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Sample records for armed replicating adenoviruses

  1. Adenovirus DNA Replication

    PubMed Central

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease. PMID:23388625

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-01

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy. PMID:27195521

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

    PubMed

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

    2016-06-01

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

  5. The Dual Nature of Nek9 in Adenovirus Replication

    PubMed Central

    Jung, Richard; Radko, Sandi

    2015-01-01

    ABSTRACT To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks

  6. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

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

    2015-01-01

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

  7. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

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

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2016-07-15

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

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

    PubMed Central

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

    1968-01-01

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

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

    PubMed Central

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

    2015-01-01

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents. PMID:26280277

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

    SciTech Connect

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

    2007-12-05

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

  13. Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

    PubMed Central

    Wides, R J; Challberg, M D; Rawlins, D R; Kelly, T J

    1987-01-01

    The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions. Images PMID:3821730

  14. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  15. Switching a replication-defective adenoviral vector into a replication-competent, oncolytic adenovirus.

    PubMed

    Nakashima, Hiroshi; Chiocca, E Antonio

    2014-01-01

    The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad

  16. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector

    PubMed Central

    Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Sylte, Matt J.; Pfeiffer, Jennifer; Van Kampen, Kent R.

    2009-01-01

    Protective immunity against avian influenza virus was elicited in chickens by single-dose in ovo vaccination with a non-replicating human adenovirus vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 (89% hemagglutinin homology; 68% protection) and H5N2 (94% hemagglutinin homology; 100% protection) highly pathogenic avian influenza virus challenges. Mass-administration of this bird flu vaccine can be streamlined with available robotic in ovo injectors. In addition, adenovirus-vectored vaccines can be produced rapidly and the safety margin of a non-replicating vector is superior to that of a replicating counterpart. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural avian influenza virus infections. PMID:17055126

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. A New Type of Adenovirus Vector That Utilizes Homologous Recombination To Achieve Tumor-Specific Replication

    PubMed Central

    Bernt, Kathrin; Liang, Min; Ye, Xun; Ni, Shaoheng; Li, Zong-Yi; Ye, Sheng Long; Hu, Fang; Lieber, André

    2002-01-01

    We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application. PMID:12368342

  19. Amplified and Persistent Immune Responses Generated by Single-Cycle Replicating Adenovirus Vaccines

    PubMed Central

    Crosby, Catherine M.; Nehete, Pramod; Sastry, K. Jagannadha

    2014-01-01

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

  20. Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells

    PubMed Central

    Radko, Sandi; Jung, Richard; Olanubi, Oladunni; Pelka, Peter

    2015-01-01

    Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts. PMID:26448631

  1. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  2. Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells.

    PubMed

    Guo, Ju-Tao; Zhou, Tianlun; Guo, Haitao; Block, Timothy M

    2007-12-01

    Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.

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

    SciTech Connect

    Liu, Ran-yi; Zhou, Ling; Zhang, Yan-ling; Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min; Li, Li-xia; Fu, Xiang; Wu, Jiang-xue; Huang, Wenlin

    2013-12-13

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

  4. Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

    PubMed

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2015-04-01

    Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

  5. 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

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

    PubMed

    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

    2015-03-01

    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.

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

    PubMed

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

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future. PMID:27557721

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

    PubMed Central

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

    2007-01-01

    Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; viral infectivity and tumor specificity have been poor. Herein we report on two CRAd agents, CRAd-S.RGD and CRAd-S.F5/3, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD or F5/3) in the adenovirus fiber region. These CRAd agents effectively target human mesothelioma cell lines, induce strong cytoxicity in these cells in vitro, and viral replication in a H226 murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting mesothelioma with low host toxicity. These agents should provide important insights into the identification of novel therapeutic strategies for mesothelioma. PMID:17409940

  9. The adenovirus terminal protein influences binding of replication proteins and changes the origin structure.

    PubMed Central

    Pronk, R; van der Vliet, P C

    1993-01-01

    The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins. Images PMID:8506126

  10. A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents.

    PubMed

    Dobbins, G Clement; Ugai, Hideyo; Curiel, David T; Gillespie, G Yancey

    2015-01-01

    Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis. PMID:26689910

  11. A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents

    PubMed Central

    Dobbins, G. Clement; Ugai, Hideyo; Curiel, David T.; Gillespie, G. Yancey

    2015-01-01

    Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis. PMID:26689910

  12. Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

    PubMed

    Bachmann, Mandy; Breitwieser, Theresa; Lipps, Christoph; Wirth, Dagmar; Jordan, Ingo; Reichl, Udo; Frensing, Timo

    2016-02-01

    Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

  13. Endotoxins and prednisolone alter replication of type 5 adenovirus and its temperature sensitive mutants.

    PubMed

    Ongrádi, J; Bertók, L; Farkas, J; Nász, I; Bendinelli, M

    1994-01-01

    Latency, replication or transformation by adenoviruses require cooperation between their gene products and cellular factors, which are controlled by external stimuli. Clinical observations suggest that bacterial endotoxin (LPS) and steroid hormones have direct effects on the viral permissivity and activation. Therefore, HEp-2 cultures were infected with low multiplicity of wild type (WT) human adenovirus 5 (Ad-5) and its temperature-sensitive mutants ts18 and ts19 damaged in the phosphorylation of structural polypeptides VI and X at 39 degrees C, respectively. Cultures were treated at permissive (32 degrees C) and nonpermissive (39 degrees C) temperatures with native and radio-detoxified (RD) LPS, alpha-tocopherol and prednisolone alone or in combination. Titration of virus yields showed dose-dependent activation and enhanced replication of latent WT and ts mutants by both LPS preparations. alpha-Tocopherol diminished these processes. LPS was likewise unable to augment virus producing capacity of cells. Prednisolone, although activating no latent virus, resulted in augmenting Ad-5 production at 32 degrees C. No compounds activated mutants at 39 degrees C except synergistic effect of LPS and prednisolone resulted in limited but significant replication of mutant ts19. Deliberation of lysosomal enzymes, enhanced cGMP and tumour necrosis factor alpha (TNF-alpha) productions by LPS as well as interaction of Ad early gene expression with prednisolone-utilized cellular transcription factors including AP-1 (c-jun, c-fos) are implicated in these processes. Excluding the role in activation of Ad proteinase processing viral structural polypeptides draws attention to the importance of cellular factors in virus replication.

  14. Infection by retroviral vectors outside of their host range in the presence of replication-defective adenovirus.

    PubMed Central

    Adams, R M; Wang, M; Steffen, D; Ledley, F D

    1995-01-01

    Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease. PMID:7853530

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

    PubMed

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

    2006-07-01

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

  16. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

    PubMed Central

    Pettit, S C; Horwitz, M S; Engler, J A

    1988-01-01

    Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP. Images PMID:3336069

  19. Conditionally replicating adenovirus prevents pluripotent stem cell–derived teratoma by specifically eliminating undifferentiated cells

    PubMed Central

    Mitsui, Kaoru; Ide, Kanako; Takayama, Akiko; Wada, Tadahisa; Irie, Rie; Kosai, Ken-ichiro

    2015-01-01

    Incomplete abolition of tumorigenicity creates potential safety concerns in clinical trials of regenerative medicine based on human pluripotent stem cells (hPSCs). Here, we demonstrate that conditionally replicating adenoviruses that specifically target cancers using multiple factors (m-CRAs), originally developed as anticancer drugs, may also be useful as novel antitumorigenic agents in hPSC-based therapy. The survivin promoter was more active in undifferentiated hPSCs than the telomerase reverse transcriptase (TERT) promoter, whereas both promoters were minimally active in differentiated normal cells. Accordingly, survivin-responsive m-CRA (Surv.m-CRA) killed undifferentiated hPSCs more efficiently than TERT-responsive m-CRAs (Tert.m-CRA); both m-CRAs exhibited efficient viral replication and cytotoxicity in undifferentiated hPSCs, but not in cocultured differentiated normal cells. Pre-infection of hPSCs with Surv.m-CRA or Tert.m-CRA abolished in vivo teratoma formation in a dose-dependent manner following hPSC implantation into mice. Thus, m-CRAs, and in particular Surv.m-CRAs, represent novel antitumorigenic agents that could facilitate safe clinical applications of hPSC-based regenerative medicine. PMID:26269798

  20. The TGGCA protein binds to the MMTV-LTR, the adenovirus origin of replication, and the BK virus enhancer.

    PubMed Central

    Nowock, J; Borgmeyer, U; Püschel, A W; Rupp, R A; Sippel, A E

    1985-01-01

    TGGCA-binding proteins are nuclear proteins with high affinity for double-stranded DNA homologous to the prototype recognition sequence 5'YTGGCANNNTGCCAR 3'. Their ubiquitous tissue distribution in higher vertebrates characterizes them as a class of highly conserved proteins which may exert a basic function. To obtain clues to this function, specific binding sites were mapped on three viral genomes. Recognition sites were identified in the enhancer region of the BK virus, in the LTR of the mouse mammary tumor virus, and in the origin of replication of adenovirus 12. The TGGCA-binding protein from HeLa cells appears to be identical to nuclear factor I described by others, which stimulates initiation of adenovirus DNA replication in vitro. However, data from MMTV, BKV, and from cellular genes suggest that this specific protein-DNA interaction may also be involved in the control of gene activity. Images PMID:2987840

  1. Augmenting the antitumor effect of TRAIL by SOCS3 with double-regulated replicating oncolytic adenovirus in hepatocellular carcinoma.

    PubMed

    Wei, Rui-Cheng; Cao, Xin; Gui, Jing-Hua; Zhou, Xiu-Mei; Zhong, Dan; Yan, Qiao-Lin; Huang, Wei-Dan; Qian, Qi-Jun; Zhao, Feng-Li; Liu, Xin-Yuan

    2011-09-01

    Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy. PMID:21361790

  2. Eliminating established tumor in nu/nu nude mice by a TRAIL-armed oncolytic adenovirus

    PubMed Central

    Dong, Fengqin; Wang, Li; Davis, John J.; Hu, Wenxian; Zhang, Lidong; Guo, Wei; Teraishi, Fuminori; Ji, Lin; Fang, Bingliang

    2006-01-01

    Purpose The tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector. Experimental Design We constructed a TRAIL-expressing oncolytic adenovector designated Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors. Results Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing replication-defective adenovector or an oncolytic adenovector expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all subcutaneous xenograft tumors established from a human non-small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term tumor-free survival. Furthermore, we found no treatment-related toxicity. Conclusions Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy. PMID:16951242

  3. Adenovirus E1A Targets the DREF Nuclear Factor To Regulate Virus Gene Expression, DNA Replication, and Growth

    PubMed Central

    Radko, Sandi; Koleva, Maria; James, Kris M. D.; Jung, Richard; Mymryk, Joe S.

    2014-01-01

    ABSTRACT The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication. IMPORTANCE This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to

  4. Transgene delivery to cultured keratinocytes via replication-deficient adenovirus vectors.

    PubMed

    Ramirez, Vincent P; Aneskievich, Brian J

    2014-01-01

    Transient transgene expression can facilitate investigation of that gene-product function or effect on keratinocyte biology. Several chemical and biologic delivery systems are available, and among them adenoviruses offer particular advantages in efficiency and transgene capacity. Here we describe the advantages of bicistronic adenovirus and inclusion of the polycation hexadimethrine bromide to aid in the detection of positively transduced cells and enhance transduction efficiency. PMID:24281865

  5. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

    PubMed

    Toth, Karoly; Lee, Sang R; Ying, Baoling; Spencer, Jacqueline F; Tollefson, Ann E; Sagartz, John E; Kong, Il-Keun; Wang, Zhongde; Wold, William S M

    2015-08-01

    Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.

  6. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control

    PubMed Central

    Spencer, Jacqueline F.; Tollefson, Ann E.; Sagartz, John E.; Kong, Il-Keun; Wang, Zhongde; Wold, William S. M.

    2015-01-01

    Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models. PMID

  7. Endonuclease G depletion may improve efficiency of first generation adenovirus vector DNA replication in HeLa cells.

    PubMed

    Misic, V; El-Mogy, M; Haj-Ahmad, Y

    2015-01-01

    First generation adenovirus (Ad5 ΔE1,E3) vectors are able to replicate their DNA in many tumour cells and can be used for oncotherapy. Highest rates of viral DNA replication occur in the G2/M transition of the cell cycle. In this study, we tried to increase the efficiency of Ad5 ΔE1,E3 DNA replication in the cervical carcinoma HeLa cells by using RNA interference (RNAi) to target endonuclease G (EndoG) whose depletion leads to an accumulation of cells in the G2/M transition. Targeting of EndoG by an shRNA encoded on an Ad5 ΔE1,E3 vector resulted in an early proliferation defect of cervical carcinoma HeLa cells. This effect coincided with enhanced DNA replication and encoded transgene expression of an Ad5 ΔE1,E3 vector. Applied in high concentrations, the EndoG-targeting Ad5 ΔE1,E3 vector showed enhanced HeLa cell killing ability relative to control Ad5 ΔE1,E3 vectors. These effects are most likely the result of EndoG depletion, which causes cells to accumulate in the G2/M transition of the cell cycle and extends favourable cellular conditions for Ad5 ΔE1,E3 DNA replication. Targeting of EndoG by RNAi may be a viable strategy for improving both the levels of transgene expression and the oncolytic properties of first generation adenovirus vectors.

  8. Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

    PubMed Central

    Brison, O; Kedinger, C; Wilhelm, J

    1977-01-01

    When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class. PMID:916022

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

    PubMed

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

    2015-08-01

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

  10. Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection.

    PubMed

    Eggerding, F A; Pierce, W C

    1986-01-15

    As an initial step toward understanding the mechanisms underlying host cell restriction of adenovirus 2 (Ad2) replication, we have studied various cell lines derived from hamster (CHO-K1), rat (CREF, NRK-49F, C-3, C-9), and mouse (3T3-Swiss) tissues to determine their degree of permissivity to Ad2 replication. For each cell line tested, the time course of Ad2 growth was determined; the yield of infectious virus, as measured by titration on HeLa cell monolayers, was reduced 3 to 5 logs. This result is independent of the multiplicity of infection at multiplicities between 4 and 100 plaque-forming units (PFU) per cell. The Western immunoblotting technique was used to quantitate the amounts of early proteins (E1A 45-54K proteins, E1B 21 and 58K proteins, E2A 72K DNA binding protein) and late structural proteins (hexon, fiber) produced during restricted infections. All cell lines expressed 72K DNA binding protein and variable levels of other early proteins. C-3, C-9, and NRK-49F cells expressed hexon as well as low, but detectable levels of fiber protein. Mouse 3T3-Swiss cells failed to synthesize any detectable levels of late structural proteins. DNA synthesis analysis indicated all rodent cell lines were capable of replicating viral DNA. A decreased rate of viral DNA synthesis was observed in CREF cells. Evidence is presented which suggests newly synthesized viral DNA is unstable in 3T3-Swiss cells.

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

    PubMed

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

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  12. Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2008-04-25

    Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

  13. Early dissociation of nuclear factor I from the origin during initiation of adenovirus DNA replication studied by origin immobilization.

    PubMed

    Coenjaerts, F E; van der Vliet, P C

    1994-12-11

    The DNA-binding domain of Nuclear Factor I (NFIBD) enhances initiation of adenovirus DNA replication up to 50-fold by binding to the auxiliary region of the origin and positioning the viral DNA polymerase. To study if and when NFIBD dissociates from the template, we immobilized origin DNA to glutathione-agarose beads by means of a GST-NFIBD fusion protein. This immobilized template is active in replication. By analyzing the release of prelabeled templates from the beads under different conditions, we show that NFIBD dissociates already early during initiation. During preinitiation NFIBD remains bound, but as soon as dCTP, dATP or dTTP are added, efficient dissociation occurs. A much lower dissociation level was induced by addition of dGTP. Since dCTP, dATP and dTTP are required for formation of a pTP-CAT initiation intermediate, we explain our results by conformational changes occurring in the polymerase during initiation leading to disruption of both the interaction between the polymerase and NFI as well as the interaction between NFI and the DNA.

  14. Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

    PubMed Central

    Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J.; Farness, Peggy; Avanzini, Jenny B.; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J.; Mayall, Tim

    2012-01-01

    Background Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. Methodology/Principal Findings The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Conclusions/Significance Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine. PMID:22363572

  15. The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle.

    PubMed Central

    Goodrum, F D; Ornelles, D A

    1997-01-01

    The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle. PMID:8985383

  16. Targeting the replication of adenovirus to p53-defective thyroid carcinoma with a p53-regulated Cre/loxP system.

    PubMed

    Nagayama, Y; Nishihara, E; Namba, H; Yokoi, H; Hasegawa, M; Mizuguchi, H; Hayakawa, T; Hamada, H; Yamashita, S; Niwa, M

    2001-01-01

    In this article, we evaluated the feasibility of the restricted replication-competent adenoviruses for treatment of anaplastic thyroid carcinomas (ATCs), which are very aggressive and difficult to treat. Because ATCs very often harbor p53 mutations, we used wt-p53 as a regulatory factor to restrict virus replication and cytopathic effect to p53-mutated cells. The recently reported "gene inactivation strategy" using p53-regulated Cre/loxP system was employed; this system consists of two recombinant adenoviruses. One has an expression unit of the synthetic p53 - responsive promoter and the Cre recombinase gene (Axyp53RECre), and another contains two expression units; the first consists of E1A gene flanked by a pair of loxP sites downstream of the constitutive CAG promoter and the second E1B19K gene under the control of the CMV promoter (AdCALE1AL). We expected that coinfection of these two adenoviruses into the cells with wt-p53 would lead to expression of the Cre, which excises E1A gene and switches off E1A expression resulting in no virus replication, whereas in the cells with mutant p53 E1A could be expressed that leads to virus replication and cell lysis. Our in vitro data demonstrate that although infection of AdCALE1AL alone led to E1A expression, viral replication and cytolysis in all the thyroid cells examined irrespective of their p53 status, the double infection did so in FRO cells (p53-null ATC) but not in FRO cells stably expressing wt-p53 and normal thyroid cells with wt-p53. These data indicate that our double infection method may have a potential for treatment of ATC and probably also other p53-defective cancer cells. PMID:11219492

  17. A heparan sulfate-targeted conditionally replicative adenovirus, Ad5.pk7-Δ24, for the treatment of advanced breast cancer

    PubMed Central

    Ranki, T; Kanerva, A; Ristimäki, A; Hakkarainen, T; Särkioja, M; Kangasniemi, L; Raki, M; Laakkonen, P; Goodison, S; Hemminki, A

    2012-01-01

    Conditionally replicating adenoviruses (CRAds) that replicate in tumor but less in normal cells are promising anticancer agents. A major determinant of their potency is their capacity for infecting target cells. The primary receptor for serotype 5 adenovirus (Ad5), the most widely used serotype in gene therapy, is the coxsackie-adenovirus receptor (CAR). CAR is expressed variably and often at low levels in various tumor types including advanced breast cancer. We generated a novel p16/retinoblastoma pathway-dependent CRAd, Ad5.pK7-Δ24, with a polylysine motif in the fiber C-terminus, enabling CAR-independent binding to heparan sulfate proteoglycans (HSPG). Ad5.pK7-Δ24 mediated effective oncolysis of all breast cancer cell lines tested. Further, we utilized noninvasive, fluorescent imaging for analysis of antitumor efficacy in an orthotopic model of advanced hormone refractory breast cancer. A therapeutic benefit was seen following both intratumoral and intravenous delivery. Murine biodistribution similar to Ad5, proven safe in trials, suggests feasibility of clinical safety testing. Interestingly, upregulation of CAR was seen in low-CAR M4A4-LM3 breast cancer cells in vivo, which resulted in better than expected efficacy also with an isogenic CRAd with an unmodified capsid. These results suggest utility of Ad5.pK7-Δ24 and the orthotopic model for further translational studies. PMID:16900223

  18. Protective MCMV immunity by vaccination of the salivary gland via Wharton's duct: replication-deficient recombinant adenovirus expressing individual MCMV genes elicits protection similar to that of MCMV.

    PubMed

    Liu, Guangliang; Zhang, Fangfang; Wang, Ruixue; London, Lucille; London, Steven D

    2014-04-01

    Salivary glands, a major component of the mucosal immune system, confer antigen-specific immunity to mucosally acquired pathogens. We investigated whether a physiological route of inoculation and a subunit vaccine approach elicited MCMV-specific and protective immunity. Mice were inoculated by retrograde perfusion of the submandibular salivary glands via Wharton's duct with tcMCMV or MCMV proteins focused to the salivary gland via replication-deficient adenovirus expressing individual MCMV genes (gB, gH, IE1; controls: saline and replication deficient adenovirus without MCMV inserts). Mice were evaluated for MCMV-specific antibodies, T-cell responses, germinal center formation, and protection against a lethal MCMV challenge. Retrograde perfusion with tcMCMV or adenovirus expressed MCMV proteins induced a 2- to 6-fold increase in systemic and mucosal MCMV-specific antibodies, a 3- to 6-fold increase in GC marker expression, and protection against a lethal systemic challenge, as evidenced by up to 80% increased survival, decreased splenic pathology, and decreased viral titers from 10(6) pfu to undetectable levels. Thus, a focused salivary gland immunization via a physiological route with a protein antigen induced systemic and mucosal protective immune responses. Therefore, salivary gland immunization can serve as an alternative mucosal route for administering vaccines, which is directly applicable for use in humans.

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

    PubMed

    Hirvinen, Mari; Rajecki, Maria; Kapanen, Mika; Parviainen, Suvi; Rouvinen-Lagerström, Noora; Diaconu, Iulia; Nokisalmi, Petri; Tenhunen, Mikko; Hemminki, Akseli; Cerullo, Vincenzo

    2015-03-01

    For long it has been recognized that tumor necrosis factor alpha (TNFa) has anticancer characteristics, and its use as a cancer therapeutic was proposed already in the 1980s. However, its systemic toxicity has limited its usability. Oncolytic viruses, selectively cancer-killing viruses, have shown great potency, and one of their most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local versus systemic concentrations. Therefore, the overall magnitude of tumor cell killing results from the combination of oncolysis, transgene-mediated direct effect such as TNFa-mediated apoptosis, and, perhaps most significantly, from activation of the host immune system against the tumor. We generated a novel chimeric oncolytic adenovirus expressing human TNFa, Ad5/3-D24-hTNFa, whose efficacy and immunogenicity were tested in vitro and in vivo. The hTNFa-expressing adenovirus showed increased cancer-eradicating potency, which was shown to be because of elevated apoptosis and necrosis rates and induction of various immune responses. Interestingly, we saw increase in immunogenic cell death markers in Ad5/3-d24-hTNFa-treated cells. Moreover, tumors treated with Ad5/3-D24-hTNFa displayed enhanced presence of OVA-specific cytotoxic T cells. We thus can conclude that tumor eradication and antitumor immune responses mediated by Ad5/3-d24-hTNFa offer a new potential drug candidate for cancer therapy.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  1. [Construction of a Recombinant Replication-defective Human Adenovirus Type 5 Expressing G Protein of Irkut Virus and the Immune Test in Mouse].

    PubMed

    Wang, Yuying; Chen, Qi; Liu, Ye; Hu, Rongliang; Zhang, Lecui

    2015-11-01

    To develop a safe and effective new generation vaccine for IRKV-THChina12 prevention, we constructed a non-replicative recombinant human adenovirus carrying the IRKV-THChina12 G gene, named as rAd5-IRKV-G. The IRKV-THChina12 G protein expressed by the recombinant human adenovirus in 293AD cells was detected by western blot and indirect immunofluorescence test. To evaluate the immunogenicity of the recombinant, mice were immunized with rAd5-IRKV-G by intramuscular (i. m.) or intraperitoneal (i. p.) route and with non-exogenous gene expressing wild type adenovirus wt-rAd5 as a control. Results showed that the rAd5-IRKV-G could induce continuous and statistically significant (P ≤ 0.05) anti-IRKV neutralizing antibody (NA) production in immunized mice by i. m. or i. p. route. In particular, no significant difference (P > 0.05) of the NA titers between the two administration routes were observed, that provides an alternative choice for animal immunization method in the future application. PMID:26951008

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

    SciTech Connect

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

    2013-09-01

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

  3. 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.

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

    PubMed

    Subramanian, T; Zhao, Ling-Jun; Chinnadurai, G

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. PMID:23747199

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

    PubMed

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

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies. PMID:16482201

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

    PubMed

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

    2006-06-01

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

  7. Innate Immunity to Adenovirus

    PubMed Central

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

    2014-01-01

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

  8. A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

    PubMed

    Sha, Jian; Kirtley, Michelle L; Klages, Curtis; Erova, Tatiana E; Telepnev, Maxim; Ponnusamy, Duraisamy; Fitts, Eric C; Baze, Wallace B; Sivasubramani, Satheesh K; Lawrence, William S; Patrikeev, Igor; Peel, Jennifer E; Andersson, Jourdan A; Kozlova, Elena V; Tiner, Bethany L; Peterson, Johnny W; McWilliams, David; Patel, Snehal; Rothe, Eric; Motin, Vladimir L; Chopra, Ashok K

    2016-07-01

    Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. PMID:27170642

  9. Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.

    PubMed

    Sakurai, Fuminori; Narii, Nobuhiro; Tomita, Kyoko; Togo, Shinsaku; Takahashi, Kazuhisa; Machitani, Mitsuhiro; Tachibana, Masashi; Ouchi, Masaaki; Katagiri, Nobuyoshi; Urata, Yasuo; Fujiwara, Toshiyoshi; Mizuguchi, Hiroyuki

    2016-01-01

    Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.

  10. A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

    PubMed

    Sha, Jian; Kirtley, Michelle L; Klages, Curtis; Erova, Tatiana E; Telepnev, Maxim; Ponnusamy, Duraisamy; Fitts, Eric C; Baze, Wallace B; Sivasubramani, Satheesh K; Lawrence, William S; Patrikeev, Igor; Peel, Jennifer E; Andersson, Jourdan A; Kozlova, Elena V; Tiner, Bethany L; Peterson, Johnny W; McWilliams, David; Patel, Snehal; Rothe, Eric; Motin, Vladimir L; Chopra, Ashok K

    2016-07-01

    Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

  11. Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus

    PubMed Central

    Sakurai, Fuminori; Narii, Nobuhiro; Tomita, Kyoko; Togo, Shinsaku; Takahashi, Kazuhisa; Machitani, Mitsuhiro; Tachibana, Masashi; Ouchi, Masaaki; Katagiri, Nobuyoshi; Urata, Yasuo; Fujiwara, Toshiyoshi; Mizuguchi, Hiroyuki

    2016-01-01

    Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)–expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus–adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell–specific microRNA, miR-142-3p, were incorporated into the 3′-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells. PMID:26966699

  12. Efficient antitumor effects of carrier cells loaded with a fiber-substituted conditionally replicating adenovirus on CAR-negative tumor cells.

