for global rise in sea level

    NASA Astrophysics Data System (ADS)

    An, Kaixuan; Chen, Hanlin; Lin, Xiubin; Wang, Fang; Yang, Shufeng; Wen, Zhixin; Wang, Zhaoming; Zhang, Guangya; Tong, Xiaoguang

    2017-12-01

    The global rise in sea level during the Late Cretaceous has been an issue under discussion by the international geological community. Despite the significance, its impact on the deposition of continental basins is not well known. This paper presents the systematic review on stratigraphy and sedimentary facies compiled from 22 continental basins in northern Africa. The results indicate that the region was dominated by sediments of continental facies during Early Cretaceous, which were replaced by deposits of marine facies in Late Cretaceous. The spatio-temporal distribution of sedimentary facies suggests marine facies deposition reached as far south as Taoudeni-Iullemmeden-Chad-Al Kufra-Upper Egypt basins during Turonian to Campanian. These results indicate that northern Africa underwent significant transgression during Late Cretaceous reaching its peak during Turonian to Coniacian. This significant transgression has been attributed to the global high sea-level during this time. Previous studies show that global rise in sea level in Late Cretaceous may have been driven by an increase in the volume of ocean water (attributed to high CO2 concentration and subsequently warm climate) and a decrease in the volume of the ocean basin (attributed to rapid production of oceanic crust and seamounts). Tectonic mechanism of rapid production of oceanic crust and seamounts could play a fundamental role in driving the global rise in sea level and subsequent transgression in northern Africa during Late Cretaceous.

  14. Correlates and prevalence of hypogonadism in patients with early- and late-onset type 2 diabetes.

    PubMed

    Li, Y; Zhang, M; Liu, X; Cui, W; Rampersad, S; Li, F; Lin, Z; Yang, P; Li, H; Sheng, C; Cheng, X; Qu, S

    2017-07-01

    This study aims to compare the prevalence of hypogonadism between male patients with early-onset type 2 diabetes mellitus (T2DM) and late-onset type 2 diabetes. A total of 122 male patients with early-onset T2DM (diagnosis age ≤40 years) and 100 male patients with late-onset T2DM (diagnosis age >40 years) were recruited from our in-patient department between 1 January 2013 and 28 December 2015. Serum FSH, LH, testosterone, lipid profile, uric acid, HbA1c, and beta-cell function were determined in blood samples. The diagnosis of hypogonadism was based on the levels of LH, FSH, and total testosterone. The mean onset age was 29.86 ± 6.31 and 54.47 ± 9.97 years old in the early-onset group and late-onset group, respectively. Compared with late-onset T2DM, those with early-onset T2DM had a higher proportion of new-onset diabetes, were more likely to be obese, and had worse glycemic control, lipid control, and lower sex hormone-binding globulin (SHBG). The prevalence of hypogonadism was much higher in the early-onset group than in the late-onset group (48.0% vs. 26.7%, p < 0.05). The rate of secondary hypogonadism in the early-onset group and late-onset group were 44.3% and 25.0%, respectively (p < 0.05). Obesity, waist circumference, and SHBG were significantly associated with serum total testosterone level in all, early-onset, and late-onset T2DM. Both all and early-onset T2DM groups had positive correlations between total testosterone and fasting C-peptide, total cholesterol, triglycerides, and uric acid. Our results indicate that in a population of admission to a large urban hospital in China, the prevalence of hypogonadism was higher in the patients with early-onset T2DM than that of late-onset T2DM. This prevalence might be attributable to greater obesity, worse lipid control, and lower SHBG levels in those patients. © 2017 American Society of Andrology and European Academy of Andrology.

  15. Serotype-Specific Neutralizing Antibody Epitopes of Human Adenovirus Type 3 (HAdV-3) and HAdV-7 Reside in Multiple Hexon Hypervariable Regions

    PubMed Central

    Qiu, Hongling; Li, Xiao; Tian, Xingui; Zhou, Zhichao; Xing, Ke; Li, Haitao; Tang, Ni; Liu, Wenkuan; Bai, Peisheng

    2012-01-01

    Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAdΔE3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo. Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAdΔE3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors. PMID:22623776

  16. Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

    PubMed Central

    Scala, Stefania; Portella, Giuseppe; Fedele, Monica; Chiappetta, Gennaro; Fusco, Alfredo

    2000-01-01

    High mobility group I (HMGI) proteins are overexpressed in several human malignant tumors. We previously demonstrated that inhibition of HMGI synthesis prevents thyroid cell transformation. Here, we report that an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas) induced programmed cell death of two human thyroid anaplastic carcinoma cell lines (ARO and FB-1), but not normal thyroid cells. The Ad-Yas virus led to death of lung, colon, and breast carcinoma cells. A control adenovirus carrying the lacZ gene did not inhibit the growth of either normal or neoplastic cells. Ad-Yas treatment of tumors induced in athymic mice by ARO cells caused a drastic reduction in tumor size. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may be a useful treatment strategy in a variety of human malignant neoplasias, in which HMGI(Y) gene overexpression is a general event. PMID:10759549

  17. Time course for memory dysfunction in early-life and late-life major depression: a longitudinal study from the Juntendo University Mood Disorder Project.

