Adeno-associated Virus as a Mammalian DNA Vector

PubMed Central

SALGANIK, MAX; HIRSCH, MATTHEW L.; SAMULSKI, RICHARD JUDE

2015-01-01

In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells. PMID:26350320

Adeno-associated virus-mediated gene transfer.

PubMed

Srivastava, Arun

2008-09-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

PubMed

Koo, Taeyoung; Okada, Takashi; Athanasopoulos, Takis; Foster, Helen; Takeda, Shin'ichi; Dickson, George

2011-09-01

Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+)  T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD. Copyright © 2011 John Wiley & Sons, Ltd.

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

PubMed

Barajas, Daniel; Aponte-Ubillus, Juan Jose; Akeefe, Hassibullah; Cinek, Tomas; Peltier, Joseph; Gold, Daniel

2017-01-01

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

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

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

PubMed

Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

2010-09-01

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

NASA Astrophysics Data System (ADS)

Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

1997-12-01

About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

PubMed Central

Janovitz, Tyler; Klein, Isaac A.; Oliveira, Thiago; Mukherjee, Piali; Nussenzweig, Michel C.; Sadelain, Michel

2013-01-01

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics. PMID:23720718

Crystal structure of the SF3 helicase from adeno-associated virus type 2.

PubMed

James, J Anson; Escalante, Carlos R; Yoon-Robarts, Miran; Edwards, Thomas A; Linden, R Michael; Aggarwal, Aneel K

2003-08-01

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

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

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira

2012-01-06

Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors,more » therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.« less

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

PubMed

van Lieshout, Laura P; Soule, Geoff; Sorensen, Debra; Frost, Kathy L; He, Shihua; Tierney, Kevin; Safronetz, David; Booth, Stephanie A; Kobinger, Gary P; Qiu, Xiangguo; Wootton, Sarah K

2018-03-05

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

NASA Astrophysics Data System (ADS)

Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

2007-05-01

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.

PubMed

Marsic, Damien; Méndez-Gómez, Héctor R; Zolotukhin, Sergei

2015-01-01

Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.

Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System

PubMed Central

Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe

2015-01-01

Abstract Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

Toward exascale production of recombinant adeno-associated virus for gene transfer applications.

PubMed

Cecchini, S; Negrete, A; Kotin, R M

2008-06-01

To gain acceptance as a medical treatment, adeno-associated virus (AAV) vectors require a scalable and economical production method. Recent developments indicate that recombinant AAV (rAAV) production in insect cells is compatible with current good manufacturing practice production on an industrial scale. This platform can fully support development of rAAV therapeutics from tissue culture to small animal models, to large animal models, to toxicology studies, to Phase I clinical trials and beyond. Efforts to characterize, optimize and develop insect cell-based rAAV production have culminated in successful bioreactor-scale production of rAAV, with total yields potentially capable of approaching the exa-(10(18)) scale. These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases such as Duchenne muscular dystrophy, for which large amounts of recombinant vector are essential for successful outcome.

Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.

PubMed

Nash, Kevin R; Gordon, Marcia N

2016-01-01

Recombinant adeno-associated virus (rAAV) has become an extremely useful tool for the study of gene over expression or knockdown in the central nervous system of experimental animals. One disadvantage of intracranial injections of rAAV vectors into the brain parenchyma has been restricted distribution to relatively small volumes of the brain. Convection enhanced delivery (CED) is a method for delivery of clinically relevant amounts of therapeutic agents to large areas of the brain in a direct intracranial injection procedure. CED uses bulk flow to increase the hydrostatic pressure and thus improve volume distribution. The CED method has shown robust gene transfer and increased distribution within the CNS and can be successfully used for different serotypes of rAAV for increased transduction of the mouse CNS. This chapter details the surgical injection of rAAV by CED into a mouse brain.

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

PubMed Central

Li, Chengwen; He, Yi; Nicolson, Sarah; Hirsch, Matt; Weinberg, Marc S.; Zhang, Ping; Kafri, Tal; Samulski, R. Jude

2013-01-01

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials. PMID:23454772

Production, Purification and Preliminary X-ray Crystallographic Studies of Adeno-Associated Virus Serotype 9

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

Mitchell, M.; Nam, H; Carter, A

2009-01-01

Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetricmore » unit capsid have been determined by molecular-replacement methods and structure determination is in progress.« less

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Adeno-associated virus type 2 binding study on model heparan sulfate surface

NASA Astrophysics Data System (ADS)

Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

2003-11-01

Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

Adeno-associated virus inverted terminal repeats stimulate gene editing.