    PubMed

    Iguchi, K; Sakurai, F; Tomita, K; Katayama, K; Yamaguchi, T; Kawabata, K; Tagawa, M; Kawabata, M; Shirakawa, T; Mizuguchi, H

    2012-02-01

    Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  14. Human adenoviruses: propagation, purification, quantification, and storage.

    PubMed

    Green, Maurice; Loewenstein, Paul M

    2006-01-01

    Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

  15. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

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

  16. Adenovirus Early Proteins and Host Sumoylation.

    PubMed

    Sohn, Sook-Young; Hearing, Patrick

    2016-01-01

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

  17. Immune Responses Induced by Replication-Defective Adenovirus Expressing the C-Terminal Portion of the Mycoplasma hyopneumoniae P97 Adhesin▿

    PubMed Central

    Okamba, F. R.; Moreau, E.; Cheikh Saad Bouh, K.; Gagnon, C. A.; Massie, B.; Arella, M.

    2007-01-01

    Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine, causing significant economic losses to swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of this disease. The goal of this study was to design and evaluate a replication-defective recombinant adenovirus, rAdP97c, expressing the C-terminal portion of P97 adhesin (P97c), an important pathogenesis-associated protein of M. hyopneumoniae, as a new vaccine candidate against M. hyopneumoniae infection. P97c-specific immune responses were evaluated in BALB/c mice following intranasal and intramuscular inoculation with rAdP97c. Mice inoculated by both routes of immunization produced significant levels of specific immunoglobulin G (IgG) antibodies in the serum and in bronchoalveolar lavage fluids (BALs). Animals immunized intranasally also produced a significant level of P97c-specific IgA in BALs. Intramuscular inoculation of rAdP97c induced a systemic and mucosal Th1-type biased response, evidenced by the predominance of IgG2a in the serum and BALs, whereas intranasal inoculation resulted in a mixed Th1/Th2-type response (balanced levels of IgG1 and IgG2a) in both sytemic and mucosal compartments. P97c-specific antibodies were able to inhibit the growth of M. hyopneumoniae cells in vitro. These data suggest that rAdP97c vaccine may represent a new strategy for controlling infection by M. hyopneumoniae. PMID:17409219

  18. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

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

  19. Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

    PubMed Central

    Alexander, Jeff; Mendy, Jason; Vang, Lo; Avanzini, Jenny B.; Garduno, Fermin; Manayani, Darly J.; Ishioka, Glenn; Farness, Peggy; Ping, Li-Hua; Swanstrom, Ronald; Parks, Robert; Liao, Hua-Xin; Haynes, Barton F.; Montefiori, David C.; LaBranche, Celia; Smith, Jonathan; Gurwith, Marc; Mayall, Tim

    2013-01-01

    Background There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. Methods The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. Results Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. Conclusions The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical

  20. Hepatitis B Virus (HBV) Virion and Covalently Closed Circular DNA Formation in Primary Tupaia Hepatocytes and Human Hepatoma Cell Lines upon HBV Genome Transduction with Replication-Defective Adenovirus Vectors

    PubMed Central

    Ren, Shaotang; Nassal, Michael

    2001-01-01

    Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range, there is as yet no practical small-animal system for HBV infection. The hosts of the few related animal viruses, including woodchuck hepatitis B virus and duck hepatitis B virus, are either difficult to keep or only distantly related to humans. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To bypass restrictions during the initial entry phase, we used recombinant replication-defective adenovirus vectors, either with or without a green fluorescent protein marker gene, to deliver complete HBV genomes into primary tupaia hepatocytes. Here we show that these cells, like the human hepatoma cell lines HepG2 and Huh7, are efficiently transduced by the vectors and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. We further demonstrate that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. These data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection. PMID:11152483

  1. 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.

  2. 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.

  3. 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. PMID:27467954

  4. IL-17 contributes to neutrophil recruitment but not to control of viral replication during acute mouse adenovirus type 1 respiratory infection.

    PubMed

    McCarthy, Mary K; Zhu, Lingqiao; Procario, Megan C; Weinberg, Jason B

    2014-05-01

    IL-17-producing CD4(+) helper T cells (Th17 cells) promote inflammatory responses to many pathogens. We used mouse adenovirus type 1 (MAV-1) to determine contributions of IL-17 to adenovirus pathogenesis. MAV-1 infection of C57BL/6 mice upregulated lung expression of IL-17 and the Th17-associated factors IL-23 and RORγt. Only CD4(+)T cells were associated with virus-specific IL-17 production. Fewer neutrophils were recruited to airways of IL-17(-/-) mice following MAV-1 infection, but there were no other differences in pulmonary inflammation between IL-17(+/+) and IL-17(-/-) mice. Mice depleted of neutrophils using anti-Gr-1 antibody had greater lung viral loads than controls. Despite impaired neutrophil recruitment, there were no differences between IL-17(+/+) and IL-17(-/-) mice in peak lung viral loads, clearance of virus from the lungs, or establishment of protective immunity. We demonstrate robust Th17 responses during MAV-1 respiratory infection, but these responses are not essential for control of virus infection or for virus-induced pulmonary inflammation. PMID:24889245

  5. Activated recombinant adenovirus proteinases

    SciTech Connect

    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.

  6. 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.

  7. Adenovirus-associated deaths in US military during postvaccination period, 1999-2010.

    PubMed

    Potter, Robert N; Cantrell, Joyce A; Mallak, Craig T; Gaydos, Joel C

    2012-03-01

    Adenoviruses are frequent causes of respiratory disease in the US military population. A successful immunization program against adenovirus types 4 and 7 was terminated in 1999. Review of records in the Mortality Surveillance Division, Armed Forces Medical Examiner System, identified 8 deaths attributed to adenovirus infections in service members during 1999-2010.

  8. Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs.

    PubMed

    Horikawa, Y; Wang, Y; Nagano, S; Kamizono, J; Ikeda, M; Komiya, S; Kosai, K-i

    2011-10-01

    Although previous studies modified two components of conditionally replicating adenoviruses (CRAs), which selectively replicate in and kill cancer cells, the most accurate ways to achieve increased cancer specificity (that is, safety) without reducing the anticancer (that is, therapeutic) effects are unknown. Here, we generated two types of survivin-responsive m-CRAs (Surv.m-CRAs), Surv.m-CRA-CMVp and Surv.m-CRA-OCp, which use two and three different mechanisms to target cancer, that is, early region 1A (E1A) regulated by the survivin promoter and mutated E1BΔ55K regulated by the ubiquitously active cytomegalovirus promoter and cancer/tissue-specific osteocalcin promoter, respectively, and carefully examined their safety and anticancer effects. Endogenous osteocalcin mRNA was expressed and further enhanced by vitamin D(3) in all osteosarcoma and prostate cancer cell lines and human osteoblasts, but not in human fibroblasts. The osteocalcin promoter activity was weak even with vitamin D(3) treatment in these osteocalcin-expressing cancers, leading to low E1BΔ55K expression after Surv.m-CRA-OCp infection. Nevertheless, Surv.m-CRA-OCp had significantly increased cancer specificity without reduced anticancer effects in both in vitro and in vivo experiments. The unexpected but favorable fact that strong activity of an altered E1B promoter is unnecessary indicates that the majority of cancer/tissue-specific promoters may be used to generate ideal m-CRAs and will advance the development of m-CRA-based cancer therapies.

  9. Replication-Competent Adenovirus Formation in 293 Cells: the Recombination-Based Rate Is Influenced by Structure and Location of the Transgene Cassette and Not Increased by Overproduction of HsRad51, Rad51-Interacting, or E2F Family Proteins

    PubMed Central

    Duigou, Gregory J.; Young, C. S. H.

    2005-01-01

    Propagation of E1 region replacement adenovirus vectors in 293 cells results in the rare appearance of replication-competent adenovirus (RCA). The RCA genome contains E1 DNA acquired from the 293 cellular genome. The Luria-Delbrück fluctuation test was adapted to measure RCA formation rates. To test if structure affected rate, we measured rates during the production of adenovirus vectors with genomes containing three different expression cassette arrangements. The vectors had different extents of sequence identity with integrated Ad5 DNA of 293 cells and had different distributions of identity flanking the expression cassettes. Empty cassette vector RCA rates ranged from 2.5 × 10−8 to 5.6 × 10−10. The extent of sequence identity was not an accurate RCA rate predictor. The vector with the highest RCA rate also had the least overall sequence identity. To define factors controlling RCA generation, adenovirus vectors expressing E2F family proteins, known to modulate recombination gene expression, and overexpressing the human Rad51 recombination protein were analyzed. Compared to their corresponding empty vectors, RCA rates were not increased but were slightly decreased. Initial results suggested expression cassette orientation and/or transcription direction as potential RCA rate modifiers. Testing adenovirus vectors with identical transgene cassettes oriented in opposite directions suggested that transcription direction was not the basis of these rate differences. Thus, the overall structure and location of the transgene cassette had the largest effect on RCA rate. The RCA fluctuation test should be useful for investigators who require accurate measurements of targeted recombination and the probability of RCA formation during stock production. PMID:15827158

  10. Core labeling of adenovirus with EGFP

    SciTech Connect

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

    2006-08-01

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

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

    PubMed

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

    2010-07-01

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

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

    PubMed Central

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

    2016-01-01

    Gene therapy on the basis of oncolytic adenovirus is a novel approach for human cancer therapeutics. We aim to investigate whether it will synergistically reinforce their antiovarian cancer activities when the combined use of ZD55-manganese superoxide dismutase (MnSOD) and cisplatin was performed. The experiments in vitro showed that ZD55-MnSOD enhances cisplatin-induced apoptosis and causes remarkable ovarian cancer cell death. Apoptosis induction by treatment with ZD55-MnSOD and/or cisplatin was detected in SKOV-3 by apoptotic cell staining, flow cytometry, and western blot analysis. In addition, the cytotoxicity caused by ZD55-MnSOD to normal cells was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and western blot analysis. Animal experiment further confirmed that combination of ZD55-MnSOD and cisplatin achieved significant inhibition of SKOV-3 ovarian tumor xenografted growth. In summary, we have demonstrated that ZD55-MnSOD can sensitize human ovarian cancer cells to cisplatin-induced cell death and apoptosis in vitro and in vivo. These findings indicate that the combined treatment with ZD55-MnSOD and cisplatin could represent a rational approach for antiovarian cancer therapy. PMID:27799786

  13. 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.

  14. 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.

  15. Safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study

    PubMed Central

    Gurwith, Marc; Lock, Michael; Taylor, Eve M; Ishioka, Glenn; Alexander, Jeff; Mayall, Tim; Ervin, John E; Greenberg, Richard N; Strout, Cynthia; Treanor, John J; Webby, Richard; Wright, Peter F

    2013-01-01

    Summary Background Replication-competent virus vector vaccines might have advantages compared with non-replicating vector vaccines. We tested the safety and immunogenicity of an oral adenovirus serotype 4 vector vaccine candidate (Ad4-H5-Vtn) expressing the haemagglutinin from an avian influenza A H5N1 virus. Methods We did this phase 1 study at four sites in the USA. We used a computer-generated randomisation list (block size eight, stratified by site) to assign healthy volunteers aged 18–40 years to receive one of five doses of Ad4-H5-Vtn (107 viral particles [VP], 108 VP, 109 VP, 1010 VP, 1011 VP) or placebo (3:1). Vaccine or placebo was given on three occasions, about 56 days apart. Participants, investigators, and study-site personnel were masked to assignment throughout the study. Subsequently, volunteers received a boost dose with 90 μg of an inactivated parenteral H5N1 vaccine. Primary immunogenicity endpoints were seroconversion by haemagglutination-inhibition (HAI), defined as a four-times rise compared with baseline titre, and HAI geometric mean titre (GMT). We solicited symptoms of reactogenicity daily for 7 days after each vaccination and recorded symptoms that persisted beyond 7 days as adverse events. Primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01006798. Findings We enrolled 166 participants (125 vaccine; 41 placebo) between Oct 19, 2009, and Sept 9, 2010. HAI responses were low: 13 of 123 vaccinees (11%, 95% CI 6–17) and three of 41 placebo recipients (7%, 2–20) seroconverted. HAI GMT was 6 (95% CI 5–7) for vaccinees, and 5 (5–6) for placebo recipients. However, when inactivated H5N1 vaccine became available, one H5N1 boost was offered to all participants. In this substudy, HAI seroconversion occurred in 19 of 19 participants in the 1011 VP cohort (100%; 95% CI 82–100) and eight of 22 placebo recipients (36%; 17–59); 17 of 19 participants in the 1011 VP cohort (89%; 67–99) achieved

  16. Rapid generation of fowl adenovirus 9 vectors.

    PubMed

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

    2015-10-01

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

  17. Transductional targeting with recombinant adenovirus vectors.

    PubMed

    Legrand, Valerie; Leissner, Philippe; Winter, Arend; Mehtali, Majid; Lusky, Monika

    2002-09-01

    Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed. PMID:12189719

  18. Effects of cold atmospheric plasmas on adenoviruses in solution

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

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

    PubMed

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

    2015-11-01

    Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus. PMID:26189043

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

    PubMed

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

    2015-11-01

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

  1. Co-factor activated recombinant adenovirus proteinases

    SciTech Connect

    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.

  2. 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.

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

    PubMed

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

    2002-09-20

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

  4. 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.

  5. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

  6. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker. PMID:26723876

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

    PubMed

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

    2012-05-01

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

  8. The Utility of a Tissue Slice Model System to Determine Breast Cancer Infectivity by Oncolytic Adenoviruses

    PubMed Central

    Pennington, Krista; Chu, Quyen D.; Curiel, David T.; Li, Benjamin D.L.; Mathis, J. Michael

    2010-01-01

    Background Due to advances in viral design, oncolytic adenoviruses have emerged as a promising approach for treatment of breast cancer. Tumor tissue slices offer a stringent model system for preclinical evaluation of adenovirus therapies, since the slices retain a morphology and phenotype that more closely resembles the in vivo setting than cell line cultures, and it has been shown to have utility in the evaluation of viral infectivity and replication. In this study, we evaluated the efficacy of viral infection and replication using a tropism-modified oncolytic adenovirus. Methods Breast tumor tissue slices were infected with a tropism-modified oncolytic adenovirus, and a wild-type adenovirus for comparison. Efficiency of infection was evaluated using fluorescent microscopy, as the viruses used have been modified to express red fluorescent protein. Replication of the viruses was evaluated with quantitative real-time PCR to assay viral E4 genome copy number, a surrogate indicator for the number of virions. The breast tumor tissue slices were evaluated for the expression of CD46 expression by immunohistochemistry. Results Infection and replication of our tropism modified oncolytic virus has been observed in breast cancer tissue slice model system and is comparative to wild-type virus. A qualitative increase in the number of cells showing RFP expression was observed correlating with increasing multiplicity of infection. Higher relative infectivity of the virus was observed in tumor tissue compared with normal breast tissue. Replication of the virus was demonstrated through increases in E4 copy number at 48 and 72 hours after infection in human breast tumor slices. Conclusions We have shown that a tropism modified oncolytic oncolytic adenovirus can infect and replicate in breast cancer tissue slices, which may be an important preclinical indicator for its therapeutic utility. PMID:20691986

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

    SciTech Connect

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

    1981-02-01

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

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

    PubMed

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

    2016-01-01

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

  11. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Manischewitz, Jody; King, Lisa R; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

  12. Construction of mouse adenovirus type 1 mutants.

    PubMed

    Cauthen, Angela N; Welton, Amanda R; Spindler, Katherine R

    2007-01-01

    Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wild-type mouse adenovirus, production of viral DNA and viral DNA-protein complex, and transfection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of the mutant viruses are also presented.

  13. CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells

    PubMed Central

    Wang, Yaohe; Gangeswaran, Rathi; Zhao, Xingbo; Wang, Pengju; Tysome, James; Bhakta, Vipul; Yuan, Ming; Chikkanna-Gowda, C.P.; Jiang, Guozhong; Gao, Dongling; Cao, Fengyu; Francis, Jennelle; Yu, Jinxia; Liu, Kangdong; Yang, Hongyan; Zhang, Yunhan; Zang, Weidong; Chelala, Claude; Dong, Ziming; Lemoine, Nick

    2009-01-01

    The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics. PMID:19411761

  14. A novel Golgi protein (GOLPH2)-regulated oncolytic adenovirus exhibits potent antitumor efficacy in hepatocellular carcinoma

    PubMed Central

    Wang, Yigang; Zhao, Hongfang; Zhang, Rong; Ma, Buyun; Chen, Kan; Huang, Fang; Zhou, Xiumei; Cui, Caixia; Liu, Xinyuan

    2015-01-01

    Golgi apparatus is the organelle mainly functioning as protein processing and secretion. GOLPH2 is a resident Golgi glycoprotein, usually called GP73. Recent data displayed that GOLPH2 is a superb hepatocellular carcinoma (HCC) marker candidate, and even its specificity is better than liver cancer marker AFP. Oncolytic adenoviruses are broadly used for targeting cancer therapy due to their selective tumor-killing effect. However, it was reported that traditionally oncolytic adenovirus lack the HCC specificity. In this study, a novel dual-regulated oncolytic adenovirus GD55 targeting HCC was first constructed based on our cancer targeted gene-viral therapeutic strategy. To verify the targeting and effectiveness of GOLPH2-regulated oncolytic adenovirus GD55 in HCC, the anticancer capacity was investigated in HCC cell lines and animal model. The results proved that the novel GOLPH2-regulated GD55 conferred higher adenovirus replication and infectivity for liver cancer cells than oncolytic adenovirus ZD55. The GOLPH2-regulated GD55 exerted a significant grow-suppressing effect on HCC cells in vitro but little damage to normal liver cells. In animal experiment, antitumor effect of GD55 was more effective in HCC xenograft of nude mice than that of ZD55. Thus GOLPH2-regulated GD55 may be a promising oncolytic virus agent for future liver cancer treatment. PMID:25980438

  15. Replicating nucleosomes

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2015-01-01

    Eukaryotic replication disrupts each nucleosome as the fork passes, followed by reassembly of disrupted nucleosomes and incorporation of newly synthesized histones into nucleosomes in the daughter genomes. In this review, we examine this process of replication-coupled nucleosome assembly to understand how characteristic steady-state nucleosome landscapes are attained. Recent studies have begun to elucidate mechanisms involved in histone transfer during replication and maturation of the nucleosome landscape after disruption by replication. A fuller understanding of replication-coupled nucleosome assembly will be needed to explain how epigenetic information is replicated at every cell division. PMID:26269799

  16. Etoposide enhances antitumor efficacy of MDR1-driven oncolytic adenovirus through autoupregulation of the MDR1 promoter activity

    PubMed Central

    Tseng, Yau-Lin; Shiau, Ai-Li; Wu, Chao-Liang

    2015-01-01

    Conditionally replicating adenoviruses (CRAds), or oncolytic adenoviruses, such as E1B55K-deleted adenovirus, are attractive anticancer agents. However, the therapeutic efficacy of E1B55K-deleted adenovirus for refractory solid tumors has been limited. Environmental stress conditions may induce nuclear accumulation of YB-1, which occurs in multidrug-resistant and adenovirus-infected cancer cells. Overexpression and nuclear localization of YB-1 are associated with poor prognosis and tumor recurrence in various cancers. Nuclear YB-1 transactivates the multidrug resistance 1 (MDR1) genes through the Y-box. Here, we developed a novel E1B55K-deleted adenovirus driven by the MDR1 promoter, designed Ad5GS3. We tested the feasibility of using YB-1 to transcriptionally regulate Ad5GS3 replication in cancer cells and thereby to enhance antitumor efficacy. We evaluated synergistic antitumor effects of oncolytic virotherapy in combination with chemotherapy. Our results show that adenovirus E1A induced E2F-1 activity to augment YB-1 expression, which shut down host protein synthesis in cancer cells during adenovirus replication. In cancer cells infected with Ad5WS1, an E1B55K-deleted adenovirus driven by the E1 promoter, E1A enhanced YB-1 expression, and then further phosphorylated Akt, which, in turn, triggered nuclear translocation of YB-1. Ad5GS3 in combination with chemotherapeutic agents facilitated nuclear localization of YB-1 and, in turn, upregulated the MDR1 promoter activity and enhanced Ad5GS3 replication in cancer cells. Thus, E1A, YB-1, and the MDR1 promoter form a positive feedback loop to promote Ad5GS3 replication in cancer cells, and this regulation can be further augmented when chemotherapeutic agents are added. In the in vivo study, Ad5GS3 in combination with etoposide synergistically suppressed tumor growth and prolonged survival in NOD/SCID mice bearing human lung tumor xenografts. More importantly, Ad5GS3 exerted potent oncolytic activity against clinical

  17. Treatment of Pancreatic Cancer With an Oncolytic Adenovirus Expressing Interleukin-12 in Syrian Hamsters

    PubMed Central

    Bortolanza, Sergia; Bunuales, Maria; Otano, Itziar; Gonzalez-Aseguinolaza, Gloria; Ortiz-de-Solorzano, Carlos; Perez, Daniel; Prieto, Jesus; Hernandez-Alcoceba, Ruben

    2009-01-01

    Pancreatic cancer is an aggressive malignancy resistant to most conventional and experimental therapies, including conditionally replicative adenoviruses (CRAds). The incorporation of immunostimulatory genes such as interleukin-12 (IL-12) in these viruses may overcome some of their limitations, but evaluation of such vectors requires suitable preclinical models. We describe a CRAd in which replication is dependent on hypoxia-inducible factor (HIF) activity and alterations of the pRB pathway in cancer cells. Transgenes (luciferase or IL-12) were incorporated into E3 region of the virus using a selective 6.7K/gp19K deletion. A novel permissive model of pancreatic cancer developed in immunocompetent Syrian hamsters was used for in vivo analysis. We show that, in contrast with nonreplicating adenoviruses (NR-Ad), active viral production and enhanced transgene expression took place in vivo. A single intratumor inoculation of the CRAd expressing IL-12 (Ad-DHscIL12) achieved a potent antitumor effect, whereas higher doses of replication-competent adenoviruses carrying luciferase did not. Compared to a standard NR-Ad expressing IL-12, Ad-DHscIL12 was less toxic in hamsters, with more selective tumor expression and shorter systemic exposure to the cytokine. We conclude that the expression of IL-12 in the context of a hypoxia-inducible oncolytic adenovirus is effective against pancreatic cancer in a relevant animal model. PMID:19223865

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

    SciTech Connect

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

    1989-09-01

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

  19. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.

    PubMed Central

    Newman, K D; Dunn, P F; Owens, J W; Schulick, A H; Virmani, R; Sukhova, G; Libby, P; Dichek, D A

    1995-01-01

    Adenovirus vectors are capable of high efficiency in vivo arterial gene transfer, and are currently in use as therapeutic agents in animal models of vascular disease. However, despite substantial data on the ability of viruses to cause vascular inflammation and proliferation, and the presence in current adenovirus vectors of viral open reading frames that are translated in vivo, no study has examined the effect of adenovirus vectors alone on the arterial phenotype. In a rabbit model of gene transfer into a normal artery, we examined potential vascular cell activation, inflammation, and neointimal proliferation resulting from exposure to replication-defective adenovirus. Exposure of normal arteries to adenovirus vectors resulted in: (a) pronounced infiltration of T cells throughout the artery wall; (b) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (c) neointimal hyperplasia. These findings were present both 10 and 30 d after gene transfer, with no evidence of a decline in severity over time. Adenovirus vectors have pleiotropic effects on the arterial wall and cause significant pathology. Interpretation of experimental protocols that use adenovirus vectors to address either biological or therapeutic issues should take these observations into account. These observations should also prompt the design of more inert gene transfer vectors. Images PMID:8675667

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

    PubMed

    Gallaher, Sean D; Berk, Arnold J

    2013-09-01

    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.

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

    PubMed

    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

  2. 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

  3. Hyaluronidase expression by an oncolytic adenovirus enhances its intratumoral spread and suppresses tumor growth.

    PubMed

    Guedan, Sonia; Rojas, Juan José; Gros, Alena; Mercade, Elena; Cascallo, Manel; Alemany, Ramon

    2010-07-01

    Successful virotherapy requires efficient virus spread within tumors. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix (ECM), could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in mice-bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (MLP) (AdwtRGD-PH20). Intratumoral treatment of human melanoma xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan (HA), enhanced viral distribution, and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus that selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR17) was compared to that of the parental virus ICOVIR15. ICOVIR17 showed more antitumor efficacy following intratumoral and systemic administration in mice with prestablished tumors, along with an improved spread of the virus within the tumor. Importantly, a single intravenous dose of ICOVIR17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR17 is a promising candidate for clinical testing.

  4. A genetic fiber modification to achieve matrix-metalloprotease-activated infectivity of oncolytic adenovirus.

    PubMed

    José, Anabel; Rovira-Rigau, Maria; Luna, Jeroni; Giménez-Alejandre, Marta; Vaquero, Eva; García de la Torre, Beatriz; Andreu, David; Alemany, Ramon; Fillat, Cristina

    2014-10-28

    Selective tumor targeting of oncolytic adenovirus at the level of cell entry remains a major challenge to improve efficacy and safety. Matrix metalloproteases (MMPs) are overexpressed in a variety of tumors and in particular in pancreatic cancer. In the current work, we have exploited the expression of MMPs together with the penetration capabilities of a TAT-like peptide to engineer tumor selective adenoviruses. We have generated adenoviruses containing CAR-binding ablated fibers further modified with a C-terminus TAT-like peptide linked to a blocking domain by an MMP-cleavable sequence. This linker resulted in a MMP-dependent cell transduction of the reporter MMP-activatable virus AdTATMMP and in efficient transduction of neoplastic cells and cancer-associated fibroblasts. Intravenous and intraductal administration of AdTATMMP into mice showed very low AdTATMMP activity in the normal pancreas, whereas increased transduction was observed in pancreatic tumors of transgenic Ela-myc mice. Intraductal administration of AdTATMMP into mice bearing orthotopic tumors led to a 25-fold increase in tumor targeting compared to the wild type fiber control. A replication competent adenovirus, Ad(RC)MMP, with the MMP-activatable fiber showed oncolytic efficacy and increased antitumor activity compared to Adwt in a pancreatic orthotopic model. Reduced local and distant metastases were observed in Ad(RC)MMP treated-mice. Moreover, no signs of pancreatic toxicity were detected. We conclude that MMP-activatable adenovirus may be beneficial for pancreatic cancer treatment.