    PubMed

    Maeshima, Hitoshi; Baba, Hajime; Nakano, Yoshiyuki; Satomura, Emi; Namekawa, Yuki; Takebayashi, Naoko; Nomoto, Hiroshi; Suzuki, Toshihito; Mimura, Masaru; Arai, Heii

    2013-10-01

    Previous studies have demonstrated that patients with depression also have memory dysfunctions during depressive episodes. These dysfunctions partially remain immediately after remission from a depressive state; however, it is unclear whether these residual memory dysfunctions may disappear through long-term remission from depression. The present study compared patients during early-life (age<60) and late-life (age ≥ 60) depression while in their remitted stage with healthy controls to elucidate the impact of a long-term course on memory. Logical memory from the Wechsler Memory Scale-Revised was administered to 67 patients with major depressive disorder (MDD) (47 patients with early-life depression and residual 20 patients with late-life depression) and 50 healthy controls. MDD patients received memory assessments at the time of their initial remission and at a follow-up three years after remission. At the time of initial remission, scores for logical memory were significantly lower in both patient groups compared to matched controls. At follow-up, memory dysfunction for early-life MDD patients disappeared, whereas scores in the late-life MDD group remained significantly lower than those of matched controls. All patients in the present study were on antidepressant medications. Our findings suggested that the progress of memory performance in late-life MDD patients may be different from early-life MDD patients. © 2013 Elsevier B.V. All rights reserved.

  18. Molecular epidemiology of a post-influenza pandemic outbreak of acute respiratory infections in Korea caused by human adenovirus type 3.

    PubMed

    Lee, Wan-Ji; Jung, Hee-Dong; Cheong, Hyang-Min; Kim, Kisoon

    2015-01-01

    An outbreak of upper respiratory tract infections associated with human adenovirus (HAdV) occurred on a national scale in Korea from September to December 2010, following a major H1N1 influenza pandemic. Data from the Korea Influenza and Respiratory Surveillance System (KINRESS) showed an unusually high positive rate accounting for up to 20% of all diagnosed cases. To determine the principal cause of the outbreak, direct polymerase chain reaction (PCR) amplification followed by sequence analysis targeting parts of the hexon gene of HAdV was performed. Serotypes of 1,007 PCR-diagnosed HAdV-positive samples from patients with an acute upper respiratory tract illness were determined and epidemiological characteristics including major aged group and clinical symptoms were analyzed. The principal symptom of HAdV infections was fever and the vulnerable aged group was 1-5 years old. Based on sequence analysis, HAdV-3 was the predominant serotype in the outbreak, with an incidence of 74.3%. From the beginning of 2010 until May, the major serotypes were HAdV-1, 2, and 5 (70-100%) in any given period. However, an outbreak dominated by HAdV-3 started between July and August and peaked in September. Phylogenetic analysis revealed that there was no genetic variation in HAdV-3. The results demonstrated that an outbreak of upper respiratory illness followed by H1N1 influenza pandemic in Korea was caused mainly by emerged HAdV-3. J. © 2014 Wiley Periodicals, Inc.

  19. ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice

    PubMed Central

    Reddi, HV; Madde, P; Reichert-Eberhardt, AJ; Galanis, EC; Copland, JA; McIver, B; Grebe, SKG; Eberhardt, NL

    2011-01-01

    Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1–2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials. PMID:18583996

  20. Oncolytic adenovirus encoding tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits the growth and metastasis of triple-negative breast cancer

    PubMed Central

    Zhu, Wei; Zhang, Hongwei; Shi, Yi; Song, Mangen; Zhu, Bijun; Wei, Lai

    2013-01-01

    Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising cancer therapeutic target due to its selective apoptosis-inducing effect in cancer cells. To efficiently deliver TRAIL to the tumor cells, an oncolytic adenovirus (p55-hTERT-HRE-TRAIL) carrying the TRAIL coding sequence was constructed. In the present study, we aimed to investigate the effect of p55-hTERT-HRE-TRAIL on the growth and metastasis of triple-negative breast cancer (TNBC). We observed that infection of the recombinant adenovirus resulted in expression of TRAIL and massive cell death in a TNBC cell line MDA-MB-231. This effect is much weaker in MCF-10A, which is a normal breast cell line. Administration of P55-HTERT-HRE-TRAIL significantly reduced orthotopic breast tumor growth and extended survival in a metastatic model. Our results suggest the oncolytic adenovirus armed with P55-HTERT-HRE-TRAIL, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of TNBC. PMID:24025362