PubMed

Hirsch, M L

2015-02-01

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

Adeno-associated virus vector-mediated transduction in the cat brain.

PubMed

Vite, Charles H; Passini, Marco A; Haskins, Mark E; Wolfe, John H

2003-10-01

Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. In this study, we evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme beta-glucuronidase (GUSB) was used as a reporter gene, because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cortex, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of 8-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain.

Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.

PubMed

Wang, Zhong; Zhu, Tong; Qiao, Chunping; Zhou, Liqiao; Wang, Bing; Zhang, Jian; Chen, Chunlian; Li, Juan; Xiao, Xiao

2005-03-01

Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventions to enforce broad vector distribution. We compared the efficiency of multiple adeno-associated virus (AAV) vectors after a single injection via intraperitoneal or intravenous routes without additional intervention. We show that AAV8 is the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles of mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier. Gene expression persisted in muscle and heart, but diminished in tissues undergoing rapid cell division, such as neonatal liver. This technology should prove useful for muscle-directed systemic gene therapy.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

PubMed

Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

2009-08-01

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

Production and validation of recombinant adeno-associated virus for channelrhodopsin expression in neurons.

PubMed

Lin, John Y

2013-01-01

Recent discovery of the light-activated ion channel, channelrhodopsin (ChR), has provided researchers a powerful and convenient tool to manipulate the membrane potential of specific cells with light. With genetic targeting of these channels and illumination of light to a specific location, the experimenter can selectively activate the voltage-gated ion channels (VGICs) of ChR-expressing cells, initiating electrical signaling in temporally and spatially precise manners. In neuroscience research, this can be used to study electrical signal processing within one neuron at the cellular level, or the synaptic connectivity between neurons at the circuitry level. To conduct experiments with ChRs, these exogenous channels need to be introduced into the cells of interest, commonly through a viral approach. This chapter provides an overview of the design, production, and validation of recombinant adeno-associated virus (rAAV) for ChR expression that can be used in vitro or in vivo to infect neurons. The virus produced can be used to conduct "optogenetic" experiments in behaving animals, in vitro preparations and cultured cells, and can be used to study signal transduction and processing at a cellular or circuitry level.

Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses.

PubMed

Tse, Longping V; Moller-Tank, Sven; Meganck, Rita M; Asokan, Aravind

2018-04-25

Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly. Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a

Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

PubMed

Hastie, Eric; Samulski, R Jude

2015-05-01

Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

PubMed

Asavarut, Paladd; O'Neill, Kevin; Syed, Nelofer; Hajitou, Amin

2014-01-01

The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences.

PubMed

Nony, P; Tessier, J; Chadeuf, G; Ward, P; Giraud, A; Dugast, M; Linden, R M; Moullier, P; Salvetti, A

2001-10-01

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

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

Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

2012-02-05

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

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

Lerch, Thomas F.; Chapman, Michael S.

2012-05-24

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

PubMed

White, K; Büning, H; Kritz, A; Janicki, H; McVey, J; Perabo, L; Murphy, G; Odenthal, M; Work, L M; Hallek, M; Nicklin, S A; Baker, A H

2008-03-01

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus.

PubMed

Watson, G L; Sayles, J N; Chen, C; Elliger, S S; Elliger, C A; Raju, N R; Kurtzman, G J; Podsakoff, G M

1998-12-01

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a genetic deficiency of beta-glucuronidase (GUS). We used a recombinant adeno-associated virus vector (AAV-GUS) to deliver GUS cDNA to MPS VII mice. The route of vector administration had a dramatic effect on the extent and distribution of GUS activity. Intramuscular injection of AAV-GUS resulted in high, localized production of GUS, while intravenous administration produced low GUS activity in several tissues. This latter treatment of MPS VII mice reduced glycosaminoglycan levels in the liver to normal and reduced storage granules dramatically. We show that a single administration of AAV-GUS can provide sustained expression of GUS in a variety of cell types and is sufficient to reverse the disease phenotype at least in the liver.

Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

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

McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.

2012-09-15

The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon,more » covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.« less

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

PubMed

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

Molecular design for recombinant adeno-associated virus (rAAV) vector production.