  5. Canine adenovirus downstream processing protocol.

    PubMed

    Puig, Meritxell; Piedra, Jose; Miravet, Susana; Segura, María Mercedes

    2014-01-01

    Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %. PMID:24132487

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

    PubMed Central

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

    2016-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Simultaneous detection of infectious human echoviruses and adenoviruses by an in situ nuclease-resistant molecular beacon-based assay.

    PubMed

    Dunams, Daniela; Sarkar, Payal; Chen, Wilfred; Yates, Marylynn V

    2012-03-01

    A multiplex methodology using two nuclease-resistant molecular beacons that target specific genomic regions of adenovirus 2 and echovirus 17 during simultaneous infection in A549 cells is presented. Using fluorescence microscopy, visualization of enteroviral and adenoviral replication was possible within 3 h postinfection.

  9. Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor

    PubMed Central

    Arnberg, Niklas; Edlund, Karin; Kidd, Alistair H.; Wadell, Göran

    2000-01-01

    Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2. PMID:10590089

  10. Acid-Soluble Material of Adenovirus

    PubMed Central

    Boulanger, P. A.; Jaume, F.; Flamencourt, P.; Biserte, G.

    1970-01-01

    Two methods are described for adenovirus capsid disruption and extraction of acid-soluble proteins from the viral core. The acid-soluble material of adenovirus consisted of three major proteins, one of them being selectively extracted after mild disruption of the virus particle. Some chemical properties of these proteins are reported. Images PMID:4986288

  11. MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

    PubMed Central

    Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

    2015-01-01

    Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

  12. Characterization of transgenic mice containing adenovirus early region 3 genomic DNA.

    PubMed Central

    Fejer, G; Gyory, I; Tufariello, J; Horwitz, M S

    1994-01-01

    Human adenoviruses (Ad) contain a complex transcription region (E3) which codes for proteins that interact with several arms of the immune system. However, E3 genes are not essential for replication in tissue culture. An E3-encoded 19,000-molecular-weight (19K) glycoprotein (gp19K) binds to the class I major histocompatibility complex (MHC) in the endoplasmic reticulum and prevents MHC transport to the cell surface. Three other E3 proteins are involved in the inhibition of apoptosis by tumor necrosis factor alpha. The entire E3 genomic DNA was utilized to produce transgenic mice to study the effect of the E3 proteins on pathogenesis of various infectious agents and to investigate the in vivo synthesis and processing of the multiple E3 mRNAs and proteins. There was basal expression of the E3 promoter in the thymus, kidneys, uterus, and testes and at all levels of the gastrointestinal tract. In addition, the E3 promoter of the transgene could be activated in some other organs, including the liver, by infection of these animals with an E3-deficient Ad (Ad7001) which contains a functional E1A region. Transactivation in vivo could also be demonstrated by infusion of bacterial lipopolysaccharide. There appeared to be differential ratios of expression between several of the E3 mRNAs in transgenic lung fibroblasts and primary kidney cells cultured from the transgenic animals. This observation suggested that there was differential mRNA splicing that was organ specific. These transgenic animals should provide a useful model for studying the effects of the E3 proteins on the immune system and on diseases affected either by control of MHC or by selected functions of tumor necrosis factor that are inhibitable by Ad E3 proteins. Images PMID:8057467

  13. Combination of oncolytic adenovirus and endostatin inhibits human retinoblastoma in an in vivo mouse model.

    PubMed

    Wang, Huiping; Wei, Fang; Li, Huiming; Ji, Xunda; Li, Shuxia; Chen, Xiafang

    2013-02-01

    There is a critical need for new paradigms in retinoblastoma (RB) treatment that would more efficiently inhibit tumor growth while sparing the vision of patients. Oncolytic adenoviruses with the ability to selectively replicate and kill tumor cells are a promising strategy for cancer gene therapy. Exploration of a novel targeting strategy for RB utilizing combined oncolytic adenovirus and anti-angiogenesis therapy was applied over the course of the current study with positive results. The oncolytic adenoviruses Ad-E2F1 p-E1A and Ad-TERT p-E1 were constructed. The E1 region was regulated by the E2F-1 promoter or the human telomerase reverse transcriptase (hTERT) promoter, respectively. Effects on both replication and promotion of enhanced green fluorescent protein (EGFP) expression were observed in the replication-defective adenovirus Ad-EGFP in diverse cancer cell lines, HXO-RB44, Y79, Hep3B, NCIH460, MCF-7 and HLF. The cancer cell death induced by these agents was also explored. The in situ RB model demonstrated that mice with tumors treated with the oncolytic adenovirus and replication-defective adenovirus Ad-endostatin exhibited notable cancer cell death. This anticancer effect was further examined by stereo microscope, and the survival rate of experimental mice was determined. Both Ad-E2F1 p-E1A and Ad-TERT p-E1 replicated specifically in cancer cells in vitro and promoted EGFP expression in Ad-EGFP, although Ad-E2F1 p-E1A demonstrated superior EGFP promotion activity than Ad-TERT p-E1. In Hep3B, NCIH460 and MCF-7 cells, the number of Ad-TERT p-E1 copies was observed to exceed of the number of Ad-E2F1 p-E1A copies by a minimum of 10-fold. Furthermore, Ad-TERT p-E1 demonstrated significantly superior oncolytic effects in the RB mouse model, and Ad-endostatin effectively suppressed tumor growth and extended the overall lifespan of subjects; however, the Ad-E2F1 p-E1A was clearly less effective in attaining these goals. Most notably, the antitumor effect and

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

    PubMed

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

    2016-08-31

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

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

    DOE PAGES

    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; et al

    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

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

    SciTech Connect

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

    2014-11-19

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

  17. Clinical and Virologic Characteristics May Aid Distinction of Acute Adenovirus Disease from Kawasaki Disease with Incidental Adenovirus Detection.

    PubMed

    Song, Eunkyung; Kajon, Adriana E; Wang, Huanyu; Salamon, Doug; Texter, Karen; Ramilo, Octavio; Leber, Amy; Jaggi, Preeti

    2016-03-01

    Incidental adenovirus detection in Kawasaki disease (KD) is important to differentiate from acute adenovirus disease. Twenty-four of 25 children with adenovirus disease and mimicking features of KD had <4 KD-like features, predominance of species B or E, and higher viral burden compared with those with KD and incidental adenovirus detection. PMID:26707621

  18. RAD51 and BRCA2 enhance oncolytic adenovirus type 5 activity in ovarian cancer

    PubMed Central

    Tookman, Laura A.; Browne, Ashley K.; Connell, Claire M.; Bridge, Gemma; Ingemarsdotter, Carin K.; Dowson, Suzanne; Shibata, Atsushi; Lockley, Michelle; Martin, Sarah A.; McNeish, Iain A.

    2015-01-01

    Homologous Recombination (HR) function is critically important in High Grade Serous Ovarian Cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall anti-tumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2 mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. Implications Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function. PMID:26452665

  19. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  20. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

    Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adenoviruses one of (if not the) best models for studying viral evolution on a larger time scale. Phylogenetic calculation can infer the evolutionary distance among adenovirus strains on serotype, species, and genus levels, thus helping the establishment of a correct taxonomy on the one hand, and speeding up the process of typing new isolates on the other. Initially, four major lineages corresponding to four genera were recognized. Later, the demarcation criteria of lower taxon levels, such as species or types, could also be defined with phylogenetic calculations. A limited number of possible host switches have been hypothesized and convincingly supported. Application of the web-based BLAST and MultAlin programs and the freely available PHYLIP package, along with the TreeView program, enables everyone to make correct calculations. In addition to step-by-step instruction on how to perform phylogenetic analysis, critical points where typical mistakes or misinterpretation of the results might occur will be identified and hints for their avoidance will be provided. PMID:17656792

  1. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

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

  2. Replicating vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

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

    2015-01-01

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

  4. Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

    PubMed Central

    Aparicio, Oscar; Razquin, Nerea; Zaratiegui, Mikel; Narvaiza, Iñigo; Fortes, Puri

    2006-01-01

    Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression. PMID:16415015

  5. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

    PubMed

    Imler, J L; Chartier, C; Dreyer, D; Dieterle, A; Sainte-Marie, M; Faure, T; Pavirani, A; Mehtali, M

    1996-01-01

    Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus. PMID:8929914

  6. Infectious entry pathway of adenovirus type 2.

    PubMed Central

    Varga, M J; Weibull, C; Everitt, E

    1991-01-01

    Internalization of the infectious fraction of human adenovirus type 2 into HeLa cells was followed by a quantitative internalization assay. Treatments known to selectively block receptor-mediated endocytosis reduced the internalization of infectious virus to an extent close to the reduction of endocytosis of transferrin. This suggests that one of the first steps in the infectious cycle of adenovirus type 2 is internalization by the coated-pit and -vesicle pathway. Images PMID:1920625

  7. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses. PMID:14654922

  8. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

  9. Current issues and future directions of oncolytic adenoviruses.

    PubMed

    Yamamoto, Masato; Curiel, David T

    2010-02-01

    Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy.

  10. Current Issues and Future Directions of Oncolytic Adenoviruses

    PubMed Central

    Yamamoto, Masato; Curiel, David T

    2009-01-01

    Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy. PMID:19935777

  11. 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.

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

    SciTech Connect

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

    2014-05-15

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

  13. human adenoviruses role in ophthalmic pterygium formation

    PubMed Central

    Kelishadi, Mishar; Kelishadi, Mandana; Moradi, Abdolvahab; Javid, Naeme; Bazouri, Masoud; Tabarraei, Alijan

    2015-01-01

    Background: Ophthalmic pterygium is a common benign lesion of unknown origin and the pathogenesis might be vision-threatening. This problem is often associated with exposure to solar light. Recent evidence suggests that potentially oncogenic viruses such as human papillomavirus and Epstein-Barr virus may be involved in the pathogenesis of pterygia. Expression of specific adenovirus genes such as E1A and E1B, which potentially have many functions, may contribute to their oncogenic activity as well as relevance to cellular immortalization. Objectives: For the first time, we aimed to investigate involvement of adenoviruses in pterygium formation. Patients and Methods: Fifty tissue specimens of pterygium from patients undergoing pterygium surgery (as cases), 50 conjunctival swab samples from the same patients and 10 conjunctival biopsy specimens from individuals without pterygium such as patients undergoing cataract surgery (as controls) were analyzed for evidence of adenovirus infection with polymerase chain reaction using specific primers chosen from the moderately conserved region of the hexon gene. Furthermore, β-globin primers were used to access the quality of extracted DNA. Data was analyzed using SPSS (version 16) software. Results: Of 50 patients, 20 were men and 30 women with mean age of 61.1 ± 16.9 years ranged between 22 and 85 years. All samples of pterygia had positive results for adenoviruses DNA with polymerase chain reaction, but none of the negative control groups displayed adenoviruses. The pterygium group and the control groups were β-globin positive. Direct sequencing of PCR products confirmed Adenovirus infection. Conclusions: Adenoviruses might act as a possible cause of pterygium formation and other factors could play a synergistic role in the development. However, further larger studies are required to confirm this hypothesis. PMID:26034543

  14. Spleno-enteritis caused by adenovirus in psittacine birds: a pathological study.

    PubMed

    Gomez-Vtllamandos, J C; Muias, J M; Hervas, J; De Lara, F C; Perez, J; Mozos, E

    1995-09-01

    Psittacine birds with haemorrhagic enteritis were submitted for pathological study. Histopathological examination revealed haemorrhagic enteritis and necrotic areas in the spleen, with amphophilic and basophilic intranuclear inclusion bodies in mononuclear cells of the spleen, intestinal cell infiltrate, endothelial cells of small vessels in spleen, intestine, lung and liver, and in a few hepatocytes. Thrombi were observed in intestinal capillaries. The clinical and pathological features of this outbreak resembled haemorrhagic enteritis of turkeys and immunohistochemical staining showed positive immunoreaction to group II avian adenovirus antigens in the intranuclear inclusion bodies. The possible relationship between the virus replication in endothelial cells and intestinal haemorrhages in avian adenovirus infection is discussed. This outbreak could represent a new pathological process in psittacine birds.

  15. A new animal model for human respiratory tract disease due to adenovirus.

    PubMed

    Pacini, D L; Dubovi, E J; Clyde, W A

    1984-07-01

    Cotton rats (Sigmodon hispidus) were tested as a model for human respiratory tract infection due to adenovirus. After intranasal instillation of 10(6.1) 50% tissue culture infectious doses (TCID50) of adenovirus type 5 into one-month-old cotton rats, groups were killed at intervals for nasal and lung titration of virus and lung histopathology. In lung, eclipse occurred at 8 hr followed by peak viral titer (10(7.5) TCID50/g of lung) on day 5. Titers fell to 10(3.2) TCID50/g by day 10 and persisted at that level through the remainder of the study (day 28) despite appearance of serum neutralizing antibody after day 6. Interstitial pneumonia paralleled viral growth, and peribronchial mononuclear infiltration followed one to two days later. Titers in nasal mucosa peaked on day 3 but were undetectable beyond day 21. Pulmonary histopathology and viral replicative patterns paralleled findings in natural human disease.

  16. Application of a microtiter cell-culture method to characterization of avian adenoviruses.

    PubMed

    Grimes, T M; King, D J; Kleven, S H

    1976-01-01

    A microtiter cell-culture method was developed and used to titrate virus isolates for characterization. Virus dilutions and chicken kidney cell suspensions were dispensed into the wells of disposable microculture plates, with infectivity endpoints being determined microscopically on the fifth or sixth day, or by reading crystal-violet-stained monolayers on day 6. With this method, 37 candidate avian adenoviruses isolated from diagnostic accessions were characterized as avian adenoviruses (AAV). The criteria used for characterization were production of round-cell cytopathic effect, resistance to chloroform treatment, inhibition by 5-bromodeoxyuridine, and the presence of an antigen showing identity with a known AAV by precipitation in agar gel. Statistical anlaysis of eight replicate titrations of three AAV indicated that the titration method was highly reproducible. Use of the microculture method for titrations gave substantial savings in indicator cells, media, incubator space, culture dishes, and time.

  17. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-08-10

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

  1. Components of Adenovirus Genome Packaging

    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

  2. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  3. Robotic arm

    DOEpatents

    Kwech, Horst

    1989-04-18

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

  4. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  5. The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3.

    PubMed

    Lapointe, J M; Hedges, J F; Woods, L W; Reubel, G H; MacLachlan, N J

    1999-01-01

    DNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct.

  6. 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

  7. Viral and Cellular Components of AAV2 Replication Compartments

    PubMed Central

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  8. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

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

    PubMed

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

    2012-01-01

    The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. PMID:23093934

  10. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  11. Structure and Uncoating of Immature Adenovirus

    SciTech Connect

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

    2009-09-18

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

  12. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

    PubMed Central

    Pascual, E.; Avia, M.; Rangel, G.; de Molina, A.; Alejo, A.; Sevilla, N.

    2016-01-01

    Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. PMID:26739058

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

    PubMed

    Xie, Yinli; Gao, Peng; Li, Zhiyong

    2016-01-01

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

  14. 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

  15. Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1998-03-01

    Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

  16. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

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

    PubMed

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

    2015-05-01

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

  18. Replication fork collapse at replication terminator sequences.

    PubMed

    Bidnenko, Vladimir; Ehrlich, S Dusko; Michel, Bénédicte

    2002-07-15

    Replication fork arrest is a source of genome re arrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the first blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently.

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

    PubMed

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

    2014-01-01

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

  20. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

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

    2008-02-01

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

  1. Isolation and Characterization of an Equine Adenovirus

    PubMed Central

    Ardans, Alexander A.; Pritchett, Randall F.; Zee, Yuan Chung

    1973-01-01

    A viral agent was isolated from lung tissue obtained upon necropsy of an Arabian foal which had exhibited clinical signs of pneumonia. The virus is 75 nm in diameter, cubic in symmetry, and resistant to chloroform and low pH (3.0). It contains deoxyribonucleic acid and has a buoyant density of 1.31 g/cm3 in cesium chloride. These findings indicate that the virus is a member of the adenovirus group. Images PMID:16558078

  2. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  3. Vector systems for prenatal gene therapy: principles of adenovirus design and production.

    PubMed

    Alba, Raul; Baker, Andrew H; Nicklin, Stuart A

    2012-01-01

    Adenoviruses have many attributes, which have made them one of the most widely investigated vectors for gene therapy applications. These include ease of genetic manipulation to produce replication-deficient vectors, ability to readily generate high titer stocks, efficiency of gene delivery into many cell types, and ability to encode large genetic inserts. Recent advances in adenoviral vector engineering have included the ability to genetically manipulate the tropism of the vector by engineering of the major capsid proteins, particularly fiber and hexon. Furthermore, simple replication-deficient adenoviral vectors deleted for expression of a single gene have been complemented by the development of systems in which the majority of adenoviral genes are deleted, generating sophisticated Ad vectors which can mediate sustained transgene expression following a single delivery. This chapter outlines methods for developing simple transgene over expressing Ad vectors and detailed strategies to engineer mutations into the major capsid proteins.

  4. Mouse Adenovirus Type 1 Infection of Natural Killer Cell-Deficient Mice

    PubMed Central

    Welton, Amanda R.; Gralinski, Lisa E.; Spindler, Katherine R.

    2008-01-01

    Natural killer (NK) cells contribute to the initial nonspecific response to viral infection, and viruses exhibit a range of sensitivities to NK cells in vivo. We investigated the role of NK cells in infection of mice by mouse adenovirus type 1 (MAV-1) using antibody-mediated depletion and knockout mice. MAV-1 causes encephalomyelitis and replicates to highest levels in brains. NK cell-depleted mice infected with MAV-1 showed brain viral loads 8-20 days p.i. that were similar to wild-type control non-depleted mice. Mice genetically deficient for NK cells behaved similarly to wild-type control mice with respect to brain viral loads and survival. We conclude that NK cells are not required to control virus replication in the brains of MAV-1-infected mice. PMID:18155121

  5. Coacervate microspheres as carriers of recombinant adenoviruses.

    PubMed

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

    1999-01-01

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

  6. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    SciTech Connect

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

    2001-08-01

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

  7. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  8. Structure, function and dynamics in adenovirus maturation

    DOE PAGES

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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

  11. Chemical modification with high molecular weight polyethylene glycol reduces transduction of hepatocytes and increases efficacy of intravenously delivered oncolytic adenovirus.

    PubMed

    Doronin, Konstantin; Shashkova, Elena V; May, Shannon M; Hofherr, Sean E; Barry, Michael A

    2009-09-01

    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.

  12. Gene therapy for human colorectal cancer cell lines with recombinant adenovirus 5 based on loss of the insulin-like growth factor 2 imprinting.

    PubMed

    Sun, Huiling; Pan, Yuqin; He, Bangshun; Deng, Qiwen; Li, Rui; Xu, Yeqiong; Chen, Jie; Gao, Tianyi; Ying, Houqun; Wang, Feng; Liu, Xian; Wang, Shukui

    2015-04-01

    The recombinant oncolytic adenovirus is a novel anticancer agent to replicate selectively in colon cancer cell lines. Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon. We utilized the IGF2 LOI in gene therapy for the malignant tumor cell lines. We investigated the tumoricidal effects of IGF2 LOI on four cell lines by oncolytic adenovirus, and constructed novel adenovirus vectors Ad312-E1A and Ad312-EGFP. The expression of E1A was monitored by real-time PCR and western blot analysis. The viability and apoptosis of colorectal cells infected with Ad312-E1A were tested by CCK-8 and flow cytometry. In addition, we established a colorectal cancer model in nude mice. The results showed that HCT-8 and HT-29 with IGF2 LOI were infected with Ad312-EGFP and then produced the EGFP. Nevertheless, SW480 and GES-1, which were IGF2 MOI, did not produce the EGFP. The Ad312-E1A obviously reduced the cell viability and induced apoptosis in HCT-8 and HT-29 in vitro, and successfully suppressed tumor growth in HT-29 xenografts in nude mice. In summary, the conditionally replicative adenovirus with loss of IGF2 imprinting system has a positive effect on gene therapy.

  13. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  14. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  15. 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.

  16. Time-dependent biodistribution and transgene expression of a recombinant human adenovirus serotype 5-luciferase vector as a surrogate agent for rAd5-FMDV vaccines in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Replication-defective recombinant adenovirus 5 (rAd5) vectors carrying foot-and-mouth disease virus (FMDV) transgenes elicit a robust immune response to FMDV challenge in cattle; however vaccine function mechanisms are incompletely understood. Recent efforts addressing critical interactions of rAd5 ...

  17. Capturing and concentrating adenovirus using magnetic anionic nanobeads.

    PubMed

    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.

  18. 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

  19. 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

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

    PubMed

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

    2014-05-07

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

  1. Experimental adenovirus hemorrhagic disease in yearling black-tailed deer.

    PubMed

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

    1997-10-01

    An apparently novel adenovirus was associated with an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California (USA) during 1993-1994. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. Six black-tailed yearling deer (O. hemionus columbianus) were inoculated with purified adenovirus isolated from a black-tailed fawn that died of acute adenovirus hemorrhagic disease during the epizootic. Three of six inoculated deer also received intramuscular injections of dexamethasone sodium phosphate every 3 days during the study. Eight days post-inoculation, one deer (without dexamethasone) developed bloody diarrhea and died. Necropsy and histopathologic findings were identical to lesions in free-ranging animals that died of the natural disease. Hemorrhagic enteropathy and pulmonary edema were the significant necropsy findings and there was microscopic vascular damage and endothelial intranuclear inclusion bodies in the vessels of the intestines and lungs. Adenovirus was identified in necrotic endothelial cells in the lungs by fluorescent antibody staining, immunohistochemistry and by transmission electron microscopy. Adenovirus was reisolated from tissues of the animal that died of experimental adenovirus hemorrhagic disease. Similar gross and microscopic lesions were absent in four of six adenovirus-inoculated deer and in the negative control animal which were necropsied at variable intervals during the 14 wk study. One deer was inoculated with purified adenovirus a second time, 12 wk after the first inoculation. Fifteen days after the second inoculation, this deer developed severe ulceration of the tongue, pharynx and rumen and necrotizing osteomyelitis of the mandible which was associated with

  2. Surveying genome replication

    PubMed Central

    Kearsey, Stephen

    2002-01-01

    Two recent studies have added microarrays to the toolkit used to analyze the origins of replication in yeast chromosomes, providing a fuller picture of how genomic DNA replication is organized. PMID:12093380

  3. Neutralization of adenoviruses: kinetics, stoichiometry, and mechanisms.

    PubMed Central

    Wohlfart, C

    1988-01-01

    Kinetic curves for neutralization of adenovirus type 2 with anti-hexon serum revealed no lag periods even when the serum was highly diluted or when the temperature was lowered to 4 degrees C, thus indicating a single-hit mechanism. Multiplicity curves determined with anti-hexon serum displayed a linear correlation between the degree of neutralization and dilution of antiserum. Neutralization values experimentally obtained under steady-state conditions fully fitted a single-hit model based on Poisson calculations. Quantitation of the amount of 125I-labeled type-specific anti-hexon antibodies needed for full neutralization of adenovirus showed that 1.4 antibodies were attached per virion under such conditions. Virions already attached to HeLa cells at 4 degrees C were, to a large extent, neutralizable by anti-hexon serum, whereas anti-fiber and anti-penton base antisera were negative. It is suggested that adenovirus may be neutralized by two pathways: aggregation of the virions (extracellular neutralization) as performed by anti-fiber antibodies and blocking of virion entrance from the acidic endosomes into the cytoplasm (intracellular neutralization). The latter effect could be obtained by (i) covering of the penton bases, as performed by anti-penton base antibodies, thereby preventing interaction between the penton bases and the endosomal membrane, which results in trapping of virions within endosomes, and (ii) inhibition of the low-pH-induced conformational change of the viral capsid, which seems to occur in the endosomes and is necessary for proper exposure of the penton bases, as performed by anti-hexon antibodies. Images PMID:3373570

  4. Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge.

    PubMed

    Fischer, Laurent; Tronel, Jean Phillipe; Pardo-David, Camilla; Tanner, Patrick; Colombet, Guy; Minke, Jules; Audonnet, Jean-Christophe

    2002-10-01

    None of the currently available distemper vaccines provides a satisfactory solution for the immunization of very young carnivores in the face of maternal-derived immunity. Since mucosal immunization with replication-competent adenovirus-based vaccines has been proven effective in the face of passive immunity against the vector, it has the potential to provide a solution for the vaccination of young puppies born to canine distemper virus (CDV)-immune dams. We report the engineering and the characterization of two replication-competent canine adenovirus type 2 (CAV2)-based vaccines expressing, respectively, the CDV hemagglutinin (HA) and fusion (F) antigens. We first demonstrated that the intranasal vaccination with a mixture of both recombinant CAV2s provides an excellent level of protection in seronegative puppies, confirming the value of replication-competent adenovirus-based vectors for mucosal vaccination. In contrast, intranasal immunization with the same vaccine of puppies born to CDV- and CAV2-immune dams, failed to activate specific and protective immune responses. We hypothesized that an active CAV2 infection occurred while puppies were in close contact with the vaccinated dams in the breeding units and that the resulting active mucosal immunity interfered with the intranasal administration of CAV2-based CDV vaccine. However, when puppies born to CDV- and CAV2-immune dams were vaccinated subcutaneously with the CAV2-based CDV vaccine, significant seroconversion and solid protective immunity were triggered despite pre-existing systemic immunity to the vector. This latter result is surprising and suggests that subcutaneous vaccination with a replication-competent recombinant CAV2 may be an efficient strategy to overcome both passive and active adenovirus specific immunity in the dog. From a practical point of view, this could pave the way for an original strategy to vaccinate young puppies in the face of maternal-derived immunity. PMID:12297394

  5. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  6. 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.

  7. PEGylated Adenoviruses: From Mice to Monkeys

    PubMed Central

    Wonganan, Piyanuch; Croyle, Maria A.

    2010-01-01

    Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models. PMID:21994645

  8. E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

    PubMed

    Zheng, Yueting; Stamminger, Thomas; Hearing, Patrick

    2016-01-01

    Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection. PMID:26809031

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

    PubMed Central

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

    2013-01-01

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

  10. Anomalous Arms

    NASA Technical Reports Server (NTRS)

    2007-01-01

    In this composite image of spiral galaxy M106 (NGC 4258), optical data from the Digitized Sky Survey is shown as yellow, radio data from the Very Large Array appears as purple, X-ray data from Chandra is coded blue, and infrared data from the Spitzer Space Telescope appears red. Two anomalous arms, which aren't visible at optical wavelengths, appear as purple and blue emission.

  11. Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy.

    PubMed

    Choi, Joung-Woo; Park, Ji Won; Na, Youjin; Jung, Soo-Jung; Hwang, June Kyu; Choi, Dongho; Lee, Kyeong Geun; Yun, Chae-Ok

    2015-10-01

    Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy. PMID:26164117

  12. Actin-resistant DNAse I Expression From Oncolytic Adenovirus Enadenotucirev Enhances Its Intratumoral Spread and Reduces Tumor Growth.

    PubMed

    Tedcastle, Alison; Illingworth, Sam; Brown, Alice; Seymour, Leonard W; Fisher, Kerry D

    2016-04-01

    Spread of oncolytic viruses through tumor tissue is essential to effective virotherapy. Interstitial matrix is thought to be a significant barrier to virus particle convection between "islands" of tumor cells. One way to address this is to encode matrix-degrading enzymes within oncolytic viruses, for secretion from infected cells. To test the hypothesis that extracellular DNA provides an important barrier, we assessed the ability of DNase to promote virus spread. Nonreplicating Ad5 vectors expressing actin-resistant DNase (aDNAse I), proteinase K (PK), hyaluronidase (rhPH20), and chondroitinase ABC (CABC) were injected into established DLD human colorectal adenocarcinoma xenografts, transcomplemented with a replicating Ad5 virus. Each enzyme improved oncolysis by the replicating adenovirus, with no evidence of tumor cells being shed into the bloodstream. aDNAse I and rhPH20 hyaluronidase were then cloned into conditionally-replicating group B adenovirus, Enadenotucirev (EnAd). EnAd encoding each enzyme showed significantly better antitumor efficacy than the parental virus, with the aDNAse I-expressing virus showing improved spread. Both DNase and hyaluronidase activity was still measurable 32 days postinfection. This is the first time that extracellular DNA has been implicated as a barrier for interstitial virus spread, and suggests that oncolytic viruses expressing aDNAse I may be promising candidates for clinical translation. PMID:26708004

  13. Actin-resistant DNAse I Expression From Oncolytic Adenovirus Enadenotucirev Enhances Its Intratumoral Spread and Reduces Tumor Growth.

    PubMed

    Tedcastle, Alison; Illingworth, Sam; Brown, Alice; Seymour, Leonard W; Fisher, Kerry D

    2016-04-01

    Spread of oncolytic viruses through tumor tissue is essential to effective virotherapy. Interstitial matrix is thought to be a significant barrier to virus particle convection between "islands" of tumor cells. One way to address this is to encode matrix-degrading enzymes within oncolytic viruses, for secretion from infected cells. To test the hypothesis that extracellular DNA provides an important barrier, we assessed the ability of DNase to promote virus spread. Nonreplicating Ad5 vectors expressing actin-resistant DNase (aDNAse I), proteinase K (PK), hyaluronidase (rhPH20), and chondroitinase ABC (CABC) were injected into established DLD human colorectal adenocarcinoma xenografts, transcomplemented with a replicating Ad5 virus. Each enzyme improved oncolysis by the replicating adenovirus, with no evidence of tumor cells being shed into the bloodstream. aDNAse I and rhPH20 hyaluronidase were then cloned into conditionally-replicating group B adenovirus, Enadenotucirev (EnAd). EnAd encoding each enzyme showed significantly better antitumor efficacy than the parental virus, with the aDNAse I-expressing virus showing improved spread. Both DNase and hyaluronidase activity was still measurable 32 days postinfection. This is the first time that extracellular DNA has been implicated as a barrier for interstitial virus spread, and suggests that oncolytic viruses expressing aDNAse I may be promising candidates for clinical translation.

  14. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

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

    PubMed

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

    2006-10-01

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

  16. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGES

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; et al

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  17. 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.

  18. 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

  19. 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. PMID:16537624

  20. Isolation and Epidemiology of Falcon Adenovirus

    PubMed Central

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

    2005-01-01

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

  1. Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses II. Relationship of Adenovirus 2 Deoxyribonucleic Acid and Simian Virus 40 Deoxyribonucleic Acid in the Ad2+ND1 Genome

    PubMed Central

    Levin, Myron J.; Crumpacker, Clyde S.; Lewis, Andrew M.; Oxman, Michael N.; Henry, Patrick H.; Rowe, Wallace P.

    1971-01-01

    A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2+ND1, has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2+ND1 virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2+ND1 molecule. From the results obtained, it was estimated that 1% of the Ad2+ND1 DNA consists of SV40 nucleotide sequences. PMID:4323709

  2. 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.

  3. Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

    PubMed Central

    Venkatesh, L K; Arens, M Q; Subramanian, T; Chinnadurai, G

    1990-01-01

    The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis. Images PMID:2247444

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

    PubMed Central

    Vera, Beatriz; Martínez-Vélez, Naiara; Xipell, Enric; Acanda de la Rocha, Arlet; Patiño-García, Ana; Saez-Castresana, Javier; Gonzalez-Huarriz, Marisol; Cascallo, Manel; Alemany, Ramón; Alonso, Marta M.

    2016-01-01

    Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease. PMID:26808201

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

    PubMed

    Vera, Beatriz; Martínez-Vélez, Naiara; Xipell, Enric; Acanda de la Rocha, Arlet; Patiño-García, Ana; Saez-Castresana, Javier; Gonzalez-Huarriz, Marisol; Cascallo, Manel; Alemany, Ramón; Alonso, Marta M

    2016-01-01

    Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease. PMID:26808201

  6. The evolution of replicators.

    PubMed Central

    Szathmáry, E

    2000-01-01

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators. PMID:11127914

  7. Enhanced Viral Replication by Cellular Replicative Senescence

    PubMed Central

    Kim, Ji-Ae; Seong, Rak-Kyun

    2016-01-01

    Cellular replicative senescence is a major contributing factor to aging and to the development and progression of aging-associated diseases. In this study, we sought to determine viral replication efficiency of influenza virus (IFV) and Varicella Zoster Virus (VZV) infection in senescent cells. Primary human bronchial epithelial cells (HBE) or human dermal fibroblasts (HDF) were allowed to undergo numbers of passages to induce replicative senescence. Induction of replicative senescence in cells was validated by positive senescence-associated β-galactosidase staining. Increased susceptibility to both IFV and VZV infection was observed in senescent HBE and HDF cells, respectively, resulting in higher numbers of plaque formation, along with the upregulation of major viral antigen expression than that in the non-senescent cells. Interestingly, mRNA fold induction level of virus-induced type I interferon (IFN) was attenuated by senescence, whereas IFN-mediated antiviral effect remained robust and potent in virus-infected senescent cells. Additionally, we show that a longevity-promoting gene, sirtuin 1 (SIRT1), has antiviral role against influenza virus infection. In conclusion, our data indicate that enhanced viral replication by cellular senescence could be due to senescence-mediated reduction of virus-induced type I IFN expression. PMID:27799874

  8. Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

    PubMed Central

    Hasson, T B; Soloway, P D; Ornelles, D A; Doerfler, W; Shenk, T

    1989-01-01

    A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles. Images PMID:2760976

  9. Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro

    PubMed Central

    Lasek, Witold; Janyst, Michał; Młynarczyk, Grażyna

    2016-01-01

    There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells. PMID:26862302

  10. Enteric adenovirus type 40: complementation of the E4 defect in Ad2 dl808.

    PubMed

    Mautner, V; Mackay, N

    1991-07-01

    The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 dl808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements dl808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 dl808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

  11. Tamoxifen improves cytopathic effect of oncolytic adenovirus in primary glioblastoma cells mediated through autophagy

    PubMed Central

    Ulasov, Ilya V.; Shah, Nameeta; Kaverina, Natalya V.; Lee, Hwahyang; Lin, Biaoyang; Lieber, Andre; Kadagidze, Zaira G.; Yoon, Jae-Guen; Schroeder, Brett; Hothi, Parvinder; Ghosh, Dhimankrishna; Baryshnikov, Anatoly Y.; Cobbs, Charles S.

    2015-01-01

    Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy. PMID:25738357

  12. A simplified system for generating recombinant E3-deleted canine adenovirus-2.

    PubMed

    Yu, Zuo; Jiang, Qian; Liu, Jiasen; Guo, Dongchun; Quan, Chuansong; Li, Botao; Qu, Liandong

    2015-01-01

    Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector. PMID:25450764

  13. Adenovirus serotype 30 fiber does not mediate transduction via the coxsackie-adenovirus receptor.

    PubMed

    Law, Lane K; Davidson, Beverly L

    2002-01-01

    Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR. PMID:11752156

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

    PubMed

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

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine. PMID:19178894

  15. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  16. Differential immunogenicity between HAdV-5 and chimpanzee adenovirus vector ChAdOx1 is independent of fiber and penton RGD loop sequences in mice

    PubMed Central

    Dicks, Matthew D. J.; Spencer, Alexandra J.; Coughlan, Lynda; Bauza, Karolis; Gilbert, Sarah C.; Hill, Adrian V. S.; Cottingham, Matthew G.

    2015-01-01

    Replication defective adenoviruses are promising vectors for the delivery of vaccine antigens. However, the potential of a vector to elicit transgene-specific adaptive immune responses is largely dependent on the viral serotype used. HAdV-5 (Human adenovirus C) vectors are more immunogenic than chimpanzee adenovirus vectors from species Human adenovirus E (ChAdOx1 and AdC68) in mice, though the mechanisms responsible for these differences in immunogenicity remain poorly understood. In this study, superior immunogenicity was associated with markedly higher levels of transgene expression in vivo, particularly within draining lymph nodes. To investigate the viral factors contributing to these phenotypes, we generated recombinant ChAdOx1 vectors by exchanging components of the viral capsid reported to be principally involved in cell entry with the corresponding sequences from HAdV-5. Remarkably, pseudotyping with the HAdV-5 fiber and/or penton RGD loop had little to no effect on in vivo transgene expression or transgene-specific adaptive immune responses despite considerable species-specific sequence heterogeneity in these components. Our results suggest that mechanisms governing vector transduction after intramuscular administration in mice may be different from those described in vitro. PMID:26576856

  17. Targeting of adenovirus vectors carrying a tumor cell-specific peptide: in vitro and in vivo studies.

    PubMed

    Rittner, K; Schreiber, V; Erbs, P; Lusky, M

    2007-05-01

    Previously, we have identified a tumor cell-specific peptide, HEW, by panning of phage display libraries on the human colorectal cancer cell line WiDr. In this report we demonstrate that this peptide can modify the infection properties of adenovirus vectors. Increased infectivity of replication-deficient adenovirus 5 vectors in WiDr cells was observed upon genetic insertion of the HEW peptide in the HI loop of the fiber knob. Moreover, whereas the coxsackie and adenovirus receptor (CAR)-ablating fiber mutation S408E abolished apparent infection in CAR-positive WiDr cells, the insertion of HEW completely restored infectivity toward these cells in vitro. To assess whether the de- and re-targeted infection profile was maintained in vivo, the fiber-modified adenovirus vectors were injected intratumorally or intravenously in WiDr tumor-bearing Swiss nu/nu mice. No significant differences in efficiency of infection could be observed suggesting alternative viral uptake mechanisms in vivo. Next, we have included the fiber shaft mutation S(*) in our studies, which was described to confer a de-targeted phenotype in vivo. Reduced gene transfer due to the S(*) mutation both in vitro and in vivo could be confirmed. Insertion of HEW in the HI knob loop of shaft-mutated fiber, however, did not rescue infectivity in target cells neither in vitro nor in vivo. We demonstrate the efficient ligand-mediated re-targeting of adenoviral vector infection to the human cancer cell line WiDr. The lack of apparent re-targeting in the in vivo situation is described. PMID:17318198

  18. Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus

    PubMed Central

    Robinson, Christopher M.; Zhou, Xiaohong; Rajaiya, Jaya; Yousuf, Mohammad A.; Singh, Gurdeep; DeSerres, Joshua J.; Walsh, Michael P.; Wong, Sallene; Seto, Donald; Dyer, David W.; Chodosh, James; Jones, Morris S.

    2013-01-01

    ABSTRACT For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses. PMID:23572555

  19. Directed evolution of mutator adenoviruses resistant to antibody neutralization.

    PubMed

    Myers, Nicolle D; Skorohodova, Ksenia V; Gounder, Anshu P; Smith, Jason G

    2013-05-01

    We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

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

    PubMed

    Matoq, Amr; Salahuddin, Asma

    2016-01-01

    Adenoviruses are a common cause of respiratory infection, pharyngitis, and conjunctivitis in infants and young children. They are known to cause hepatitis and liver failure in immunocompromised patients; they are a rare cause of hepatitis in immunocompetent patients and have been known to cause fulminant hepatic failure. We present a 23-month-old immunocompetent infant who presented with acute noncholestatic hepatitis, hypoalbuminemia, generalized anasarca, and pancytopenia secondary to adenovirus infection. PMID:27340581

  1. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  2. Adenovirus Infections in Immunocompetent and Immunocompromised Patients

    PubMed Central

    2014-01-01

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

  3. Polymeric oncolytic adenovirus for cancer gene therapy

    PubMed Central

    Choi, Joung-Woo; Lee, Young Sook; Yun, Chae-Ok; Kim, Sung Wan

    2015-01-01

    Oncolytic adenovirus (Ad) vectors present a promising modality to treat cancer. Many clinical trials have been done with either naked oncolytic Ad or combination with chemotherapies. However, the systemic injection of oncolytic Ad in clinical applications is restricted due to significant liver toxicity and immunogenicity. To overcome these issues, Ad has been engineered physically or chemically with numerous polymers for shielding the Ad surface, accomplishing extended blood circulation time and reduced immunogenicity as well as hepatotoxicity. In this review, we describe and classify the characteristics of polymer modified oncolytic Ad following each strategy for cancer treatment. Furthermore, this review concludes with the highlights of various polymer-coated Ads and their prospects, and directions for future research. PMID:26453806

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

    SciTech Connect

    Freytag, Svend O.; Stricker, Hans; Lu, Mei; Elshaikh, Mohamed; Aref, Ibrahim; Pradhan, Deepak; Levin, Kenneth; Kim, Jae Ho; Peabody, James; Siddiqui, Farzan; Barton, Kenneth; Pegg, Jan; Zhang, Yingshu; Cheng, Jingfang; Oja-Tebbe, Nancy; Bourgeois, Renee; Gupta, Nilesh; Lane, Zhaoli; Rodriguez, Ron; DeWeese, Theodore; and others

    2014-06-01

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

  5. Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups.

    PubMed

    Green, M; Brackmann, K; Wold, W S; Cartas, M; Thornton, H; Elder, J H

    1979-09-01

    The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.

  6. Who Needs Replication?

    ERIC Educational Resources Information Center

    Porte, Graeme

    2013-01-01

    In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

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

    PubMed Central

    Kumar, Ramesh; Sreenivasa, B. P.; Tamilselvan, R. P.

    2015-01-01

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

  8. 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.

  9. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells

    PubMed Central

    Lenman, Annasara; Liaci, A. Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S.; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-01-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy. PMID:25674795

  10. Adenovirus uncoating and nuclear establishment are not affected by weak base amines.

    PubMed Central

    Rodríguez, E; Everitt, E

    1996-01-01

    We have used four established lysosomotropic agents, ammonium chloride, amantadine, chloroquine, and methylamine, to monitor the possible interference with an early low-pH-dependent step during adenovirus replication. Two concentrations of each of the different agents were selected; one was essentially nontoxic to uninfected HeLa cells, and the other resulted in some toxicity as measured by trypan blue staining and by interference with cell monolayer establishment, cell proliferation, and radioisotope labelling. It was separately determined that these concentrations displayed pH-raising effects of the same magnitude as higher concentrations previously used in similar studies. Adenovirus uncoating in vivo, normally reaching its maximum within 1 h after infection, was not affected by any of the agents. The subsequent levels of successful nuclear entry events by the parental genomes were monitored by measuring the extent of transcription of an mRNA species coding for the early 72-kDa DNA-binding protein at 10 to 12 h postinfection. In HeLa, KB, HEp-2, and A549 cells, none of the agents were able to affect the levels of early transcription after administration at the point of infection or at 3 h after infection. The cumulative synthesis of the hexon antigen was assessed late in infection, and inhibitory effects were revealed upon administration of 10, 20, and 40 mM ammonium chloride, 10 mM methylamine, and 0.5 mM amantadine, irrespective of the time point of addition. Ammonium chloride at 5 mM reduced the hexon yield by 20% at the most when added within 50 min after infection. Chloroquine at concentrations of 2.5 and 5 microM specifically reduced the hexon yields by 30 to 40% when administered within the first 50 min of infection. On the basis of the lack of effects of nontoxic concentrations of the four agents on the early virus-cell interactive event of uncoating and the early virus-specified transcription, we conclude that a low-pH-dependent step early in the

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

    PubMed

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

    2016-07-01

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

  12. Development of a replication defective adenovirus 5 vector expressing porcine interleukin-18 and a mutated analog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell-mediated immune responses against swine pathogens are sometimes necessary to elicit durable protective immunity. Cell mediated or Th1 immunity is dependent on the coordinated expression of several cytokines, including interferon-gamma to assist in the production of antigen-specific cytotoxic T...

  13. Biosynthesis of adenovirus type 2 i-leader protein.

    PubMed Central

    Symington, J S; Lucher, L A; Brackmann, K H; Virtanen, A; Pettersson, U; Green, M

    1986-01-01

    The i-leader is a 440-base-pair sequence located between 21.8 and 23.0 map units on the adenovirus type 2 genome and is spliced between the second and third segments of the major tripartite leader in certain viral mRNA molecules. The i-leader contains an open translational reading frame for a hypothetical protein of Mr about 16,600, and a 16,000-Mr polypeptide (16K protein) has been translated in vitro on mRNA selected with DNA containing the i-leader (A. Virtanen, P. Aleström, H. Persson, M. G. Katze, and U. Pettersson, Nucleic Acids Res. 10:2539-2548, 1982). To determine whether the i-leader protein is synthesized during productive infection and to provide an immunological reagent to study the properties and functions of the i-leader protein, we prepared antipeptide antibodies directed to a 16-amino acid synthetic peptide which is encoded near the N terminus of the hypothetical i-leader protein and contains a high acidic amino acid and proline content. Antipeptide antibodies immunoprecipitated from extracts of adenovirus type 2-infected cells a major 16K protein that comigrated with a 16K protein translated in vitro. Partial N-terminal amino acid sequence analysis by Edman degradation of radiolabeled 16K antigen showed that methionine is present at residue 1 and leucine is present at residues 8 and 10, as predicted from the DNA sequence, establishing that the 16K protein precipitated by this antibody is indeed the i-leader protein. Thus, the i-leader protein is a prominent species that is synthesized during productive infection. The i-leader protein is often seen as a doublet on polyacrylamide gels, suggesting that either two related forms of i-leader protein are synthesized in infected cells or that a posttranslational modification occurs. Time course studies using immunoprecipitation analysis with antipeptide antibodies revealed that the E1A 289R T antigen and the E1B-19K (175R) T antigen are synthesized beginning at 2 to 3 and 4 to 5 h postinfection

  14. A combinatory strategy for detection of live CTCs using microfiltration and a new telomerase-selective adenovirus

    PubMed Central

    Ma, Yanchun; Hao, Sijie; Wang, Shuwen; Zhao, Yuanjun; Lim, Bora; Lei, Ming; Spector, David J.; El-Deiry, Wafik S.; Zheng, Si-yang; Zhu, Jiyue

    2015-01-01

    Circulating tumor cells (CTCs) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of cancer patients. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared to the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays (FMSAs) and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of breast and pancreatic cancer patients, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. PMID:25589497

  15. Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

    PubMed Central

    Teodoro, J G; Halliday, T; Whalen, S G; Takayesu, D; Graham, F L; Branton, P E

    1994-01-01

    The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation. Images PMID:8289381

  16. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

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

    PubMed

    Vitelli, Alessandra; Quirion, Mary R; Lo, Chia-Yun; Misplon, Julia A; Grabowska, Agnieszka K; Pierantoni, Angiolo; Ammendola, Virginia; Price, Graeme E; Soboleski, Mark R; Cortese, Riccardo; Colloca, Stefano; Nicosia, Alfredo; Epstein, Suzanne L

    2013-01-01

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

  18. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

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

  19. Abiotic self-replication.

    PubMed

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

    2012-12-18

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

  20. Abiotic self-replication.

    PubMed

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

    2012-12-18

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

  1. DNA replication origins.

    PubMed

    Leonard, Alan C; Méchali, Marcel

    2013-10-01

    The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.

  2. Replication of lightweight mirrors

    NASA Astrophysics Data System (ADS)

    Chen, Ming Y.; Matson, Lawrence E.; Lee, Heedong; Chen, Chenggang

    2009-08-01

    The fabrication of lightweight mirror assemblages via a replication technique offers great potential for eliminating the high cost and schedule associated with the grinding and polishing steps needed for conventional glass or SiC mirrors. A replication mandrel is polished to an inverse figure shape and to the desired finish quality. It is then, coated with a release layer, the appropriate reflective layer, and followed by a laminate for coefficient of thermal expansion (CTE) tailorability and strength. This optical membrane is adhered to a mirror structural substrate with a low shrinkage, CTE tailored adhesive. Afterwards, the whole assembly is separated from the mandrel. The mandrel is then cleaned and reused for the next replication run. The ultimate goal of replication is to preserve the surface finish and figure of the optical membrane upon its release from the mandrel. Successful replication requires a minimization of the residual stresses within the optical coating stack, the curing stresses from the adhesive and the thermal stress resulting from CTE mismatch between the structural substrate, the adhesive, and the optical membrane. In this paper, the results on replicated trials using both metal/metal and ceramic/ceramic laminates adhered to light weighted structural substrates made from syntactic foams (both inorganic and organic) will be discussed.

  3. Enhanced UV inactivation of adenoviruses under polychromatic UV lamps.

    PubMed

    Linden, Karl G; Thurston, Jeanette; Schaefer, Raymond; Malley, James P

    2007-12-01

    Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm(-2) is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm(-2) and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm(-2). The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed. PMID:17933932

  4. 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.

  5. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

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

    2016-01-01

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

  6. [Adenovirus-delivered BMI-1 shRNA].

    PubMed

    Chen, Zhen-Ping; Chen, Xiao-Li; Zhen, Jie

    2009-10-01

    Recently, some plasmid vectors that direct transcription of small hairpin RNAs have been developed, which are processed into functional siRNAs by cellular enzymes. Although these vectors possess certain advantages over synthesized siRNA, many disadvantages exist, including low and variable transfection efficiency. This study was aimed to establish an adenoviral siRNA delivery system without above-mentioned disadvantages on the basis of commercially available vectors. A vector was designed to target the human polycomb gene BMI-1. The pAd-BMI-1shRNA-CMV-GFP vector was produced by cloning a 300 bp U6-BMI-1 cassette from the pGE1BMI-1shRNA plasmid and a CMV-GFP cassette from pAdTrack CMV in pShutter vector. The adenovirus was produced from the 293A packaging cell line and then infected K562 cells. The mRNA and protein levels of Bmi-1 were detected by real time-PCR and Western blot respectively. The results showed that the adenovirus carrying the BMI-1shRNA was successfully produced. After being transfected with the adenovirus, the K562 cells dramatically down-regulated BMI-1 expression, whereas the adenoviruses carrying control shRNA had no effect on BMI-1 expression. It is concluded that the adenoviruses are efficient vectors for delivery of siRNA into mammalian cells and may become a candidate vector carrying siRNA drugs for gene therapy. PMID:19840467

  7. Adenovirus Pneumonia Complicated With Acute Respiratory Distress Syndrome

    PubMed Central

    Hung, Ka-Ho; Lin, Lung-Huang

    2015-01-01

    Abstract Severe adenovirus infection in children can manifest with acute respiratory distress syndrome (ARDS) and respiratory failure, leading to the need for prolonged mechanical support in the form of either mechanical ventilation or extracorporeal life support. Early extracorporeal membrane oxygenation (ECMO) intervention for children with ARDS should be considered if selection criteria fulfill. We report on a 9-month-old boy who had adenovirus pneumonia with rapid progression to ARDS. Real-time polymerase chain reaction tests of sputum and pleural effusion samples confirmed adenovirus serotype 7. Chest x-rays showed progressively increasing infiltrations and pleural effusions in both lung fields within 11 days. Because conventional ARDS therapies failed, we initiated ECMO with high-frequency oscillatory ventilation (HFOV) for 9 days. Chest x-rays showed gradual improvements in lung expansion. This patient was subsequently discharged after a hospital stay of 38 days. Post-ECMO and adenovirus sequelae were followed in our outpatient department. Adenovirus pneumonia in children can manifest with severe pulmonary morbidity and respiratory failure. The unique lung recruitment by HFOV can be a useful therapeutic option for severe ARDS patients when combined with sufficient lung rest provided by ECMO. PMID:25997046

  8. New Insights on Adenovirus as Vaccine Vectors

    PubMed Central

    Lasaro, Marcio O; Ertl, Hildegund CJ

    2009-01-01

    Adenovirus (Ad) vectors were initially developed for treatment of genetic diseases. Their usefulness for permanent gene replacement was limited by their high immunogenicity, which resulted in rapid elimination of transduced cells through induction of T and B cells to antigens of Ad and the transgene product. The very trait that excluded their use for sustained treatment of genetic diseases made them highly attractive as vaccine carriers. Recently though results showed that Ad vectors based on common human serotypes, such as serotype 5, may not be ideal as vaccine carriers. A recently conducted phase 2b trial, termed STEP trial, with an AdHu5-based vaccine expressing antigens of human immunodeficiency virus 1 (HIV-1) not only showed lack of efficacy in spite of the vaccine's immunogenicity, but also suggested an increased trend for HIV acquisition in individuals that had circulating AdHu5 neutralizing antibodies prior to vaccination. Alternative serotypes from humans or nonhuman primates (NHPs), to which most humans lack pre-existing immunity, have been vectored and may circumvent the problems encountered with the use of AdHu5 vectors in humans. In summary, although Ad vectors have seen their share of setbacks in recent years, they remain viable tools for prevention or treatment of a multitude of diseases. PMID:19513019

  9. Viability of human adenovirus from hospital fomites.

    PubMed

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

    2014-12-01

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

  10. Enhanced expression of adenovirus transforming proteins.

    PubMed Central

    Gaynor, R B; Tsukamoto, A; Montell, C; Berk, A J

    1982-01-01

    Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated. Images PMID:7143568

  11. Adenovirus 36 and Obesity: An Overview

    PubMed Central

    Ponterio, Eleonora; Gnessi, Lucio

    2015-01-01

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

  12. Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line.