PubMed

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

2018-02-01

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

PubMed

Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

2011-05-01

Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

[Ubiquitination of recombinant adeno-associated viral vector and its application].

PubMed

Wang, Qi-zhao; Lu, Ying-hui; Diao, Yong; Xu, Rui-an

2012-09-01

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

New World Bats Harbor Diverse Influenza A Viruses

PubMed Central

Tong, Suxiang; Zhu, Xueyong; Li, Yan; Shi, Mang; Zhang, Jing; Bourgeois, Melissa; Yang, Hua; Chen, Xianfeng; Recuenco, Sergio; Gomez, Jorge; Chen, Li-Mei; Johnson, Adam; Tao, Ying; Dreyfus, Cyrille; Yu, Wenli; McBride, Ryan; Carney, Paul J.; Gilbert, Amy T.; Chang, Jessie; Guo, Zhu; Davis, Charles T.; Paulson, James C.; Stevens, James; Rupprecht, Charles E.; Holmes, Edward C.; Wilson, Ian A.; Donis, Ruben O.

2013-01-01

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses. PMID:24130481

Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

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

DiMattia,M.; Govindasamy, L.; Levy, H.

2005-01-01

Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c =more » 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.« less

Adeno-associated virus rep protein synthesis during productive infection

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

Redemann, B.E.; Mendelson, E.; Carter, B.J.

1989-02-01

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing tomore » a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.« less

Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

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

O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

2009-03-15

Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensivemore » than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.« less

High-Efficiency Promoter-Dependent Transduction by Adeno-Associated Virus Type 6 Vectors in Mouse Lung

PubMed Central

HALBERT, CHRISTINE L.; LAM, SIU-LING; MILLER, A. DUSTY

2014-01-01

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, β-actin promoter and splice donor, and a β-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, highly efficient transduction can be achieved. PMID:17430088

Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

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

Laughlin, C.A.; Myers, M.W.; Risin, D.L.

1979-04-15

We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious butmore » interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.« less

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

PubMed Central

Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

2015-01-01

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

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

PubMed

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

2003-06-01

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

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

PubMed

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo

PubMed Central

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV

Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

PubMed

Ohi, S

2000-07-01

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

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

Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael

2005-05-10

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, couldmore » be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.« less

Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

PubMed Central

Lock, Martin; Alvira, Mauricio R.

2012-01-01

Abstract Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium. PMID:22428980

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

PubMed

Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J; Castellanos Rivera, Ruth M; Wells, Lindsey F; Havlik, L Patrick; Smith, J Kennon; Agbandje-McKenna, Mavis; Asokan, Aravind

2017-06-13

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

PubMed Central

White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

2013-01-01

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

PubMed Central

Whitford, Margaret H.; Arruda, Valder R.; Stedman, Hansell H.; Kay, Mark A.; High, Katherine A.

2015-01-01

Abstract Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

PubMed

Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

2015-03-01

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

PubMed

Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

2013-03-06

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants.

PubMed

Wiley, Luke A; Burnight, Erin R; Kaalberg, Emily E; Jiao, Chunhua; Riker, Megan J; Halder, Jennifer A; Luse, Meagan A; Han, Ian C; Russell, Stephen R; Sohn, Elliott H; Stone, Edwin M; Tucker, Budd A; Mullins, Robert F

2018-04-01

Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

PubMed Central

Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Kekilli, Demet; Samso, Monserrat

2013-01-01

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle. PMID:23152528

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

PubMed

Brown, Nolan; Song, Liujiang; Kollu, Nageswara R; Hirsch, Matthew L

2017-06-01

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque ▿

PubMed Central

Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H.; Bell, Peter; Somanathan, Suryanarayan; Wilson, James M.

2009-01-01

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8+ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8+ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses. PMID:19812149

Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

PubMed Central

Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

2014-01-01

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

PubMed

Parzych, Elizabeth M; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M; High, Katherine A; Levine, Matthew H; Ertl, Hildegund C J

2013-04-01

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Effects of Immunosuppression on Circulating Adeno-Associated Virus Capsid-Specific T cells in Humans

PubMed Central

Parzych, Elizabeth M.; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M.; High, Katherine A.; Levine, Matthew H.

2013-01-01

Abstract In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4+ T cells, whereas numbers of circulating CD8+ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients. PMID:23461589

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies.