    PubMed

    Zhu, Wu Yang; Tao, Xiao Yan; Lyu, Xin Jun; Yu, Peng Cheng; Lu, Zhuo Zhuang

    2016-03-01

    The nonstructural protein 1 (NS1) of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon (IFN)-mediated antiviral response. We engineered an NS1-expressing 293 (293-NS1) cell line with no response to IFN stimulation. Compared with the parental 293 cells, the IFN-nonresponsive 293-NS1 cells improved the growth capacity of various viruses, but the introduction of NS1 barely enhanced the propagation of Tahyna virus, a negative-strand RNA virus. In particular, fastidious enteric adenovirus that replicates poorly in 293 cells may grow more efficiently in 293-NS1 cells; thus, IFN-nonresponsive 293-NS1 cells might be of great value in diagnostic laboratories for the cultivation and isolation of human enteric adenoviruses.

  13. Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line.

    PubMed

    Zhu, Wu Yang; Tao, Xiao Yan; Lyu, Xin Jun; Yu, Peng Cheng; Lu, Zhuo Zhuang

    2016-03-01

    The nonstructural protein 1 (NS1) of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon (IFN)-mediated antiviral response. We engineered an NS1-expressing 293 (293-NS1) cell line with no response to IFN stimulation. Compared with the parental 293 cells, the IFN-nonresponsive 293-NS1 cells improved the growth capacity of various viruses, but the introduction of NS1 barely enhanced the propagation of Tahyna virus, a negative-strand RNA virus. In particular, fastidious enteric adenovirus that replicates poorly in 293 cells may grow more efficiently in 293-NS1 cells; thus, IFN-nonresponsive 293-NS1 cells might be of great value in diagnostic laboratories for the cultivation and isolation of human enteric adenoviruses. PMID:27109134

  14. Selection of nonfastidious adenovirus species in 293 cells inoculated with stool specimens containing adenovirus 40.

    PubMed

    Brown, M

    1985-08-01

    Of 35 stool specimens isolated and examined in 293 cells, 15 isolates contained adenovirus species 40 (Ad40), and 4 of these 15 isolates also contained a nonfastidious adenovirus species (Ad1 in two cases, Ad18 or Ad31) which was selected over Ad40 during serial passage in the 293 cells. The selection of Ad1 over Ad40 was examined in detail. Restriction analysis of intracellular DNA and the relative infectivity titers of Ad40 and Ad1 at each passage level after the inoculation of 293 cells with a particular stool specimen demonstrated that although the amount of Ad40 DNA synthesized far exceeded that of Ad1, the relative infectivity titer of Ad40 was low. The growth characteristics of Ad40 were then compared with those of Ad1, Ad18, and Ad41 in singly infected 293 cell cultures. One-step growth curves showed the same growth rate in each case, with a latent period of 12 h and a maximum titer at 24 to 36 h postinfection. Yields of infectious Ad40 virus were consistently 100- to 1,000-fold lower than those of Ad1. This difference was reflected by a reduced yield of total AD40 virions (p1.34) as determined by 35S labeling experiments. However, the 3- to 10-fold reduction in total yield of Ad40 virions did not account for the 100- to 1,000-fold reduction in the yield of infectious virus. PMID:2993350

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

    PubMed

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

    2016-01-01

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

  16. Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1997-08-19

    The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the

  17. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

    PubMed Central

    Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-01-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  18. The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response.

    PubMed

    Brestovitsky, Anna; Nebenzahl-Sharon, Keren; Kechker, Peter; Sharf, Rakefet; Kleinberger, Tamar

    2016-02-01

    The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition

  19. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis.

    PubMed Central

    Babiss, L E; Ginsberg, H S

    1984-01-01

    To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or

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

    PubMed

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

    2014-08-01

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

  1. Oncolytic Adenovirus Expressing Monoclonal Antibody Trastuzumab for Treatment of HER2-Positive Cancer.

    PubMed

    Liikanen, Ilkka; Tähtinen, Siri; Guse, Kilian; Gutmann, Theresia; Savola, Paula; Oksanen, Minna; Kanerva, Anna; Hemminki, Akseli

    2016-09-01

    Monoclonal anti-HER2 antibody trastuzumab has significantly improved the survival of patients with HER2-overexpressing tumors. Nevertheless, systemic antibody therapy is expensive, limited in efficacy due to physical tumor barriers, and carries the risk of severe side effects such as cardiomyopathy. Oncolytic viruses mediate cancer-selective transgene expression, kill infected cancer cells while mounting antitumor immune responses, and have recently demonstrated promising efficacy in combination treatments. Here, we armed an oncolytic adenovirus with full-length trastuzumab to achieve effective in situ antibody production coupled with progressive oncolytic cancer cell killing. We constructed an infectivity-enhanced serotype 5 oncolytic adenovirus, Ad5/3-Δ24-tras, coding for human trastuzumab antibody heavy- and light-chain genes, connected by an internal ribosome entry site. Infected cancer cells were able to assemble full-length functional antibody, as confirmed by Western blot, ELISA, and antibody-dependent cell-mediated cytotoxicity assay. Importantly, oncolysis was required for release of the antibody into tumors, providing additional spatial selectivity. Ad5/3-Δ24-tras showed potent in vitro cytotoxicity and enhanced antitumor efficacy over oncolytic control virus Ad5/3-Δ24 or commercial trastuzumab in HER2-positive cancer models in vivo (both P < 0.05). Furthermore, Ad5/3-Δ24-tras resulted in significantly higher tumor-to-systemic antibody concentrations (P < 0.001) over conventional delivery. Immunological analyses revealed dendritic cell activation and natural killer cell accumulation in tumor-draining lymph nodes. Thus, Ad5/3-Δ24-tras is an attractive anticancer approach combining oncolytic immunotherapy with local trastuzumab production, resulting in improved in vivo efficacy and immune cell activation in HER2-positive cancer. Moreover, the finding that tumor cells can produce functional antibody as directed by oncolytic virus could lead to many

  2. Crystal Structure of Enteric Adenovirus Serotype 41 Short Fiber Head

    PubMed Central

    Seiradake, Elena; Cusack, Stephen

    2005-01-01

    Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding. PMID:16254343

  3. Disseminated adenovirus infection in an immunocompromised host. Pitfalls in diagnosis.

    PubMed

    Landry, M L; Fong, C K; Neddermann, K; Solomon, L; Hsiung, G D

    1987-09-01

    In this report, a bone marrow transplant recipient with rapidly fatal gastroenteritis is presented. The presence of intranuclear inclusions on postmortem light microscopic examination of liver, lung, and small bowel tissue was considered diagnostic of cytomegalovirus infection. However, electron microscopic examination of liver tissue demonstrated adenovirus infection. This was confirmed by isolation of an adenovirus type 2 with unusual laboratory features from liver, lung, colon contents, serum, esophageal swab, and oral ulcerations. Results of a complement fixation test for antibodies to adenovirus performed on postmortem serum samples were negative, and a titer of 1:4 was noted for antibody against cytomegalovirus. This case illustrates the diagnostic pitfalls that may be encountered in establishing a specific viral diagnosis in severely ill patients. PMID:2821806

  4. 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. PMID:26094312

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

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

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

  6. The Prevalence of Rotavirus and Adenovirus in the Childhood Gastroenteritis

    PubMed Central

    Ozsari, Tamer; Bora, Gulhan; Kaya, Bulent; Yakut, Kahraman

    2016-01-01

    Background Acute gastroenteritis stemming from viral causes is very common during the childhood period. Rotavirus and enteric adenovirus are the most common factors of acute gastroenteritis encountered in infants and children. However, the epidemiology of rotavirus and enteric adenovirus gastroenteritis in the east Anatolia region is not well-known. Objectives We aimed to evaluate the relationship between the distribution of antigen positivity in rotavirus and enteric adenovirus antigen tests required cases and demographic data retrospectively in pediatric patients admitted to our hospital. Patients and Methods The records of stool sample analyses for 1154 patients admitted to our hospital from June 2011 to December 2011 with complaints of diarrhea were retrospectively examined. The presence of rotavirus and enteric adenovirus antigens in stool specimens was investigated by means of an immunochromatographic test. Results Viral antigens were detected in 327 (28.3%) stool specimens out of 1154. Among the positive results, the frequency was 73.7% for rotavirus and 26.2% for adenovirus. While the detected rotavirus antigen rate was high for all age groups, it was highest for children under the age of 2, with a rate of 57.1%. Moreover, the rotavirus infections were observed at a rate of 44.3% in winter and of 24.6% in autumn. Conclusions The most important factor in childhood acute gastroenteritis in east Anatolia is the rotavirus. Rotavirus and adenovirus antigens should be routinely investigated as a factor in fresh stool samples for the accurate diagnosis and treatment of gastroenteritis in children in the winter and autumn months. PMID:27635215

  7. Modeling DNA Replication Intermediates

    SciTech Connect

    Broyde, S.; Roy, D.; Shapiro, R.

    1997-06-01

    While there is now available a great deal of information on double stranded DNA from X-ray crystallography, high resolution NMR and computer modeling, very little is known about structures that are representative of the DNA core of replication intermediates. DNA replication occurs at a single strand/double strand junction and bulged out intermediates near the junction can lead to frameshift mutations. The single stranded domains are particularly challenging. Our interest is focused on strategies for modeling the DNA of these types of replication intermediates. Modeling such structures presents special problems in addressing the multiple minimum problem and in treating the electrostatic component of the force field. We are testing a number of search strategies for locating low energy structures of these types and we are also investigating two different distance dependent dielectric functions in the coulombic term of the force field. We are studying both unmodified DNA and DNA damaged by aromatic amines, carcinogens present in the environment in tobacco smoke, barbecued meats and automobile exhaust. The nature of the structure adopted by the carcinogen modified DNA at the replication fork plays a key role in determining whether the carcinogen will cause a mutation during replication that can initiate the carcinogenic process. In the present work results are presented for unmodified DNA.

  8. An evaluation of ARM radiosonde operational performance

    SciTech Connect

    Lesht, B.M.

    1995-06-01

    Because the ARM (Atmospheric Radiation Measurement) program uses data from radiosondes for real-time quality control and sensitive modeling applications, it is important to have a quantitative measure of the quality of the radiosonde data themselves. Two methods have been tried for estimating the quality of radiosonde data: comparisons with known standards before launch and examination of pseudo-replicate samples by single sensors aloft. The ground check procedure showed that the ARM radiosondes are within manufacturer`s specifications for measuring relative humidity; procedural artifacts prevented verification for temperature. Pseudo-replicates from ascent and descent suggest that the temperature measurement is within the specified {minus_plus}0.2 C. On average ascent and descent data are similar, but detailed structure may be obscured on descent by loss of sampling density, and the descent involves other uncertainties.

  9. Arms control and the arms race

    SciTech Connect

    Not Available

    1985-01-01

    A collection of 16 articles from the Scientific American discusses the evolution of nuclear weapons since 1945 and the attempts to control the nuclear arms race through national action and international negotiations. The articles and commentaries by political scientists Bruce M. Russett and Fred Chernoff combine technical information on weapons and deployment systems with political analysis of current arms strategies and diplomacy. The articles are grouped under three major topics: SALT and the history of arms control negotiations, current strategic arms negotiations, and European security. A separate abstract was written for each of the 16 articles selected for the Energy Data Base. 226 references.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1982-12-01

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

  12. Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5.

    PubMed Central

    Pettit, S C; Horwitz, M S; Engler, J A

    1989-01-01

    Using a series of transient expression plasmids and adenovirus-specific DNA replication assays for both initiation and elongation, we measured the relative activities of mutant polypeptides of the precursor to the terminal protein (pTP) in vitro. Mutations that removed two to six amino acids of the amino terminus gradually decreased pTP activity; a deletion of 18 amino acids was completely inactive. Replacement of cysteine at residue 8 with a serine had little effect on pTP activity. Two amino-terminal in-frame linker insertion mutant polypeptides previously characterized in vivo as either replication defective or temperature sensitive had considerable activity at the permissive temperature in vitro. For one mutant pTP with a temperature-sensitive phenotype in vivo, elongation activity was decreased more than initiation in vitro, suggesting a role for this protein after the initiation step. Replacement mutations of serine 580, the site of covalent attachment of dCTP, completely abolished pTP function for both initiation and elongation. Images PMID:2511338

  13. Functional prediction of hypothetical proteins in human adenoviruses.

    PubMed

    Dorden, Shane; Mahadevan, Padmanabhan

    2015-01-01

    Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins. PMID:26664031

  14. Human Adenovirus Type 2 but Not Adenovirus Type 12 Is Mutagenic at the Hypoxanthine Phosphoribosyltransferase Locus of Cloned Rat Liver Epithelial Cells

    PubMed Central

    Paraskeva, Christos; Roberts, Carl; Biggs, Paul; Gallimore, Phillip H.

    1983-01-01

    Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose. PMID:6572280

  15. Bovine adenovirus type 10 identified in fatal cases of adenovirus-associated enteric disease in cattle by in situ hybridization.

    PubMed Central

    Smyth, J A; Benkö, M; Moffett, D A; Harrach, B

    1996-01-01

    A severe, naturally occurring enteric disease of cattle in which adenovirus inclusions are present in the intestinal vascular endothelium has been recognized in several countries; three different adenovirus serotypes have been isolated from affected animals. An in situ hybridization technique for the detection of bovine adenoviral DNA was developed and applied to tissue from 13 cattle in Northern Ireland diagnosed to have the disease. Bovine adenovirus serotype 10 (BAV-10) was identified in the vascular inclusions of all cattle, providing strong evidence that adenoviral enteric vascular disease in cattle is associated with this serotype. The existence of BAV-10 has only recently been recognized. The first molecular biology-based technique for the diagnosis of BAV-10 infection is described. The animals in the present study are the first in which BAV-10 has had a confirmed role in a pathologic process. PMID:8727916

  16. CD46 Is a Cellular Receptor for All Species B Adenoviruses except Types 3 and 7

    PubMed Central

    Marttila, Marko; Persson, David; Gustafsson, Dan; Liszewski, M. Kathryn; Atkinson, John P.; Wadell, Göran; Arnberg, Niklas

    2005-01-01

    The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7. PMID:16254377

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

    SciTech Connect

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

    2013-05-25

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

  18. Transport of human adenoviruses in porous media

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  19. Delivery of avian cytokines by adenovirus vectors.

    PubMed

    Johnson, M A; Pooley, C; Lowenthal, J W

    2000-01-01

    A fowl adenovirus serotype 8 (FAV-8) recombinant was constructed by inserting an expression cassette consisting of the FAV major late promoter/splice leader sequences (MLP/SL), the chicken interferon-gamma (ChIFN-gamma) gene and SV40 polyA into sites in the right hand end of the FAV-8 genome. One recombinant (A3-13) was constructed by an insertion of ChIFN-gamma into a 1.3 kilobase pair (kbp) deletion which removed a putative open reading frame (ORF) with identity to the CELO (FAV serotype 1) 36 kDa homologue. A second recombinant (S4) removed a further 0.9 kbp and a third recombinant (AA1) was constructed in a small 50 base pair (bp) SpeI deletion. The recombinants displayed differing growth characteristics in CK monolayers. A3-13 grew slowly and only attained a titre of 10(5) pfu/ml, S4 had intermediate growth and AA1 showed wild type growth kinetics. These differing growth properties indicated that removal of the 36 kDa homologue had an effect on growth in vitro. Supernatants from CK monolayers infected with the recombinant virus were assayed for the production of ChIFN-gamma. Detectable levels of ChIFN-gamma were observed in supernatants as early as 24 h post infection (p.i.), peaked at 48 h p.i. and this level was maintained for at least 10 days. The level of production of ChIFN-gamma correlated with each recombinant's growth characteristics in vitro. Chickens treated with rFAV-ChIFN-gamma showed increased weight gains compared to controls and suffered reduced weight loss when challenged with the coccidial parasite Eimeria acervulina. PMID:10717297

  20. Adenovirus Respiratory Tract Infections in Peru

    PubMed Central

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

    2012-01-01

    Background Currently, there is a paucity of data regarding human adenovirus (HAdv) circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. Methods/Principal Findings Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI) or severe acute respiratory infection (SARI) were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. Conclusions/Significance HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness. PMID:23056519

  1. Adenovirus Dodecahedron, as a Drug Delivery Vector

    PubMed Central

    Zochowska, Monika; Paca, Agnieszka; Schoehn, Guy; Andrieu, Jean-Pierre; Chroboczek, Jadwiga; Dublet, Bernard; Szolajska, Ewa

    2009-01-01

    Background Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. Principal Findings Dodecahedron (Dd) structure is preserved at up to about 50°C at pH 7–8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37°C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. Conclusions/Significance Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs. PMID:19440379

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2013-05-25

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

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

    PubMed

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

    2015-11-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections. PMID:26342465

  5. Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults.

    PubMed

    Green, Christopher A; Scarselli, Elisa; Sande, Charles J; Thompson, Amber J; de Lara, Catherine M; Taylor, Kathryn S; Haworth, Kathryn; Del Sorbo, Mariarosaria; Angus, Brian; Siani, Loredana; Di Marco, Stefania; Traboni, Cinzia; Folgori, Antonella; Colloca, Stefano; Capone, Stefania; Vitelli, Alessandra; Cortese, Riccardo; Klenerman, Paul; Nicosia, Alfredo; Pollard, Andrew J

    2015-08-12

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. PMID:26268313

  6. Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults.

    PubMed

    Green, Christopher A; Scarselli, Elisa; Sande, Charles J; Thompson, Amber J; de Lara, Catherine M; Taylor, Kathryn S; Haworth, Kathryn; Del Sorbo, Mariarosaria; Angus, Brian; Siani, Loredana; Di Marco, Stefania; Traboni, Cinzia; Folgori, Antonella; Colloca, Stefano; Capone, Stefania; Vitelli, Alessandra; Cortese, Riccardo; Klenerman, Paul; Nicosia, Alfredo; Pollard, Andrew J

    2015-08-12

    Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease.

  7. Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

    PubMed

    Fernandes, P; Simão, D; Guerreiro, M R; Kremer, E J; Coroadinha, A S; Alves, P M

    2015-01-01

    Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

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

    PubMed

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

    2014-10-28

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

  9. Avian Retroviral Replication

    PubMed Central

    Justice, James; Beemon, Karen L.

    2013-01-01

    Avian retroviruses have undergone intense study since the beginning of the 20th century. They were originally identified as cancer-inducing filterable agents in chicken neoplasms. Since their discovery, the study of these simple retroviruses has contributed greatly to our understanding of retroviral replication and cancer. Avian retroviruses are continuing to evolve and have great economic importance in the poultry industry worldwide. The aim of this review is to provide a broad overview of the genome, pathology, and replication of avian retroviruses. Notable gaps in our current knowledge are highlighted, and areas where avian retroviruses differ from other retrovirus are also emphasized. PMID:24011707

  10. Replicated Composite Optics Development

    NASA Technical Reports Server (NTRS)

    Engelhaupt, Darell

    1997-01-01

    Advanced optical systems for applications such as grazing incidence Wolter I x-ray mirror assemblies require extraordinary mirror surfaces in ten-ns of fine surface finish and figure. The impeccable mirror surface is on the inside of the rotational mirror form. One practical method of producing devices with these requirements is to first fabricate an exterior surface for the optical device then replicate that surface to have the inverse component with lightweight characteristics. The replicate optic is not better than the master or mandrel from which it is made. This task is a continuance of previous studies to identify methods and materials for forming these extremely low roughness optical components.

  11. The Adenovirus L4-33K Protein Regulates both Late Gene Expression Patterns and Viral DNA Packaging

    PubMed Central

    Wu, Kai; Guimet, Diana

    2013-01-01

    The adenovirus (Ad) L4-33K protein has been linked to disparate functions during infection. L4-33K is a virus-encoded alternative RNA splicing factor which activates splicing of viral late gene transcripts that contain weak 3′ splice sites. Additionally, L4-33K has been indicated to play a role in adenovirus assembly. We generated and characterized an Ad5 L4-33K mutant virus to further explore its function(s) during infection. Infectivity, viral genome replication, and most viral gene expression of the L4-33K mutant virus are comparable to those of the wild-type virus, except for a prominent decrease in the levels of the late proteins IIIa and pVI. The L4-33K mutant virus produces only empty capsids, indicating a defect in viral DNA packaging. We demonstrate that L4-33K does not preferentially bind to viral packaging sequences in vivo, and mutation of L4-33K does not interfere with the binding of the known viral packaging proteins IVa2, L4-22K, L1-52/55K, and IIIa to the packaging sequences in vivo. Collectively, these results demonstrate that the phenotype of an Ad5 L4-33K mutant virus is complex. The L4-33K protein regulates the accumulation of selective Ad late gene mRNAs and is involved in the proper transition of gene expression during the late phase of infection. The L4-33K protein also plays a role in adenovirus morphogenesis by promoting the packaging of the viral genome into the empty capsid. These results demonstrate the multifunctional nature of the L4-33K protein and its involvement in several different and critical aspects of viral infection. PMID:23552425

  12. 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

  13. Improved orthopedic arm joint

    NASA Technical Reports Server (NTRS)

    Dane, D. H.

    1971-01-01

    Joint permits smooth and easy movement of disabled arm and is smaller, lighter and less expensive than previous models. Device is interchangeable and may be used on either arm at the shoulder or at the elbow.

  14. Reversing the arms race

    SciTech Connect

    von Hippel, F. ); Sagdeev, R.Z. )

    1992-01-01

    This paper contains proceedings of Reversing The Arms Race. Topics covered include: Verifying Reductions of Nuclear Warheads; Verifying Limits on Nuclear-Armed Cruise Missiles; and The Technical Basis for Warhead Detection.

  15. Arm Injuries and Disorders

    MedlinePlus

    ... of muscles, joints, tendons and other connective tissue. Injuries to any of these parts of the arm ... a fall or an accident. Types of arm injuries include Tendinitis and bursitis Sprains Dislocations Broken bones ...

  16. Replicated spectrographs in astronomy

    NASA Astrophysics Data System (ADS)

    Hill, Gary J.

    2014-06-01

    As telescope apertures increase, the challenge of scaling spectrographic astronomical instruments becomes acute. The next generation of extremely large telescopes (ELTs) strain the availability of glass blanks for optics and engineering to provide sufficient mechanical stability. While breaking the relationship between telescope diameter and instrument pupil size by adaptive optics is a clear path for small fields of view, survey instruments exploiting multiplex advantages will be pressed to find cost-effective solutions. In this review we argue that exploiting the full potential of ELTs will require the barrier of the cost and engineering difficulty of monolithic instruments to be broken by the use of large-scale replication of spectrographs. The first steps in this direction have already been taken with the soon to be commissioned MUSE and VIRUS instruments for the Very Large Telescope and the Hobby-Eberly Telescope, respectively. MUSE employs 24 spectrograph channels, while VIRUS has 150 channels. We compare the information gathering power of these replicated instruments with the present state of the art in more traditional spectrographs, and with instruments under development for ELTs. Design principles for replication are explored along with lessons learned, and we look forward to future technologies that could make massively-replicated instruments even more compelling.

  17. Human Mitochondrial DNA Replication

    PubMed Central

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  18. SIMS Replications in Ontario.

    ERIC Educational Resources Information Center

    Russell, Howard

    Replication of the Second International Mathematics Study (SIMS) in Ontario, Canada, is described and assessed. The curriculum and testing program covers numerical methods, geometry, and algebra. Whereas classical studies focused on mean scores of a system's students and on percentages of teachers for opportunity to learn (OTL), SIMS aggregates…

  19. Bioaccumulation of animal adenoviruses in the pink shrimp.

    PubMed

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

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems. PMID:26413052

  20. Bioaccumulation of animal adenoviruses in the pink shrimp.

    PubMed

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

    2015-01-01

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

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. Serologic and hexon phylogenetic analysis of ruminant adenoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Feduchi, E; Carrasco, L

    1987-04-01

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

  8. Bioaccumulation of animal adenoviruses in the pink shrimp

    PubMed Central

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

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems. PMID:26413052

  9. Isolation of adenovirus from lambs with upper respiratory syndrome.

    PubMed

    Pommer, J; Schamber, G

    1991-07-01

    The role of viruses in the etiology of recurrent upper respiratory disease in newly weaned lambs was studied during 1984-1985 at the North Dakota Sheep Experiment Station. Serum samples collected from lambs at weaning, from lambs with signs of respiratory disease, and 3 weeks following the onset of clinical signs were tested for antibodies to ovine adenovirus (OAV), respiratory syncytial virus (RSV), and parainfluenza type-3 virus (PI-3). Virus isolation studies were performed on nasal secretions samples taken at the same time. Parainfluenza type-3 was isolated from 1 of 275 lambs tested, and there was 2.5% overall 4-fold increase in antibody titer to PI-3 during the 2-year study. An adenovirus with a different restriction endonuclease digestion pattern from that previously reported adenovirus strains in the United States was isolated from 13 of 275 nasal secretions collected from lambs at the time of weaning. There was a 17.6% overall 4-fold increase in seroconversion to the adenovirus isolated from the lambs with clinical disease.

  10. TCLS Arm for Space

    NASA Astrophysics Data System (ADS)

    Leroy, Benoit; Helfers, Tim; Poupat, Jean-Luc

    2015-09-01

    The TCLS ARM FOR SPACE proposal was an answer to the H2020 topic “COMPET-6-2014: Bottom-up Space Technologies at low TRL”. This paper presents this H2020 TCLS ARM FOR SPACE initiative led by Airbus DS and which aims at fostering the use of European technology such as ARM processing for Space.

  11. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  12. Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses III. Base Composition, Molecular Weight, and Conformation of the Ad2+ND1 Genome

    PubMed Central

    Crumpacker, Clyde S.; Henry, Patrick H.; Kakefuda, Tuyoski; Rowe, Wallace P.; Levin, Myron J.; Lewis, Andrew M.

    1971-01-01

    The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2+ND1, differs from the defective Ad-SV40 hybrid populations previously described, in that this hybrid virus can replicate without the aid of nonhybrid adenovirus helper. Consequently, the deoxyribonucleic acid (DNA) from this virus, which can be obtained free of nonhybrid adenovirus DNA, is well suited for biophysical studies on Ad-SV40 hybrid DNA. Such studies have been performed and demonstrate Ad2+ND1 DNA to have a buoyant density (1.715 g/cm3) and thermal denaturation profile (Tm = 75.1 C) almost identical with nonhybrid Ad2 DNA. Furthermore, its molecular weight, as determined by analytical zone sedimentation and electron microscopy, was 22 × 106 to 25 × 106 daltons, which is also very similar to that determined for Ad2. Electron micrographs showed all of the hybrid molecules to be double-stranded and linear. By using this determination of the molecular weight of Ad2+ND1 DNA and assuming that 1% of this molecule consists of covalently linked SV40 DNA (see companion paper), we calculate that the hybrid DNA molecule contains 220 × 103 to 250 × 103 daltons of SV40 DNA, or the equivalent of one-tenth of the SV40 genome. PMID:4323710

  13. High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system.

    PubMed Central

    Alkhatib, G; Briedis, D J

    1988-01-01

    The entire measles virus (MV) hemagglutinin (HA)-coding region was reconstructed from cloned cDNAs and used as part of a hybrid transcription unit to replace a region of the adenovirus type 5 genome corresponding to the entire E1a transcription unit and most of the E1b transcription unit. The resulting recombinant virus was stable and able to replicate to high titers in 293 cells (which constitutively express the complementary E1a-E1b functions) in the absence of helper virus. During infection of 293 cells, the hybrid virus expressed MV HA protein which was indistinguishable from that expressed in MV-infected cells in terms of immunoreactivity, gel mobility, glycosylation, subcellular localization, and biologic activity. Infection of 293 cells with the hybrid virus led to high-level synthesis of the MV HA protein (equivalent to 65 to 130% of the level seen in MV-infected cells). At late times after high-multiplicity hybrid virus infection of HeLa and Vero cells (which do not express E1 functions), the level of HA protein synthesis was at least 35% of that seen in 293 cells. This MV-adenovirus recombinant will be useful in the study of the biologic properties of the MV HA protein and in assessment of the potential usefulness of hybrid adenoviruses as live-virus vaccine vectors. Images PMID:3292790

  14. Compartmentalization of prokaryotic DNA replication.

    PubMed

    Bravo, Alicia; Serrano-Heras, Gemma; Salas, Margarita

    2005-01-01

    It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

  15. [Preparation of Recombinant Human Adenoviruses Labeled with miniSOG].

    PubMed

    Zou, Xiaohui; Xiao, Rong; Guo, Xiaojuan; Qu, Jianguo; Lu, Zhuozhuang; Hong, Tao

    2016-01-01

    We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM. PMID:27295881

  16. Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

    PubMed

    Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

    2016-01-01

    Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage.

  17. Highly specific transgene expression mediated by a complex adenovirus vector incorporating a prostate-specific amplification feedback loop

    PubMed Central

    Woraratanadharm, Jan; Rubinchik, Semyon; Yu, Hong; Fan, Fan; Morrow, Scotty M.; Dong., John Y.

    2007-01-01

    Summary Development of novel therapeutic agents is needed to address the problems of locally recurrent, metastatic, and advanced hormone-refractory prostate cancer. We have constructed a novel complex adenovirus (Ad) vector regulation system that incorporates both the prostate-specific ARR2PB promoter and a positive feedback loop using the TRE promoter to enhance gene expression. This regulation strategy involves the incorporation of the TRE upstream of the prostate-specific ARR2PB promoter to enhance its activity with Tet-regulation. The expressions of both GFP and tTA were placed under the control of these TRE-ARR2PB promoters, so that in the cells of prostate origin, a positive feedback loop would be generated. This design greatly enhanced GFP reporter expression in prostate cancer cells, while retaining tight control of expression in non-prostate cancer cells, even at MOI as high as 1000. This novel positive feedback loop with prostate specificity (PFLPS) regulation system we have developed may have broad applications for expressing not only high levels of toxic proteins in cancer cells but alternatively could be manipulated to regulate essential genes in a highly efficient conditionally replicative adenovirus (CRAd) vector specifically directed to prostate cancer cells. The PFLPS regulation system, therefore, serves as a promising new approach in the development of both a specific and effective vector for cancer gene therapy. PMID:15229631

  18. Virus-neutralizing antibody titers against 8 avian adenovirus serotypes in breeder hens in Georgia by a microneutralization procedure.

    PubMed

    Grimes, T M; Culver, D H; King, D J

    1977-01-01

    Serums from 16 chicken-breeder flocks in Georgia were tested for virus-neutralizing antibody to 8 serotypes of avian adenovirus. Titers to all 8 serotypes were demonstrated in 8 of the flocks, titers to 7 in 6, and titers to fewer than 7 in the other 2 flocks. Although titers were high overall to some serotypes (types 2 and 8) and low to others (types 1, 4, and 5), with statistically significant differences between many titers, the data were difficult to interpret because of possible heterotypic responses of chickens infected with avian adeno-viruses. Used for titrations of serum antibody was a microneutralization procedure that is inexpensive. It proved also to provide reproducibility since titers from replicate tests differed by less than 3 twofold dilutions. In addition, there was a strong linear relation between titers obtained with the microneutralization procedure and a conventional plaque-assay titration procedure. The microneutralization procedure was less sensitive than the conventional procedure by a factor of about 5.5, but that was considered a disadvantage only with low-titered serums.

  19. An infection of human adenovirus 31 affects the differentiation of preadipocytes into fat cells, its metabolic profile and fat accumulation.

    PubMed

    Bil-Lula, Iwona; Krzywonos-Zawadzka, Anna; Sawicki, Grzegorz; Woźniak, Mieczysław

    2016-03-01

    The primary issue undertaken in this study was to test the hypothesis that preadipocytes would have intrinsically elevated propensity to differentiate into mature adipocytes due to HAdV31 infection. To prove that, the metabolic and molecular mechanisms responsible for HAdV31-induced adipogenesis were examined. 3T3L1 cells (mouse embryonic fibroblast, adipose like cell line) were used as a surrogate model to analyze an increased proliferation, differentiation, and maturation of preadipocytes infected with human adenovirus. An expression of E4orf1, C/EBP-β, PPAR-γ, GAPDH, aP2, LEP, and fatty acid synthase genes, intracellular lipid accumulation as well as cytokine release from the fat cells were assessed. Data showed that HAdV31 increased an expression of C/EBP-β and PPAR-γ genes leading to an enhanced differentiation of preadipocytes into fat cells. Besides, overexpression of GAPDH and fatty acid synthase, and decreased expression of leptin caused an increased accumulation of intracellular lipids. Secretion of TNF-α and IL-6 from HAdV31-infected cells was strongly decreased, leading to unlimited virus replication. The results obtained from this study provided the evidences that HAdV31, likewise previously documented HAdV36, is a subsequent human adenovirus affecting the differentiation and lipid accumulation of 3T3L1 cells.

  20. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

    PubMed Central

    Cristiano, R J; Smith, L C; Kay, M A; Brinkley, B R; Woo, S L

    1993-01-01

    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies. Images Fig. 2 Fig. 4 PMID:8265587

  1. Replicators, lineages, and interactors.

    PubMed

    Taylor, Daniel J; Bryson, Joanna J

    2014-06-01

    The target article argues that whole groups can act as interactors in an evolutionary process. We believe that Smaldino's discussion would be advanced by a more thorough analysis of the appropriate replicators and lineages for this model. We show that cultural evolution is necessarily a separate process from cultural group selection, and we also illustrate that the two processes may influence each other as demonstrated by an agent-based model of communicating food-processing skills. PMID:24970423

  2. A New Replicator: A theoretical framework for analysing replication

    PubMed Central

    2010-01-01

    Background Replicators are the crucial entities in evolution. The notion of a replicator, however, is far less exact than the weight of its importance. Without identifying and classifying multiplying entities exactly, their dynamics cannot be determined appropriately. Therefore, it is importance to decide the nature and characteristics of any multiplying entity, in a detailed and formal way. Results Replication is basically an autocatalytic process which enables us to rest on the notions of formal chemistry. This statement has major implications. Simple autocatalytic cycle intermediates are considered as non-informational replicators. A consequence of which is that any autocatalytically multiplying entity is a replicator, be it simple or overly complex (even nests). A stricter definition refers to entities which can inherit acquired changes (informational replicators). Simple autocatalytic molecules (and nests) are excluded from this group. However, in turn, any entity possessing copiable information is to be named a replicator, even multicellular organisms. In order to deal with the situation, an abstract, formal framework is presented, which allows the proper identification of various types of replicators. This sheds light on the old problem of the units and levels of selection and evolution. A hierarchical classification for the partition of the replicator-continuum is provided where specific replicators are nested within more general ones. The classification should be able to be successfully applied to known replicators and also to future candidates. Conclusion This paper redefines the concept of the replicator from a bottom-up theoretical approach. The formal definition and the abstract models presented can distinguish between among all possible replicator types, based on their quantity of variable and heritable information. This allows for the exact identification of various replicator types and their underlying dynamics. The most important claim is that

  3. Complexity transmission during replication

    PubMed Central

    Davis, Brian K.

    1979-01-01

    The transmission of complexity during DNA replication has been investigated to clarify the significance of this molecular property in a deterministic process. Complexity was equated with the amount of randomness within an ordered molecular structure and measured by the entropy of a posteriori probabilities for discrete (monomer sequences, atomic bonds) and continuous (torsion angle sequences) structural parameters in polynucleotides, proteins, and ligand molecules. A theoretical analysis revealed that sequence complexity decreases during transmission from DNA to protein. It was also found that sequence complexity limits the attainable complexity in the folding of a polypeptide chain and that a protein cannot interact with a ligand moiety of higher complexity. The analysis indicated, furthermore, that in any deterministic molecular process a cause possesses more complexity than its effect. This outcome broadly complies with Curie's symmetry principle. Results from an analysis of an extensive set of experimental data are presented; they corroborate these findings. It is suggested, therefore, that complexity governs the direction of order—order molecular transformations. Two biological implications are (i) replication of DNA in a stepwise, repetitive manner by a polymerase appears to be a necessary consequence of structural constraints imposed by complexity, and (ii) during evolution, increases in complexity had to involve a nondeterministic mechanism. This latter requirement apparently applied also to development of the first replicating system on earth. PMID:287070

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

    PubMed

    Ryu, Hodon; Cashdollar, Jennifer L; Fout, G Shay; Schrantz, Karen A; Hayes, Samuel

    2015-01-01

    Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm(-2). Two-day post-inoculation incubation period and a maximum spiking level of 10(5) MPN mL(-1) were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm(2) mJ(-1)), showed that an UV dose of 172 mJ cm(-2) achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies.

  5. Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

    PubMed Central

    Torres, J M; Alonso, C; Ortega, A; Mittal, S; Graham, F; Enjuanes, L

    1996-01-01

    The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals

  6. A capture enzyme-linked immunosorbent assay for virus infectivity titrations as exemplified in an adenovirus system.

    PubMed

    Everitt, E; Varga, M J

    1993-01-01

    An enzyme-linked immunosorbent assay (ELISA), employing a capturing antihexon monoclonal antibody specifically recognizing free hexons, was developed for quantitative infectivity titration of adenovirus in a microscale titration assay. The method is based on the quantitative assessment of the total excess production of the major structural protein late in infection in samples consisting of 10(5) virus-infected HeLa cells maintained as stationary suspension cultures. Results are obtained with a coefficient of variation of 10% within 50 hours after virus infection. The method was designed for monitoring substances interfering with viral replication, e.g., neutralizing antibodies or antiviral drugs. Since it measured the total antigen content associated with cells as well as antigens possibly released into the growth medium the general approach should be applicable to any viral system where a structural protein is synthesized in excess.

  7. Replication Research and Special Education

    ERIC Educational Resources Information Center

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications

    PubMed Central

    Uusi-Kerttula, Hanni; Hulin-Curtis, Sarah; Davies, James; Parker, Alan L.

    2015-01-01

    Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies. PMID:26610547

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2013-10-01

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

  13. 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

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

    PubMed Central

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

    2007-01-01

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

  15. 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.

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

    PubMed

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

    2016-07-01

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

  17. The arms race

    SciTech Connect

    Sheehan, M.

    1983-01-01

    This book presents a comprehensive examination of the nature of the contemporary arms race, the forces that encourage arms competition, and the means by which these forces can be controlled. The author provides analyses of such specific issues as the viability of arms control agreements; the possibilities for nuclear disarmament; the means of deterrence, detection, and defense; and the methods of destruction themselves - nuclear, conventional, chemical, and space weapons.

  18. Radial arm strike rail

    DOEpatents

    McKeown, Mark H.; Beason, Steven C.

    1991-01-01

    The radial arm strike rail assembly is a system for measurement of bearings, directions, and stereophotography for geologic mapping, particularly where magnetic compasses are not appropriate. The radial arm, pivoting around a shaft axis, provides a reference direction determination for geologic mapping and bearing or direction determination. The centerable and levelable pedestal provide a base for the radial arm strike rail and the telescoping camera pedestal. The telescoping feature of the radial arm strike rail allows positioning the end of the rail for strike direction or bearing measurement with a goniometer.

  19. Oncolytic adenovirus-mediated therapy for prostate cancer.

    PubMed

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

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

  20. Oncolytic adenovirus-mediated therapy for prostate cancer

    PubMed Central

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

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

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

    PubMed

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

    2010-01-01

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

  2. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

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

    2015-01-01

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

  3. 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.

  4. Cis and trans activation of adenovirus IVa2 gene transcription.

    PubMed Central

    Natarajan, V; Salzman, N P

    1985-01-01

    The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter in front of a gene coding for bacterial chloramphenicol acetyl transferase (CATase) and estimating levels of CATase and IVa2 promoter specific RNA synthesized after transfection. To produce detectable amounts of CATase with the IVa2 promoter, an enhancer has to be present in cis. In the absence of enhancer sequences, the adenovirus E1A gene can not stimulate CATase synthesis. When cells were transfected with plasmids containing enhancer sequences and various IVa2 mutant promoters upstream of the CAT gene, we observed that CATase activity was not reduced significantly even after deletion of all sequences upstream of the RNA initiation site. Synthesis of IVa2 specific RNA was dependent on plasmids containing an enhancer (SV40 72 bp repeat) that was present in cis. In the absence of enhancer sequences, co-transfection to provide the adenovirus E1A gene in trans also stimulated IVa2 RNA synthesis. When HeLa cells were transfected with various deletion mutants with an enhancer in cis it was seen that sequences -38 to -64 base pairs upstream of the RNA initiation site are necessary for efficient transcription. The E1A gene in trans and an enhancer in cis have an additive effect on RNA synthesis from both IVa2 and major late promoters. The basis for the conflicting results between transcription and CATase synthesis is discussed. Images PMID:2989786

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

    PubMed

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

    2013-08-30

    An outbreak of fatal fulminant hepatitis at a Japanese aquarium involved 3 otariids: a California sea lion (Zalophus californianus), a South African fur seal (Arctocephalus pusillus) and a South American sea lion (Otaria flavescens). In a span of about a week in February 2012, 3 otariids showed diarrhea and were acutely low-spirited; subsequently, all three animals died within a period of 3 days. Markedly increased aspartate amino transferase and alanine amino transferase activities were observed. Necrotic hepatitis and eosinophilic intranuclear inclusion bodies in liver hepatocytes and intestinal epithelial cells were observed in the South American sea lion on histological examination. Otarine adenovirus DNA was detected from the livers of all three animals by polymerase chain reaction and determination of the sequences showed that all were identical. These results suggest that a single otarine adenovirus strain may have been the etiological agent of this outbreak of fatal fulminant hepatitis among the different otariid species, and it may be a lethal threat to wild and captive otariids. This is the first evidence of an outbreak of lethal adenovirus infection among different otariid species. PMID:23643878

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

    PubMed

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

    2006-07-19

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

  7. Structural Analysis of Adenovirus VAI RNA Defines the Mechanism of Inhibition of PKR

    PubMed Central

    Launer-Felty, Katherine; Wong, C. Jason; Cole, James L.

    2015-01-01

    Protein kinase R (PKR) is activated by dsRNA produced during virus replication and plays a major role in the innate immunity response to virus infection. In response, viruses have evolved multiple strategies to evade PKR. Adenovirus virus-associated RNA-I (VAI) is a short, noncoding transcript that functions as an RNA decoy to sequester PKR in an inactive state. VAI consists of an apical stem-loop, a highly structured central domain, and a terminal stem. Chemical probing and mutagenesis demonstrate that the central domain is stabilized by a pseudoknot. A structural model of VAI was obtained from constraints derived from chemical probing and small angle x-ray scattering (SAXS) measurements. VAI adopts a flat, extended conformation with the apical and terminal stems emanating from a protuberance in the center. This model reveals how the apical stem and central domain assemble to produce an extended duplex that is precisely tuned to bind a single PKR monomer with high affinity, thereby inhibiting activation of PKR by viral dsRNA. PMID:25650941

  8. [Morphogenetic study of human adenovirus type 41 in 293TE cells].

    PubMed

    Song, Jing-Dong; Wang, Min; Zou, Xiao-Hui; Qu, Jian-Guo; Lu, Zhuo-Zhuang; Hong, Tao

    2014-03-01

    To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41. PMID:24923169

  9. Extended Protection against Phlebovirus Infection Conferred by Recombinant Adenovirus Expressing Consensus Interferon (DEF201)

    PubMed Central

    Ennis, Jane; Sefing, Eric J.; Wong, Min-Hui; Jung, Kie-Hoon; Turner, Jeffrey D.

    2012-01-01

    Punta Toro virus (PTV; Bunyaviridae, Phlebovirus) is related to Rift Valley fever virus (RVFV), a pathogenic agent which causes severe disease in humans and livestock primarily in the sub-Saharan region of Africa. The recent range expansion of RVFV and the potential for its intentional release into naïve populations pose a significant threat to public health and agriculture. Studies modeling disease in rodents and nonhuman primates have shown that PTV and RVFV are highly sensitive to the antiviral effects of alpha interferon (IFN-α), an important component of the innate antiviral host response. While recombinant IFN-α has high therapeutic value, its utility for the treatment of neglected tropical diseases is hindered by its short in vivo half-life and costly production of longer-lasting pegylated IFNs. Here, we demonstrate extended preexposure protection against lethal PTV challenge following a single intranasal administration of DEF201, which is a replication-deficient human adenovirus type 5 vector engineered to constitutively express consensus IFN-α (cIFN-α) from transduced host cells. DEF201 was also efficacious when administered within 24 h as a postexposure countermeasure. Serum concentrations of cIFN-α could be detected as early as 8 h following treatment and persisted for more than 1 week. The prolonged antiphlebovirus prophylactic effect, low production costs, and ease of administration make DEF201 a promising agent for intervention during natural disease outbreaks and for countering possible bioterrorist acts. PMID:22615273

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

    PubMed

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

    2015-11-27

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

  11. Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

    SciTech Connect

    Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian

    2007-10-05

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10{sup 8} PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization.

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

    PubMed

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

    2008-03-01

    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients. PMID:18205221

  13. Adenovirus carrying gene encoding Haliotis discus discus sialic acid binding lectin induces cancer cell apoptosis.

    PubMed

    Yang, Xinyan; Wu, Liqin; Duan, Xuemei; Cui, Lianzhen; Luo, Jingjing; Li, Gongchu

    2014-06-30

    Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.

  14. 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. PMID:23291475

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

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

    2015-01-01

    Hepatocellular carcinoma (HCC) is a highly malignant disease, and its outcome of routine therapies is poor. Comprehensive treatment including gene therapy is an important way to improve patients’ prognosis and survival. In this study, we successfully constructed a triple-controlled cancer-selective oncolytic adenovirus, QG511-HA-Melittin, carrying melittin gene, in which the hybrid promoter, hypoxia-response element (HRE)-AFP promoter, was used to control viral E1a expression targeting AFP-positive cancer cells in hypoxia microenviroment, and the E1b-55 kDa gene was deleted in cancer cells with p53-deficiency. The cytological experiments found that the viral replication of QG511-HA-Melittin was increased to 12800-folds in Hep3B cells within 48 h, and 130-folds in SMMC-7721, but the virus did not replicate in L-02 cells. QG511-HA-Melittin had a strong inhibition effect on AFP-positive HCC cell proliferation, such as Hep3B and HepG2, whereas, there was low or no inhibition effect of QG511-HA-Melittin on AFP-negative cancer cells SMMC-7721 and normal cells L-02. In the in vivo experiment, compared with the blank control group, QG511-HA-Melittin can significantly inhibit the growth of HCC xenografts (P<0.05). The survival of mice in QG511-HA-Melittin group was much longer than that of the blank control group. Both in vitro and in vivo experiments manifested that QG511-HA-Melittin exerts an inhibitory effect on HCC cells, which may provide a new strategy for HCC biotherapy. PMID:26617748

  17. Identification of an origin of bidirectional DNA replication in mammalian chromosomes.

    PubMed

    Burhans, W C; Vassilev, L T; Caddle, M S; Heintz, N H; DePamphilis, M L

    1990-09-01

    Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.

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

    PubMed Central

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

    2013-01-01

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

  19. Quantum Replicator Dynamics

    NASA Astrophysics Data System (ADS)

    Guevara Hidalgo, Esteban

    2006-09-01

    We propose quantization relationships which would let us describe and solution problems originated by conflicting or cooperative behaviors among the members of a system from the point of view of quantum mechanical interactions. The quantum version of the replicator dynamics is the equation of evolution of mixed states from quantum statistical mechanics. A system and all its members will cooperate and rearrange its states to improve their present condition. They strive to reach the best possible state for each of them which is also the best possible state for the whole system. This led us to propose a quantum equilibrium in which a system is stable only if it maximizes the welfare of the collective above the welfare of the individual. If it is maximized the welfare of the individual above the welfare of the collective the system gets unstable and eventually it collapses.

  20. Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins.

    PubMed Central

    Harter, M L; Lewis, J B; Anderson, C W

    1979-01-01

    The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon. Images PMID:513195

  1. SUMO and KSHV Replication

    PubMed Central

    Chang, Pei-Ching; Kung, Hsing-Jien

    2014-01-01

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis. PMID:25268162

  2. SUMO and KSHV Replication.

    PubMed

    Chang, Pei-Ching; Kung, Hsing-Jien

    2014-01-01

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi's sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV's life cycle and pathogenesis. PMID:25268162

  3. ARM Mentor Selection Process

    SciTech Connect

    Sisterson, D. L.

    2015-10-01

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

  4. Supercoiling, knotting and replication fork reversal in partially replicated plasmids

    PubMed Central

    Olavarrieta, L.; Martínez-Robles, M. L.; Sogo, J. M.; Stasiak, A.; Hernández, P.; Krimer, D. B.; Schvartzman, J. B.

    2002-01-01

    To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter–TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive ΔLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. PMID:11809877

  5. Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector.

    PubMed

    Shayakhmetov, D M; Papayannopoulou, T; Stamatoyannopoulos, G; Lieber, A

    2000-03-01

    Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

  6. Crystal Structure of the Fibre Head Domain of the Atadenovirus Snake Adenovirus 1

    PubMed Central

    Singh, Abhimanyu K.; Menéndez-Conejero, Rosa; San Martín, Carmen; van Raaij, Mark J.

    2014-01-01

    Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest. PMID:25486282

  7. Hemorrhagic enteritis by adenovirus-like particles in turkeys: a possible pathogenic mechanism.

    PubMed

    Gómez-Villamandos, J C; Carranza, J; Sierra, M A; Carrasco, L; Hervás, J; Blanco, A; Fernández, A

    1994-01-01

    This paper describes an outbreak of hemorrhagic enteritis due to adenovirus in turkeys in Spain. Diagnosis of the disease was confirmed by histopathological examination and the observation of adenovirus in spleen mononuclear cells and intestinal infiltrate. Evidence was also found of intravascular coagulation, which may give rise to the bleeding considered characteristic of this disease.

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

    PubMed

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

    2015-02-01

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

  9. 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. PMID:26926035

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

    PubMed

    Hara, Michimaru; Takao, Shinichi; Shimazu, Yukie

    2015-06-01

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

  11. KSHV Genome Replication and Maintenance

    PubMed Central

    Purushothaman, Pravinkumar; Dabral, Prerna; Gupta, Namrata; Sarkar, Roni; Verma, Subhash C.

    2016-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is a major etiological agent for multiple severe malignancies in immune-compromised patients. KSHV establishes lifetime persistence in the infected individuals and displays two distinct life cycles, generally a prolonged passive latent, and a short productive or lytic cycle. During latent phase, the viral episome is tethered to the host chromosome and replicates once during every cell division. Latency-associated nuclear antigen (LANA) is a predominant multifunctional nuclear protein expressed during latency, which plays a central role in episome tethering, replication and perpetual segregation of the episomes during cell division. LANA binds cooperatively to LANA binding sites (LBS) within the terminal repeat (TR) region of the viral episome as well as to the cellular nucleosomal proteins to tether viral episome to the host chromosome. LANA has been shown to modulate multiple cellular signaling pathways and recruits various cellular proteins such as chromatin modifying enzymes, replication factors, transcription factors, and cellular mitotic framework to maintain a successful latent infection. Although, many other regions within the KSHV genome can initiate replication, KSHV TR is important for latent DNA replication and possible segregation of the replicated episomes. Binding of LANA to LBS favors the recruitment of various replication factors to initiate LANA dependent DNA replication. In this review, we discuss the molecular mechanisms relevant to KSHV genome replication, segregation, and maintenance of latency. PMID:26870016

  12. The asymmetry of telomere replication contributes to replicative senescence heterogeneity.

    PubMed

    Bourgeron, Thibault; Xu, Zhou; Doumic, Marie; Teixeira, Maria Teresa

    2015-10-15

    In eukaryotes, the absence of telomerase results in telomere shortening, eventually leading to replicative senescence, an arrested state that prevents further cell divisions. While replicative senescence is mainly controlled by telomere length, the heterogeneity of its onset is not well understood. This study proposes a mathematical model based on the molecular mechanisms of telomere replication and shortening to decipher the causes of this heterogeneity. Using simulations fitted on experimental data obtained from individual lineages of senescent Saccharomyces cerevisiae cells, we decompose the sources of senescence heterogeneity into interclonal and intraclonal components, and show that the latter is based on the asymmetry of the telomere replication mechanism. We also evidence telomere rank-switching events with distinct frequencies in short-lived versus long-lived lineages, revealing that telomere shortening dynamics display important variations. Thus, the intrinsic heterogeneity of replicative senescence and its consequences find their roots in the asymmetric structure of telomeres.