PubMed

Sun, Junjiang; Hua, Baolai; Chen, Xiaojing; Samulski, Richard J; Li, Chengwen

2017-08-01

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10 13 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

PubMed Central

Hüser, Daniela; Weger, Stefan; Heilbronn, Regine

2003-01-01

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought. PMID:12663794

Development and Evaluation of Adeno-HTLV-III Hybrid Virus and Non-Cytopathic HTLV-III Mutant for Vaccine Use.

DTIC Science & Technology

1987-10-28

34" HIV will be tested. The recombinant Adeno-HIV virus is being developed as part of this proposal. The vaccines will be tested in two species of monkeys...vaccinia-HIV viruses was investigated (Table 4). All the vaccinated monkeys developed antibodies to HIV envelope antigen. However, no neutralizing antibody...Table 5). Epstein Barr virus transformed autologous B cells infected with recon- binant vaccinia-HIV virus may be used as targets. 30 chimpanzees from

ENHANCED GENE DELIVERY IN PORCINE VASCULATURE TISSUE FOLLOWING INCORPORATION OF ADENO-ASSOCIATED VIRUS NANOPARTICLES INTO POROUS SILICON MICROPARTICLES

PubMed Central

McConnell, Kellie I.; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E.

2014-01-01

There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells. PMID:25180449

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

Regulation of adeno-associated virus DNA replication by the cellular TAF-I/set complex.

PubMed

Pegoraro, Gianluca; Marcello, Alessandro; Myers, Michael P; Giacca, Mauro

2006-07-01

The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

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

Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert

2012-09-17

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pHmore » 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.« less

Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections.

PubMed

Robert, Marc-André; Nassoury, Nasha; Chahal, Parminder S; Venne, Marie-Hélène; Racine, Trina; Qiu, Xiangguo; Kobinger, Gary; Kamen, Amine; Gilbert, Rénald; Gaillet, Bruno

2018-04-01

Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 10 10 and 13.0 × 10 10 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 10 10 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.

Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

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

Cassell, Geoffrey D.; Weitzman, Matthew D.

2004-10-01

Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha}more » interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.« less

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses

PubMed Central

Shigetomi, Eiji; Bushong, Eric A.; Haustein, Martin D.; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V.; Ellisman, Mark H.

2013-01-01

Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function. PMID:23589582

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

PubMed

Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V; Ellisman, Mark H; Khakh, Baljit S

2013-05-01

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

PubMed

Galli, A; Della Latta, V; Bologna, C; Pucciarelli, D; Cipriani, F; Backovic, A; Cervelli, T

2017-08-01

Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production. © 2017 The Society for Applied Microbiology.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

PubMed

Tadokoro, Takahiro; Miyanohara, Atsushi; Navarro, Michael; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Marsala, Silvia; Platoshyn, Oleksandr; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Lukacova, Nada; Bimbova, Katarina; Marsala, Martin

2017-07-13

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10 13 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors.

PubMed

Handa, A; Muramatsu, S; Qiu, J; Mizukami, H; Brown, K E

2000-08-01

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID(50) of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.

Release of Bioactive Adeno-Associated Virus from Fibrin Scaffolds: Effects of Fibrin Glue Concentrations

PubMed Central

Lee, Hannah H.; Haleem, Amgad M.; Yao, Veronica; Li, Juan; Xiao, Xiao

2011-01-01

Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. However, the effect of different fibrinogen concentrations on FG scaffold delivery of bioactive adeno-associated viruses (AAVs) has not been established. This study was performed to test the hypothesis that FG concentration alters AAV release profiles, which affect AAV bioavailability. Gene transfer efficiency of AAV-GFP released from FG was measured using HEK-293 cells. Bioactivity of AAV transforming growth factor-beta1 (TGF-β1) released from FG was assessed using the mink lung cell assay, and by measuring induction of cartilage-specific gene expression in human mesenchymal stem cells (hMSCs). Nondiluted FG had longer clotting times, smaller pore sizes, thicker fibers, and slower dissolution rate, resulting in reduced release of AAV. AAV release and gene transfer efficiency was higher with 25% and 50% FG than with the 75% and 100% FG. AAV-TGF-β1 released from dilute-FG transduced hMSCs, resulting in higher concentrations of bioactive TGF-β1 and greater upregulation of cartilage-specific gene expression compared with hMSC from undiluted FG. This study, showing improved release, transduction efficiency, and chondrogenic effect on hMSC of bioactive AAV-TGF-β1 released from diluted FG, provides information important to optimization of this clinically available scaffold for therapeutic gene delivery, both in cartilage regeneration and for other tissue engineering applications. PMID:21449684

Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

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

Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.