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

    PubMed

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

    2012-01-01

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

  14. Nonspecific Arm Pain

    PubMed Central

    Moradi, Ali; Ebrahimzadeh, Mohammad H; Ring, David

    2013-01-01

    Nonspecific activity-related arm pain is characterized by an absence of objective physical findings and symptoms that do not correspond with objective pathophysiology. Arm pain without strict diagnosis is often related to activity, work-related activity in particular, and is often seen in patients with physically demanding work. Psychological factors such as catastrophic thinking, symptoms of depression, and heightened illness concern determine a substantial percentage of the disability associated with puzzling hand and arm pains. Ergonomic modifications can help to control symptoms, but optimal health may require collaborative management incorporating psychosocial and psychological elements of illness. PMID:25207288

  15. Charter School Replication. Policy Guide

    ERIC Educational Resources Information Center

    Rhim, Lauren Morando

    2009-01-01

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

  16. Kinematically redundant arm formulations for coordinated multiple arm implementations

    NASA Technical Reports Server (NTRS)

    Bailey, Robert W.; Quiocho, Leslie J.; Cleghorn, Timothy F.

    1990-01-01

    Although control laws for kinematically redundant robotic arms were presented as early as 1969, redundant arms have only recently become recognized as viable solutions to limitations inherent to kinematically sufficient arms. The advantages of run-time control optimization and arm reconfiguration are becoming increasingly attractive as the complexity and criticality of robotic systems continues to progress. A generalized control law for a spatial arm with 7 or more degrees of freedom (DOF) based on Whitney's resolved rate formulation is given. Results from a simulation implementation utilizing this control law are presented. Furthermore, results from a two arm simulation are presented to demonstrate the coordinated control of multiple arms using this formulation.

  17. Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

    PubMed Central

    Grodzicker, Terri; Anderson, Carl; Sharp, Phillip A.; Sambrook, Joe

    1974-01-01

    Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2+ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2+ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2+ND1 either by the cleavage patterns produced by Escherichia coli R·RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2+ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2+ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2+ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40. Images PMID:4364898

  18. 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

  19. Interactions of human lacrimal and salivary cystatins with adenovirus endopeptidase.

    PubMed

    Ruzindana-Umunyana, A; Weber, J M

    2001-09-01

    Over 100 serotypes of adenoviruses have been implicated in a variety of human and domesticated animal pathologies and some serotypes are widely used as gene transfer vectors. Aside from the limited use of vaccines for specific serotypes, little effort has been expended in the development of antivirals. The objective here was to study the effect of cystatins from human saliva (CS) and tears (CT), two points of viral entry, on adenain, the adenovirus type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both fluids at a yield of 5 mg/l. In vitro adenain activity was inhibited to 50% at a molar ratio of 5 CS:1 adenain and 3 CT:1 adenain. By comparison, papain was inhibited to 50% at a molar ratio of 2 CS:1 papain and 1.5 CT:1 papain. Adenain differed from papain in response to CS and chicken egg white (CEW) cystatin in being stimulated at low concentrations, and in being inhibited only at very high concentrations of cystatins. The presence of cleavage consensus sites specific to adenain in the human cystatins could drive the adenain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denaturation and by highly active fresh enzyme preparations. Our experiments designed to test the nature of the interaction between adenain and cystatins suggest a docking model for the adenain-human cystatin interaction, similar to that proposed for papain and CEW. At equilibrium the dissociation constant, K(d), between adenain and CT was 1.2 nM. The kinetic parameters determined here suggest a simple reversible mechanism for the inhibition of adenain by human cystatins. We conclude that the cystatins present in tears and saliva are unlikely to play a significant role in inhibiting adenovirus infections.

  20. Characterization of a new species of adenovirus in falcons.

    PubMed

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

    2005-07-01

    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 x 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.

  1. Regulating DNA Replication in Plants

    PubMed Central

    Sanchez, Maria de la Paz; Costas, Celina; Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2012-01-01

    Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed. PMID:23209151

  2. Transductional targeting of adenovirus vectors for gene therapy

    PubMed Central

    Glasgow, JN; Everts, M; Curiel, DT

    2007-01-01

    Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions. PMID:16439993

  3. Total arm flap.

    PubMed

    Becker, D W

    1987-11-01

    The development of an unusual and rarely indicated total arm flap is described in the context of widely indicated and automatically used principles passed down by the recognized father of plastic surgery, Sir Harold G. Gillies.

  4. An elastica arm scale.

    PubMed

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

    2014-09-01

    The concept of a 'deformable arm scale' (completely different from a traditional rigid arm balance) is theoretically introduced and experimentally validated. The idea is not intuitive, but is the result of nonlinear equilibrium kinematics of rods inducing configurational forces, so that deflection of the arms becomes necessary for equilibrium, which would be impossible for a rigid system. In particular, the rigid arms of usual scales are replaced by a flexible elastic lamina, free to slide in a frictionless and inclined sliding sleeve, which can reach a unique equilibrium configuration when two vertical dead loads are applied. Prototypes designed to demonstrate the feasibility of the system show a high accuracy in the measurement of load within a certain range of use. Finally, we show that the presented results are strongly related to snaking of confined beams, with implications for locomotion of serpents, plumbing and smart oil drilling. PMID:25197248

  5. Bruising Hands and Arms

    MedlinePlus

    ... and arms is common. Dermatologists call it 'actinic purpura', 'solar purpura' or 'Bateman's purpura'. These flat blotches start out red, then turn ... flimsy looking. Mostly seen in older individuals, actinic purpura is due to the weakened state of blood ...

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

    PubMed Central

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

    2015-01-01

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

  7. Intratumoral spread of wild-type adenovirus is limited after local injection of human xenograft tumors: virus persists and spreads systemically at late time points.

    PubMed

    Sauthoff, Harald; Hu, Jing; Maca, Cielo; Goldman, Michael; Heitner, Sheila; Yee, Herman; Pipiya, Teona; Rom, William N; Hay, John G

    2003-03-20

    Oncolytic replicating adenoviruses are a promising new modality for the treatment of cancer. Despite the assumed biologic advantage of continued viral replication and spread from infected to uninfected cancer cells, early clinical trials demonstrate that the efficacy of current vectors is limited. In xenograft tumor models using immune-incompetent mice, wild-type adenovirus is also rarely able to eradicate established tumors. This suggests that innate immune mechanisms may clear the virus or that barriers within the tumor prevent viral spread. The aim of this study was to evaluate the kinetics of viral distribution and spread after intratumoral injection of virus in a human tumor xenograft model. After intratumoral injection of wild-type virus, high levels of titratable virus persisted within the xenograft tumors for at least 8 weeks. Virus distribution within the tumors as determined by immunohistochemistry was patchy, and virus-infected cells appeared to be flanked by tumor necrosis and connective tissue. The close proximity of virus-infected cells to the tumor-supporting structure, which is of murine origin, was clearly demonstrated using a DNA probe that specifically hybridizes to the B1 murine DNA repeat. Importantly, although virus was cleared from the circulation 6 hr after intratumoral injection, after 4 weeks systemic spread of virus was detected. In addition, vessels of infected tumors were surrounded by necrosis and an advancing rim of virus-infected tumor cells, suggesting reinfection of the xenograft tumor through the vasculature. These data suggest that human adenoviral spread within tumor xenografts is impaired by murine tumor-supporting structures. In addition, there is evidence for continued viral replication within the tumor, with subsequent systemic dissemination and reinfection of tumors via the tumor vasculature. Despite the limitations of immune-incompetent models, an understanding of the interactions between the virus and the tumor

  8. ARM for Platform Application

    NASA Astrophysics Data System (ADS)

    Patte, Mathieu; Poupat, Jean-Luc; Le Meur, Patrick

    2015-09-01

    The activities described in this paper are part of the CNES R&T “Study of a Cortex-R ARM based architecture” performed by Airbus DS Space System & Electronics in 2014. With the support of CNES, Airbus DS has performed the porting of a representative space application software on an ARM based demonstration platform. This paper presents the platform itself, the activities performed at software level and the first results on this evaluation study.

  9. Hello to Arms

    NASA Technical Reports Server (NTRS)

    2005-01-01

    This image highlights the hidden spiral arms (blue) that were discovered around the nearby galaxy NGC 4625 by the ultraviolet eyes of NASA's Galaxy Evolution Explorer.

    The image is composed of ultraviolet and visible-light data, from the Galaxy Evolution Explorer and the California Institute of Technology's Digitized Sky Survey, respectively. Near-ultraviolet light is colored green; far-ultraviolet light is colored blue; and optical light is colored red.

    As the image demonstrates, the lengthy spiral arms are nearly invisible when viewed in optical light while bright in ultraviolet. This is because they are bustling with hot, newborn stars that radiate primarily ultraviolet light.

    The youthful arms are also very long, stretching out to a distance four times the size of the galaxy's core. They are part of the largest ultraviolet galactic disk discovered so far.

    Located 31 million light-years away in the constellation Canes Venatici, NGC 4625 is the closest galaxy ever seen with such a young halo of arms. It is slightly smaller than our Milky Way, both in size and mass. However, the fact that this galaxy's disk is forming stars very actively suggests that it might evolve into a more massive and mature galaxy resembling our own.

    The armless companion galaxy seen below NGC 4625 is called NGC 4618. Astronomers do not know why it lacks arms but speculate that it may have triggered the development of arms in NGC 4625.

  10. Noninvasive Monitoring of mRFP1- and mCherry-Labeled Oncolytic Adenoviruses in an Orthotopic Breast Cancer Model by Spectral Imaging

    PubMed Central

    Borovjagin, Anton V.; McNally, Lacey R.; Wang, Minghui; Curiel, David T.; MacDougall, Mary J.; Zinn, Kurt R.

    2010-01-01

    Genetic capsid labeling of conditionally replicative adenoviruses (CRAds) with fluorescent tags offers a potentially more accurate monitoring of those virotherapy agents in vivo. The capsid of an infectivity-enhanced CRAd, Ad5/3, delta 24, was genetically labeled with monomeric red fluorescent protein 1 (mRFP1) or its advanced derivative, “mCherry,” to evaluate the utility of each red fluorescent reporter and the benefit of CRAd capsid labeling for noninvasive virus tracking in animal tumor models by a new spectral imaging approach. Either reporter was incorporated into the CRAd particles by genetic fusion to the viral capsid protein IX. Following intratumoral injection, localization and replication of each virus in orthotopic breast cancer xenografts were analyzed by spectral imaging and verified by quantitative polymerase chain reaction. Fluorescence in tumors increased up to 2,000-fold by day 4 and persisted for 5 to 7 weeks, showing oscillatory dynamics reflective of CRAd replication cycles. Capsid labeling in conjunction with spectral imaging thus enables direct visualization and quantification of CRAd particles in tumors prior to the reporter transgene expression. This allows for noninvasive control of CRAd delivery and distribution in tumors and facilitates quantitative assessment of viral replication. Although mCherry appeared to be superior to mRFP1 as an imaging tag, both reporters showed utility for CRAd imaging applications. PMID:20236604

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

    PubMed Central

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

    2015-01-01

    In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model. PMID:26029188

  12. Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers.

    PubMed

    Grafl, Beatrice; Prokofieva, Irina; Wernsdorf, Patricia; Steinborn, Ralf; Hess, Michael

    2014-08-01

    Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

  13. Biology of E1-Deleted Adenovirus Vectors in Nonhuman Primate Muscle

    PubMed Central

    Zoltick, Philip W.; Chirmule, Narendra; Schnell, Michael A.; Gao, Guang-ping; Hughes, Joseph V.; Wilson, James M.

    2001-01-01

    Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors. PMID:11333904

  14. 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.

  15. Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

    PubMed Central

    Stewart, P L; Chiu, C Y; Huang, S; Muir, T; Zhao, Y; Chait, B; Mathias, P; Nemerow, G R

    1997-01-01

    Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies. PMID:9135136

  16. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex.

    PubMed

    Querido, E; Blanchette, P; Yan, Q; Kamura, T; Morrison, M; Boivin, D; Kaelin, W G; Conaway, R C; Conaway, J W; Branton, P E

    2001-12-01

    Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1-Cul1/Cdc53-F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53. PMID:11731475

  17. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex

    PubMed Central

    Querido, Emmanuelle; Blanchette, Paola; Yan, Qin; Kamura, Takumi; Morrison, Megan; Boivin, Dominique; Kaelin, William G.; Conaway, Ronald C.; Conaway, Joan Weliky; Branton, Philip E.

    2001-01-01

    Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1–Cul1/Cdc53–F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53. PMID:11731475

  18. 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.

  19. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

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

    2015-08-15

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

  20. Purification of a native membrane-associated adenovirus tumor antigen.

    PubMed Central

    Persson, H; Katze, M G; Philipson, L

    1982-01-01

    A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro. Images PMID:7097863

  1. Detection of adenovirus using PCR and molecular beacon.

    PubMed

    Poddar, S K

    1999-09-01

    The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.

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

    PubMed

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

    2015-12-29

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

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

    PubMed Central

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

    2009-01-01

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

  4. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  6. Adenovirus-based vaccine against Listeria monocytogenes: extending the concept of invariant chain linkage.

    PubMed

    Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Schlüter, Dirk; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2013-10-15

    The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further, vaccination of C57BL/6 (L. monocytogenes-resistant) and BALB/c (L. monocytogenes-susceptible) mice with adenoviral vectors encoding natural L. monocytogenes-derived soluble Ags (listeriolysin O and p60) revealed that tethering of these Ags to Ii markedly improved the vaccine-induced CD8(+) T cell response to two of three epitopes studied. More importantly, Ii linkage accelerated and augmented vaccine-induced protection in both mouse strains and prolonged protection, in particular that induced by the weak Ag, p60, in L. monocytogenes-susceptible BALB/c mice.

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  8. Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients

    PubMed Central

    2014-01-01

    Background Alpha fetoprotein (AFP) is an oncofetal antigen over-expressed by many hepatocellular cancers (HCC). We previously demonstrated that HLA-A2-restricted epitopes derived from AFP are immunogenic in vitro and in vivo despite high circulating levels of this oncofetal antigen. In order to test a more broadly applicable, HLA-unrestricted, inexpensive, cell-free vaccine platform capable of activating tumor antigen-specific CD8+ and CD4+ T cells, we tested full length AFP in a plasmid DNA construct in combination with an AFP-expressing replication-deficient adenovirus (AdV) in a prime-boost vaccine strategy. Methods HCC patients who had an AFP+ tumor and previous treatment for HCC were screened and two patients received vaccination with three plasmid DNA injections followed by a single AdV injection, all delivered intramuscularly (i.m.). Results The vaccine was well tolerated and safe. Both patients showed immunologic evidence of immunization. The first patient had a weak AFP-specific T cell response, a strong AdV-specific cellular response and recurred with an AFP-expressing HCC at nine months. The second patient developed a strong AFP-specific CD8+ and CD4+ cellular response and an AdV neutralizing antibody response, and recurred at 18 months without an increase in serum AFP. Conclusions The AFP DNA prime-AdV boost vaccine was safe and immunogenic. Circulating anti-AdV neutralizing antibodies at baseline did not prohibit the development of AFP-specific cellular immunity. The patient who developed CD8+ and CD4+ AFP-specific T cell immunity had more favorable progression-free survival. The observations with these two patients support development of this vaccine strategy in a larger clinical trial. Trial registration ClinicalTrials.gov: NCT00093548 PMID:24708667

  9. Phoenix Stretches its Arm

    NASA Technical Reports Server (NTRS)

    2008-01-01

    [figure removed for brevity, see original site] Click on image for animation

    The Phoenix spacecraft is scheduled to begin raising its robotic arm up and out of its stowed configuration on the third Martian day, or Sol 3 (May 28, 2008) of the mission. This artist's animation, based on engineering models, shows how Phoenix will accomplish this task. First, its wrist actuator will rotate, releasing its launch-restraint pin. Next, the forearm moves up, releasing the elbow launch-restraint pin. The elbow will then move up and over in small steps, a process referred to as 'staircasing.' This ensures that the arm's protective biobarrier wrap, now unpeeled and lying to the side of the arm, will not get in the way of the arm's deployment.

    The arm is scheduled to straighten all the way out on Sol 4 (May 29, 2008), after engineers have reviewed images and telemetry data from the spacecraft showing that the biobarrier material has been cleared.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  10. Pediatric Arm Function Test

    PubMed Central

    Uswatte, Gitendra; Taub, Edward; Griffin, Angi; Rowe, Jan; Vogtle, Laura; Barman, Joydip

    2012-01-01

    Objective Although there are several validated upper-extremity measures in young children with cerebral palsy (CP), none primarily assess capacity to carry out actions and tasks with the more-affected arm. To address this need, we developed the Pediatric Arm Function Test (PAFT), which involves behavioral observation of how children use their more-affected arm during structured play in the laboratory or clinic. This paper evaluates the reliability and validity of the PAFT Functional Ability scale. Design In Study 1, 20 children between 2–8 years with a wide range of upper-extremity hemiparesis due to CP completed the PAFT on two occasions separated by three weeks. In Study 2, 41 children between 2–6 years with similar characteristics completed the PAFT and received a grade reflecting severity of more-affected arm motor impairment. Results In Study 1, the PAFT test-retest reliability correlation coefficient was 0.74. In Study 2, convergent validity was supported by a strong, inverse correlation (r = −0.6, p < .001) between PAFT scores and grade of impairment. Conclusions The PAFT Functional Ability scale is a reliable and valid measure of more-affected arm motor capacity in children with CP between 2–6 years. It can be employed to measure upper-extremity neurorehabilitation outcome. PMID:23103486

  11. Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading.

    PubMed

    Brüning, Jan-Gert; Myka, Kamila Katarzyna; McGlynn, Peter

    2016-03-27

    Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated

  12. Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading

    PubMed Central

    Brüning, Jan-Gert; Myka, Kamila Katarzyna; McGlynn, Peter

    2016-01-01

    Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6–DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6–DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6–DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6–DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096–22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825–831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its

  13. The Biology of Replicative Senescence

    SciTech Connect

    Campisi, J.

    1996-12-04

    Most cells cannot divide indefinitely due to a processtermed cellular or replicative senescence. Replicative senescence appearsto be a fundamental feature of somatic cells, with the exception of mosttumour cells and possibly certain stem cells. How do cells sense thenumber of divisions they have completed? Although it has not yet beencritically tested, the telomere shortening hypothesis is currentlyperhaps the best explanation for a cell division 'counting' mechanism.Why do cells irreversibly cease proliferation after completing a finitenumber of divisions? It is now known that replicative senescence altersthe expression of a few crucial growth-regulatory genes. It is not knownhow these changes in growth-regulatory gene expression are related totelomere shortening in higher eukaryotes. However, lower eukaryotes haveprovided several plausible mechanisms. Finally, what are thephysiological consequences of replicative senescence? Several lines ofevidence suggest that, at least in human cells, replicative senescence isa powerful tumour suppressive mechanism. There is also indirect evidencethat replicative senescence contributes to ageing. Taken together,current findings suggest that, at least in mammals, replicativesenescence may have evolved to curtail tumorigenesis, but may also havethe unselected effect of contributing to age-related pathologies,including cancer.

  14. Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

    PubMed Central

    Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

    2015-01-01

    SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

  15. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

    PubMed Central

    Pilder, S; Moore, M; Logan, J; Shenk, T

    1986-01-01

    The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs. Images PMID:2946932

  16. Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

    PubMed Central

    Yoshida, K; Narita, M; Fujinaga, K

    1989-01-01

    Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor. Images PMID:2532319

  17. Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

    PubMed

    Li, D L; Huang, Y; Chang, L L; DU, Q; Chen, Y; Wang, T T; Luo, X M; Zhao, X M; Tong, D W

    2016-01-01

    Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2. PMID:27640437

  18. Mystery Spiral Arms Explained?

    NASA Astrophysics Data System (ADS)

    2007-04-01

    Using a quartet of space observatories, University of Maryland astronomers may have cracked a 45-year mystery surrounding two ghostly spiral arms in the galaxy M106. The Maryland team, led by Yuxuan Yang, took advantage of the unique capabilities of NASA's Chandra X-ray Observatory, NASA's Spitzer Space Telescope, the European Space Agency's XMM-Newton X-ray observatory, and data obtained almost a decade ago with NASA's Hubble Space Telescope. NGC X-ray Image NGC 4258 X-ray Image M106 (also known as NGC 4258) is a stately spiral galaxy 23.5 million light-years away in the constellation Canes Venatici. In visible-light images, two prominent arms emanate from the bright nucleus and spiral outward. These arms are dominated by young, bright stars, which light up the gas within the arms. "But in radio and X-ray images, two additional spiral arms dominate the picture, appearing as ghostly apparitions between the main arms," says team member Andrew Wilson of the University of Maryland. These so-called "anomalous arms" consist mostly of gas. "The nature of these anomalous arms is a long-standing puzzle in astronomy," says Yang. "They have been a mystery since they were first discovered in the early 1960s." By analyzing data from XMM-Newton, Spitzer, and Chandra, Yang, Bo Li, Wilson, and Christopher Reynolds, all at the University of Maryland at College Park, have confirmed earlier suspicions that the ghostly arms represent regions of gas that are being violently heated by shock waves. Previously, some astronomers had suggested that the anomalous arms are jets of particles being ejected by a supermassive black hole in M106's nucleus. But radio observations by the National Radio Astronomy Observatory's Very Long Baseline Array, and the Very Large Array in New Mexico, later identified another pair of jets originating in the core. "It is highly unlikely that an active galactic nucleus could have more than one pair of jets," says Yang. In 2001, Wilson, Yang, and Gerald Cecil

  19. Coordination of multiple robot arms

    NASA Technical Reports Server (NTRS)

    Barker, L. K.; Soloway, D.

    1987-01-01

    Kinematic resolved-rate control from one robot arm is extended to the coordinated control of multiple robot arms in the movement of an object. The structure supports the general movement of one axis system (moving reference frame) with respect to another axis system (control reference frame) by one or more robot arms. The grippers of the robot arms do not have to be parallel or at any pre-disposed positions on the object. For multiarm control, the operator chooses the same moving and control reference frames for each of the robot arms. Consequently, each arm then moves as though it were carrying out the commanded motions by itself.

  20. E1a promotes c-Myc-dependent replicative stress

    PubMed Central

    Valero, María Llanos; Cimas, Francisco Jose; Arias, Laura; Melgar-Rojas, Pedro; García, Elena; Callejas-Valera, Juan Luis; García-Cano, Jesús; Serrano-Oviedo, Leticia; Ángel de la Cruz-Morcillo, Miguel; Sánchez-Pérez, Isabel; Sánchez-Prieto, Ricardo

    2014-01-01

    The E1a gene from adenovirus is known to be a potent inducer of chemo/radiosensitivity in a wide range of tumors. However, the molecular bases of its radiosensitizer properties are still poorly understood. In an attempt to study this effect, U87MG cells, derived from a radio-resistant tumor as glioblastoma, where infected with lentivirus carrying E1a gene developing an acute sensitivity to ionizing radiation. The induction of radiosensitivity correlated with a marked G2/M phase accumulation and a potent apoptotic response. Our findings demonstrate that c-Myc plays a pivotal role in E1a-associated radiosensitivity through the induction of a replicative stress situation, as our data support by genetic approaches, based in interference and overexpression in U87MG cells. In fact, we present evidence showing that Chk1 is a novel transcriptional target of E1a gene through the effect exerted by this adenoviral protein onto c-Myc. Moreover, c-Myc upregulation also explains the marked phosphorylation of H2AX associated to E1a expression in the absence of DNA damage. Indeed, all these observations were applicable to other experimental models, such as T98G, LN-405 and A172, rendering the same pattern in terms of radiosensitivity, cell cycle distribution, upregulation of Chk1, c-Myc, and phosphorylation pattern of H2AX. In summary, our data propose a novel mechanism to explain how E1a mediates radiosensitivity through the signaling axis E1a→c-Myc→ replicative stress situation. This novel mechanism of E1a-mediated radiosensitivity could be the key to open new possibilities in the current therapy of glioblastoma. PMID:24196438

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Robotic Arm Unwrapped

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This image, taken shortly after NASA's Phoenix Mars Lander touched down on the surface of Mars, shows the spacecraft's robotic arm in its stowed configuration, with its biobarrier successfully unpeeled. The 'elbow' of the arm can be seen at the top center of the picture, and the biobarrier is the shiny film seen to the left of the arm.

    The biobarrier is an extra precautionary measure for protecting Mars from contamination with any bacteria from Earth. While the whole spacecraft was decontaminated through cleaning, filters and heat, the robotic arm was given additional protection because it is the only spacecraft part that will directly touch the ice below the surface of Mars.

    Before the arm was heated, it was sealed in the biobarrier, which is made of a trademarked film called Tedlar that holds up to baking like a turkey-basting bag. This ensures that any new bacterial spores that might have appeared during the final steps before launch and during the journey to Mars will not contact the robotic arm.

    After Phoenix landed, springs were used to pop back the barrier, giving it room to deploy.

    The base of the lander's Meteorological Station can be seen in this picture on the upper left. Because only the base of the station is showing, this image tells engineers that the instrument deployed successfully.

    The image was taken on landing day, May 25, 2008, by the spacecraft's Surface Stereo Imager.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  3. Biosynthesis and properties of the adenovirus 2 L1-encoded 52,000- and 55,000-Mr proteins.

    PubMed Central

    Lucher, L A; Symington, J S; Green, M

    1986-01-01

    The adenovirus type 2 L1 region, which is located at 30.7 to 39.2 map units on the viral genome, is transcribed from the major late promoter during both early and late stages of virus replication, and a 52,000-Mr (52K) protein-55K protein doublet has been translated in vitro on L1-specific RNA. To investigate the biosynthesis and properties of the L1 52K and 55K proteins, we prepared antibody against a synthetic peptide encoded near the predicted N terminus. As determined by immunoprecipitation and immunoblot analysis, the antipeptide antibody recognized major 52K and 55K proteins synthesized in adenovirus type 2-infected cells that appeared to be identical to the 52K-55K doublet translated in vitro. The immunoprecipitated 52K and 55K proteins were very closely related, as shown by a peptide map analysis. Both L1 proteins were phosphorylated, and they were phosphorylated at similar sites. No precursor-product relationship was detected between the 52K and 55K proteins by a pulse-chase analysis. Biosynthesis of the L1 52K and 55K proteins began about 6 to 7 h postinfection, after biosynthesis of the early region 1A and early region 1B 19K (175R) T antigens, and reached a maximum rate at about 15 h; the maximum rate was maintained until at least 25 h postinfection. At all times, the 55K protein appeared to be synthesized at a severalfold-higher level than the 52K protein. Both proteins were quite stable and accumulated until late times after infection. Viral DNA replication was not essential for formation of the L1 proteins. Thus, the L1 52K-55K gene appears to be regulated in a manner different from the classical early and late viral genes but similar to the protein encoded by the i-leader (Symington et al., J. Virol. 57:849-856, 1986). The L1 proteins were detected in the cell nucleus by immunofluorescence microscopy with antipeptide antibody and were found to be primarily associated with the nuclear membrane by an immunoblot analysis of subcellular fractions

  4. Verification and arms control

    SciTech Connect

    Potter, W.C.

    1985-01-01

    Recent years have witnessed an increased stress upon the verification of arms control agreements, both as a technical problem and as a political issue. As one contribution here points out, the middle ground has shrunk between those who are persuaded that the Soviets are ''cheating'' and those who are willing to take some verification risks for the sake of achieving arms control. One angle, according to a Lawrence Livermore physicist who served as a member of the delegation to the various test-ban treaty negotiations, is the limited effectiveness of on-site inspection as compared to other means of verification.

  5. Titration of adenovirus by counting cells containing virus-induced inclusion bodies.

    PubMed

    Weber, J

    1972-05-01

    A new method for the titration of adenovirus types 2 and 12 based on the enumeration of viral inclusions in infected cells was devised and evaluated. The technique gave virus titers comparable to those obtained by the plaque assay procedure.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2000-07-01

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

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

    PubMed

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

    2010-02-01

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

  9. A single immunization with a recombinant canine adenovirus type 2 expressing the seoul virus Gn glycoprotein confers protective immunity against seoul virus in mice.

    PubMed

    Yuan, Zi-Guo; Li, Xiu-Ming; Mahmmod, Yasser Saad; Wang, Xiao-Hu; Xu, Hui-Juan; Zhang, Xiu-Xiang

    2009-08-20

    Seoul virus (SEOV), a member of hantavirus genus, is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) and afflicts tens of thousands of people annually. In this paper, we evaluate the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gn protein of SEOV (rCAV-2-Gn) in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against a lethal challenge with the highly virulent SEOV strain CC-2. Protective level neutralizing antibodies were maintained for at least 20 weeks. The efficacy of the recombinant was similar to that induced by a currently available inactivated HFRS vaccine. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

  10. Generation of E3-deleted canine adenovirus type 2 expressing the Gc glycoprotein of Seoul virus by gene insertion or deletion of related terminal region sequences.

    PubMed

    Yuan, Zi-Guo; Luo, Sheng-Jun; Xu, Hui-Juan; Wang, Xiao-Hu; Li, Juan; Yuan, Li-Guo; He, Le-Tian; Zhang, Xiu-Xiang

    2010-07-01

    Seoul virus (SEOV) is one of the four hantaviruses known to cause haemorrhagic fever with renal syndrome. The medium genome segment encodes the Gn/Gc glycoproteins of SEOV, which form the major structural part of the virus envelope. Gc and/or Gn are the candidate antigens of hantavirus for induction of a highly immunogenic response for hantavirus vaccine. In this study, the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gc protein of SEOV was evaluated in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against an intensive infectious challenge with approximately 1000 50 % infective doses for SEOV strain CC-2. Protective-level neutralizing antibodies were maintained for at least 20 weeks. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

  11. Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A

    PubMed Central

    Stylianou, E.; Griffiths, K.L.; Poyntz, H.C.; Harrington-Kandt, R.; Dicks, M.D.; Stockdale, L.; Betts, G.; McShane, H.

    2015-01-01

    A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further. PMID:26478198

  12. Mesangial Localization of Immune Complexes in Experimental Canine Adenovirus Glomerulonephritis

    PubMed Central

    Wright, N. G.; Morrison, W. I.; Thompson, H.; Cornwell, H. J. C.

    1974-01-01

    Each of a group of 14 dogs was infected experimentally by an intravenous dose of canine adenovirus calculated to allow survival until the initial stages of antibody production; the kidneys of infected dogs were examined during the period of 4-14 days after administration of virus. Proliferative glomerulonephritis with localization of IgG, C3 and viral antigen in mesangial regions was demonstrated. With the electron microscope, electron dense deposits were found scattered throughout the mesangium. There was proliferation of mesangial cells, infiltration into the glomerular tuft of polymorphonuclear leucocytes and, in some cases, focal glomerular necrosis with intracapsular and tubular haemorrhage. By means of an indirect immunofluorescence test, anti-viral antibody was detected in kidney eluates; anti-kidney antibody was not present. ImagesFigs. 5-8Figs. 9-10Figs. 1-4 PMID:4375485

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

    PubMed Central

    Bachenheimer, S L

    1977-01-01

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

  14. Adenovirus receptors and their implications in gene delivery

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Wiethoff, Christopher M.; Nemerow, Glen R.

    2015-01-01

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

  16. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

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

    PubMed

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

    2015-12-01

    Every year brings another round of zoonotic viral infections. Usually they fall under the radar, but the occasional lethal epidemic brings another scare to the public and new urgency to the medical community. The types of these viruses (DNA vs. RNA genomes, enveloped vs. proteinaceous) as well as the preceding host(s) vary. Over the last 20 years, bats have been identified as an enigmatic carrier for several pathogens that have jumped the species barrier and infected humans. Factors that favour the emergence of zoonotic pathogens include the increasing overlap of the human and animal habitats, cultural activities, and the host reservoir. In this context, we asked whether bat and/or nonhuman primate adenoviruses are a risk for human health. PMID:26672663

  18. Adenovirus membrane penetration: Tickling the tail of a sleeping dragon.

    PubMed

    Wiethoff, Christopher M; Nemerow, Glen R

    2015-05-01

    As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (Moyer and Nemerow, 2011, Curr. Opin. Virol., 1, 44-49), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus (HAdV).

  19. Adenovirus vectors targeting distinct cell types in the retina.

    PubMed

    Sweigard, J Harry; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-04-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5DeltaRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5DeltaRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5DeltaRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5DeltaRGD vectors.

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

    SciTech Connect

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

    2008-08-15

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

  1. Comparative investigations for adenovirus recognition and quantification: Plastic or natural antibodies?

    PubMed

    Altintas, Zeynep; Pocock, Jack; Thompson, Katy-Anne; Tothill, Ibtisam E

    2015-12-15

    Comparative and comprehensive investigations for adenovirus recognition and detection were conducted using plastic and natural antibodies to compare three different strategies. The implementation of molecularly imprinted polymer (MIP) technology for specific and sensitive recognition of viruses with the combination of biosensors was reported. Plastic antibodies (MIPs nanoparticles) were produced for adenovirus by employing a novel solid phase synthesis method. MIP receptors were then characterised using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques prior to immobilisation on a surface plasmon resonance (SPR) sensor as affinity receptor for adenovirus detection. Two different templates were also imprinted as control MIPs (vancomycin-MIP and MS2-MIP). The specific recognition of adenovirus was investigated in the concentration range of 0.01-20 pM and the limit of detection was achieved as 0.02 pM. As an alternative to MIP receptors, direct and sandwich assays were developed for adenovirus quantification using natural antibodies. The detection limit of direct and sandwich assays were found as 0.3 pM and 0.008 pM, respectively. The kinetic data analyses were performed for three different adenovirus recognition methods and cross-reactivity studies were also conducted using MS2 phage as control virus and an excellent specificity was achieved with all assays types. This work confirmed the suitability of the MIPs SPR sensor for the detection of viruses. PMID:26264266

  2. Particle Tracking of Intracellular Trafficking of Octaarginine-modified Liposomes: A Comparative Study With Adenovirus

    PubMed Central

    Akita, Hidetaka; Enoto, Kaoru; Masuda, Tomoya; Mizuguchi, Hiroyuki; Tani, Tomomi; Harashima, Hideyoshi

    2010-01-01

    It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)–tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips. PMID:20216528

  3. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

    PubMed

    Lyons, Mark; Onion, David; Green, Nicky K; Aslan, Kriss; Rajaratnam, Ratna; Bazan-Peregrino, Miriam; Phipps, Sue; Hale, Sarah; Mautner, Vivien; Seymour, Leonard W; Fisher, Kerry D

    2006-07-01

    Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access. PMID:16580883

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1984-08-01

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

  6. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

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

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  7. [Anti-adenovirus activity of a substance and medical form of ribamydil in cell culture].

    PubMed

    Nosach, L N; Diachenko, N S; Zhovnovataia, V L

    2009-01-01

    The inhibiting effect of ribamydil on adenovirus reproduction was studied under the determination of the number of cells with virus- induced DNA-containing intranucleus inclusion bodies and hexone antigen, the synthesis of adenovirus proteins and the infection virus by t he investigation. EC50 of ribamydil substance is 4-8 microg/ml, but complete suppression of adenovirus genome expression was found when adding ribamydil after the virus adsorption, in concentrations of 125-500 microg/ml. The original effect of ribamydil on the expression of adenovirus genome was found under its effect in concentration of 31 microg/ml. Intranucleus virus-induced inclusion bodies of the early type only were found under these conditions. Synthesis of the structural virus polypeptides, including hexone polypeptide (II) and non-structural polypeptide 100K, taking part in hexone trimerization, proceed intensively but without formation of immunologically active hexone. The inhibiting effect of officinal form of ribamydil was less expressed as compared with the substance (EC50: 62 microg/ml). The work results prove that the therapeutic effect of ribamydil (ribavirin) under treatment of adenovirus infections may be achieved in case when it is used in a dose excluding the expression of the adenovirus genome.

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

    PubMed Central

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

    2016-01-01

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

  9. Arm pain and erythema

    PubMed Central

    Juergens, Andrew L.

    2016-01-01

    Pyomyositis can be a difficult diagnosis to make, as it can mimic many other disease processes. Various laboratory studies can be abnormal with pyomyositis, but none are specific to the disease. Early disease can generally be treated with antibiotics alone, whereas advanced disease frequently requires emergent surgical intervention with significant resuscitation. We describe a case of pyomyositis of the right arm. PMID:27034571

  10. Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

    PubMed

    Nalbantoglu, J; Pari, G; Karpati, G; Holland, P C

    1999-04-10

    Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

  11. 32 CFR 935.134 - Arm signals.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... arm horizontally. (2) Right turn—extend left arm upward. (3) Stop or decrease speed—extend left arm downward. (c) A signal light or other device may be used in place of an arm signal prescribed in...

  12. 32 CFR 935.134 - Arm signals.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... arm horizontally. (2) Right turn—extend left arm upward. (3) Stop or decrease speed—extend left arm downward. (c) A signal light or other device may be used in place of an arm signal prescribed in...

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

    PubMed

    Koonin, Eugene V; Starokadomskyy, Petro

    2016-10-01

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

  14. Response to Multiple Radiation Doses of Human Colorectal Carcinoma Cells Infected With Recombinant Adenovirus Containing Dominant-Negative Ku70 Fragment

    SciTech Connect

    Urano, Muneyasu; He Fuqiu; Minami, Akiko; Ling, C. Clifton; Li, Gloria C.

    2010-07-01

    Purpose: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells. Methods and Materials: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals. Hypoxia was induced by flushing with 100% nitrogen gas. The cells were trypsinized 0 or 6 h after the final irradiation, and cell survival was determined by colony formation. The survival data were fitted to linear-quadratic model or exponential line. Results: Virus infection enhanced the radiation response of the HCT8 and HT29 cells. The virus enhancement ratio for single-dose irradiation at a surviving fraction of 0.1 was {approx}1.3 for oxic and hypoxic HCT8 and 1.4 and 1.1 for oxic and hypoxic HT29, respectively. A similar virus enhancement ratio of 1.2-1.3 was observed for both oxic and hypoxic cells irradiated with multiple doses; however, these values were smaller than the values found for dominant-negative Ku70-transfected Rat-1 cells. This difference has been discussed. The oxygen enhancement ratio for HCT8 and HT29 receiving fractionated doses was 1.2 and 2.0, respectively, and virus infection altered them slightly. Conclusion: Infection of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment enhanced the response of human colorectal cancer cells to single and multiple radiation doses.

  15. Mimiviruses: Replication, Purification, and Quantification.

    PubMed

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

    2016-01-01

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

  16. Layers of Experience Using "Arms"

    ERIC Educational Resources Information Center

    Brown, Laurinda; Coles, Alf; Ball, Derek; Morton, Pat; Coles, Matt; Ordman, Louise; Orr, Barry; Lam, Tung Ken

    2008-01-01

    This article presents the authors' personal accounts and their experiences in working on mathematics using "arms." "Arms" is an idea that first appeared as a program written by John Warwick and David Wooldridge in an ATM publication "Some Lessons in Mathematics with a Microcomputer," 1983. The introduction to "Arms" in the book states that it is a…

  17. Robotic Arm of Rover 1

    NASA Technical Reports Server (NTRS)

    2003-01-01

    JPL engineers examine the robotic arm of Mars Exploration Rover 1. The arm is modeled after a human arm, complete with joints, and holds four devices on its end, the Rock Abrasion Tool which can grind into Martian rocks, a microscopic imager, and two spectrometers for elemental and iron-mineral identification.

  18. Replicating systems concepts: Self-replicating lunar factory and demonstration

    NASA Technical Reports Server (NTRS)

    1982-01-01

    Automation of lunar mining and manufacturing facility maintenance and repair is addressed. Designing the factory as an automated, multiproduct, remotely controlled, reprogrammable Lunar Manufacturing Facility capable of constructing duplicates of itself which would themselves be capable of further replication is proposed.

  19. A Replication of Failure, Not a Failure to Replicate

    ERIC Educational Resources Information Center

    Holden, Gary; Barker, Kathleen; Kuppens, Sofie; Rosenberg, Gary; LeBreton, Jonathan

    2015-01-01

    Purpose: The increasing role of systematic reviews in knowledge production demands greater rigor in the literature search process. The performance of the Social Work Abstracts (SWA) database has been examined multiple times over the past three decades. The current study is a replication within this line of research. Method: Issue-level coverage…

  20. Personality and Academic Motivation: Replication, Extension, and Replication

    ERIC Educational Resources Information Center

    Jones, Martin H.; McMichael, Stephanie N.

    2015-01-01

    Previous work examines the relationships between personality traits and intrinsic/extrinsic motivation. We replicate and extend previous work to examine how personality may relate to achievement goals, efficacious beliefs, and mindset about intelligence. Approximately 200 undergraduates responded to the survey with a 150 participants replicating…

  1. Exploiting replication in distributed systems

    NASA Technical Reports Server (NTRS)

    Birman, Kenneth P.; Joseph, T. A.

    1989-01-01

    Techniques are examined for replicating data and execution in directly distributed systems: systems in which multiple processes interact directly with one another while continuously respecting constraints on their joint behavior. Directly distributed systems are often required to solve difficult problems, ranging from management of replicated data to dynamic reconfiguration in response to failures. It is shown that these problems reduce to more primitive, order-based consistency problems, which can be solved using primitives such as the reliable broadcast protocols. Moreover, given a system that implements reliable broadcast primitives, a flexible set of high-level tools can be provided for building a wide variety of directly distributed application programs.

  2. Phylogenetic Analysis and Structural Predictions of Human Adenovirus Penton Proteins as a Basis for Tissue-Specific Adenovirus Vector Design▿

    PubMed Central

    Madisch, Ijad; Hofmayer, Soeren; Moritz, Christian; Grintzalis, Alexander; Hainmueller, Jens; Pring-Akerblom, Patricia; Heim, Albert

    2007-01-01

    The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17′H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors. PMID:17522221

  3. Role of coxsackievirus and adenovirus receptor (CAR) expression and viral load of adenovirus and enterovirus in patients with dilated cardiomyopathy.

    PubMed

    Sharma, Mirnalini; Mishra, Baijayantimala; Saikia, Uma Nahar; Bahl, Ajay; Ratho, Radha Kanta; Talwar, Kewal Kishan

    2016-01-01

    Enteroviruses (EVs) and adenoviruses (AdVs) are two important etiological agents of viral myocarditis and dilated cardiomyopathy (DCM). Both these viruses share a common receptor, the coxsackievirus and adenovirus receptor (CAR), for their infection. However, the role of viral load and CAR expression in disease severity has not yet been completely elucidated. The present study aimed to determine viral load of EV and AdV in DCM patients and correlate them with the level of CAR expression in these patients. Sixty-three DCM cases and 30 controls, each of whom died of heart disease other than DCM and non-cardiac disease respectively, were included. Viral load was determined by TaqMan real-time PCR using primers and probes specific for the AdV hexon gene and the 5'UTR region of EV. The CAR mRNA level was semi-quantitated by RT-PCR, and antigen expression was studied by immunohistochemistry. A significantly high AdV load (p < 0.05) and CAR expression (p < 0.05) were observed in DCM cases versus controls, whereas the EV load showed no significant difference. The data suggests a clinical threshold of 128 AdV copies/500 ng of DNA for DCM, with 66.7 % sensitivity and 65 % specificity. A positive correlation between AdV load and CAR expression (p < 0.001) was also observed in DCM cases. The high adenoviral load and increased CAR expression in DCM and their association with adverse disease outcome indicates role of both virus and receptor in disease pathogenesis. Thus, the need for targeting both the virus and the receptor for treatment of viral myocarditis and early DCM requires further confirmation with larger studies.

  4. Complement-dependent cytotoxicity in rats bearing human adenovirus type 12-induced primary retinoblastoma-like tumor in the eye.

    PubMed

    Nishida, T; Mukai, N; Solish, S P

    1981-01-01

    Using an animal model of retinoblastoma in inbred rats and cultured human adenovirus type 12-induced retinoblastoma-like tumor cells (RAO 188), complement-dependent cytotoxicity was determined by measuring release of 3H-uridine labelled RNA. Sera from rats in which tumors did not grow after adenovirus type 12 inoculation had higher cytotoxicity against RAO 188 cells than sera from rats bearing primary adenovirus type 12-induced retinoblastoma-like tumor. These results showed that the rat which could raise antibodies against adenovirus type 12-induced retinoblastoma-like tumor cells did not allow the tumor growth in the eye after virus inoculation.

  5. Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

    PubMed

    Shin, Soochul; Lee, Jinwoo; Yoo, Sangwoon; Kulikowicz, Tomasz; Bohr, Vilhelm A; Ahn, Byungchan; Hohng, Sungchul

    2016-08-01

    The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

  6. Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

    PubMed Central

    Xie, Baode; Calabro, Valerie; Wainberg, Mark A.; Frankel, Alan D.

    2004-01-01

    The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a “chameleon,” adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions. PMID:14722301

  7. Armed conflict and child health

    PubMed Central

    Rieder, Michael; Choonara, Imti

    2012-01-01

    Summary Armed conflict has a major impact on child health throughout the world. One in six children worldwide lives in an area of armed conflict and civilians are more likely to die than soldiers as a result of the conflict. In stark contrast to the effect on children, the international arms trade results in huge profits for the large corporations involved in producing arms, weapons and munitions. Armed conflict is not inevitable but is an important health issue that should be prevented. PMID:21393303

  8. Characterization of Infectivity-Enhanced Conditionally Replicating Adenovectors for Prostate Cancer Radiovirotherapy

    PubMed Central

    Oneal, Michael J.; Trujillo, Miguel A.; Davydova, Julia; McDonough, Samantha; Yamamoto, Masato

    2012-01-01

    Abstract Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor–expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier 131I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer. PMID:22694073

  9. IL-9 Inhibits Viral Replication in Coxsackievirus B3-Induced Myocarditis

    PubMed Central

    Yu, Miao; Long, Qi; Li, Huan-Huan; Liang, Wei; Liao, Yu-Hua; Yuan, Jing; Cheng, Xiang

    2016-01-01

    Myocardial injuries in viral myocarditis (VMC) are caused by viral infection and related autoimmune disorders. Recent studies suggest that IL-9 mediated both antimicrobial immune and autoimmune responses in addition to allergic diseases. However, the role of IL-9 in viral infection and VMC remains controversial and uncertain. In this study, we infected Balb/c mice with Coxsackievirus B3 (CVB3), and found that IL-9 was enriched in the blood and hearts of VMC mice on days 5 and 7 after virus infection. Most of IL-9 was secreted by CD8+ T cells on day 5 and CD4+ T cells on day 7 in the myocardium. Further, IL-9 knockout exacerbated cardiac damage following CVB3 infection, along with a sharp increase in viral replication and IL-17a expression, as well as a decrease in TGF-β. In contrast, the repletion of IL-9 in Balb/c mice with CVB infection induced the opposite effect. Studies in vitro further revealed that IL-9 directly inhibited viral replication in cardiomyocytes by reducing coxsackie and adenovirus receptor expression, which might be associated with upregulation of TGF-β autocrine effect in these cells. However, IL-9 had no direct effect on apoptosis in cardiomyocytes. Our data indicated that IL-9 played a protective role in disease progression by inhibiting CVB3 replication in the early stages of VMC. PMID:27766098

  10. Simultaneous neutralization and innate immune detection of a replicating virus by TRIM21.

    PubMed

    Watkinson, R E; Tam, J C H; Vaysburd, M J; James, L C

    2013-07-01

    Tripartite motif-containing 21 (TRIM21) is a cytosolic immunoglobulin receptor that mediates antibody-dependent intracellular neutralization (ADIN). Here we show that TRIM21 potently inhibits the spreading infection of a replicating cytopathic virus and activates innate immunity. We used a quantitative PCR (qPCR)-based assay to measure in vitro replication of mouse adenovirus type 1 (MAV-1), a virus that causes dose-dependent hemorrhagic encephalitis in mice. Using this assay, we show that genetic ablation of TRIM21 or chemical inhibition of either the AAA ATPase p97/valosin-containing protein (VCP) or the proteasome results in a >1,000-fold increase in the relative level of infection in the presence of immune serum. Moreover, the TRIM21-mediated ability of antisera to block replication was a consistent feature of the humoral immune response in immunized mice. In the presence of immune sera and upon infection, TRIM21 also activates a proinflammatory response, resulting in secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These results demonstrate that TRIM21 provides a potent block to spreading infection and induces an antiviral state. PMID:23596308

  11. Covert Reinforcement: A Partial Replication.

    ERIC Educational Resources Information Center

    Ripstra, Constance C.; And Others

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

  12. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

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

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  13. Influence of various parameters on benzo(a)pyrene enhancement of adenovirus SA7 transformation of Syrian hamster embryo cells

    SciTech Connect

    Lubet, R.A.; Nims, R.W.; Kiss, E.; Kouri, R.E.; Putman, D.L.; Schechtman, L.M.

    1986-01-01

    Several of the major variable factors in the Syrian hamster embryo/simian adenovirus SA7 (SHE/SA7) viral enhancement assay were identified and the effects of these parameters on assay sensitivity were assessed. The extent of dose-dependent cytotoxicity and enhancement of SA7 transformation of primary SHE target cells by benzo(a)pyrene was examined through analysis of data obtained from 37 assays performed over a 2-year period. The variables analyzed for contribution to assay sensitivity included (a) the number of SA7-induced transformed SHE cell foci enumerated in ten replicate dishes in the negative control condition (background focus count) (range: 26-139); (b) the age of the SHE cell cultures at the time of exposure to benzo(a)pyrene (range: 72-144 hr postseeding); and (c) the source of the pregnant hamsters used to prepare the primary SHE cells (Wilmington colony vs Lakeview colony, Charles River Laboratories, Inc., Wilmington, MA). The benzo(a)pyrene-induced cytotoxicity and enhancement of SA7 transformation responses were found to be independent of each of these variables, within the range of values tested.

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

    PubMed

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

    2015-02-15

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

  15. Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge.

    PubMed

    Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Senina, Svetlana; Perlin, David; Crystal, Ronald G; Boyer, Julie L

    2009-04-01

    Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

  16. Chimpanzee adenovirus vector-based avian influenza vaccine completely protects mice against lethal challenge of H5N1.

    PubMed

    Cheng, Tao; Wang, Xiang; Song, Yufeng; Tang, Xinying; Zhang, Chao; Zhang, Hongbo; Jin, Xia; Zhou, Dongming

    2016-09-22

    Highly pathogenic avian H5N1 viruses may give rise to the next influenza pandemic due to their reassortment and mutation of the genome. Vaccine against this virus is important for coping with its potential threat. Chimpanzee adenovirus (Ad) vectors are a novel type of vaccine vectors that share the advantages of human serotype Ad vectors but without being affected by pre-existing human neutralizing antibody to the vaccine vector. Based on a replication-deficient chimpanzee Ad vector, AdC7, we generated a novel H5N1 vaccine candidate AdC7-H5HA that expresses H5N1 Hemagglutinin(HA). When tested in mice, the vaccine significantly reduced the virus load and pathological lesions in the lung tissues, and conferred complete protection against lethal challenge by a homologous virus. Mechanistically, the AdC7-H5HA vaccine can induce both HA-specific humoral and cell-mediated immune responses in mice. Also, sera transfer experiments demonstrated that neutralizing antibodies alone could provide protection. In conclusion, our results show that chimpanzee Ad vector expressing influenza virus HA may represent a promising vaccine candidate for H5N1 viruses and other influenza virus subtypes. PMID:27576071

  17. Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a computer model.

    PubMed

    Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J; Lütschg, Verena A; Jurgeit, Andreas; Sbalzarini, Ivo F; Greber, Urs F

    2012-09-01

    Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.

  18. Robot arm apparatus

    DOEpatents

    Nachbar, Henry D.

    1992-01-01

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

  19. Robot arm apparatus

    DOEpatents

    Nachbar, Henry D.

    1992-12-01

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

  20. ARM User Survey Report

    SciTech Connect

    Roeder, LR

    2010-06-22

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