2006-07-15

Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months inmore » plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.« less

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles.

PubMed

Smith, J Kennon; Agbandje-McKenna, Mavis

2018-05-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding ("footprints") can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein-DNA and protein-protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle-specifically, those involved with binding neutralizing antibodies (NAbs) [2-4]. This method offers a unique opportunity for visualizing antibody binding "hotspots" on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create "stealth" AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms.

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles

PubMed Central

Agbandje-McKenna, Mavis

2018-01-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding (“footprints”) can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein–DNA and protein–protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle—specifically, those involved with binding neutralizing antibodies (NAbs) [2–4]. This method offers a unique opportunity for visualizing antibody binding “hotspots” on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create “stealth” AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms. PMID:29723270

Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice

PubMed Central

Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.

2005-01-01

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817

The Interaction Between Human Papillomavirus and Other Viruses

PubMed Central

Guidry, J. T.; Scott, R. S.

2016-01-01

The etiological role of human papillomavirus (HPV) in anogenital tract and head and neck cancers is well established. However, only a low percentage of HPV-positive women develop cancer, indicating that HPV is necessary but not sufficient in carcinogenesis. Several biological and environmental cofactors have been implicated in the development of HPV-associated carcinoma that include immune status, hormonal changes, parity, dietary habits, tobacco usage, and co-infection with other sexually transmissible agents. Such cofactors likely contribute to HPV persistent infection through diverse mechanisms related to immune control, efficiency of HPV infection, and influences on tumor initiation and progression. Conversely, HPV co-infection with other factors may also harbor anti-tumor effects. Here, we review epidemiological and experimental studies investigating human immunodeficiency virus (HIV), herpes simplex virus (HSV) 1 and 2, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), BK virus (BKV) JC virus (JCV), and adeno-associated virus (AAV) as viral cofactors in or therapeutic factors against the development of genital and oral HPV-associated carcinomas. PMID:27826043

Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

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

Trempe, J.P.; Carter, B.J.

1988-01-01

The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/submore » 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.« less

[Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

PubMed

Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

2012-01-01

Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

PubMed

Shao, Wenwei; Earley, Lauriel F; Chai, Zheng; Chen, Xiaojing; Sun, Junjiang; He, Ting; Deng, Meng; Hirsch, Matthew L; Ting, Jenny; Samulski, R Jude; Li, Chengwen

2018-06-21

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

PubMed

Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

2016-07-05

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

PubMed

Brockstedt, D G; Podsakoff, G M; Fong, L; Kurtzman, G; Mueller-Ruchholtz, W; Engleman, E G

1999-07-01

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations. Copyright 1999 Academic Press.

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

PubMed

Nambiar, Bindu; Cornell Sookdeo, Cathleen; Berthelette, Patricia; Jackson, Robert; Piraino, Susan; Burnham, Brenda; Nass, Shelley; Souza, David; O'Riordan, Catherine R; Vincent, Karen A; Cheng, Seng H; Armentano, Donna; Kyostio-Moore, Sirkka

2017-02-01

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Recombination and Population Mosaic of a Multifunctional Viral Gene, Adeno-Associated Virus cap

PubMed Central

Takeuchi, Yasuhiro; Myers, Richard; Danos, Olivier

2008-01-01

Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV) cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI) revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u) region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred. PMID:18286191

Preclinical evaluation of radiation and systemic, RGD-targeted, adeno-associated virus phage-TNF gene therapy in a mouse model of spontaneously metastatic melanoma.

PubMed

Quinn, T J; Healy, N; Sara, A; Maggi, E; Claros, C S; Kabarriti, R; Scandiuzzi, L; Liu, L; Gorecka, J; Adem, A; Basu, I; Yuan, Z; Guha, C

2017-01-01

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

Pseudotyped adeno-associated virus 2/9-delivered CCL11 shRNA alleviates lung inflammation in an allergen-sensitized mouse model.

PubMed

Wu, Chia-Jen; Huang, Wen-Chung; Chen, Li-Chen; Shen, Chia-Rui; Kuo, Ming-Ling

2012-11-01

Airway infiltration by eosinophils is a major characteristic of chronic asthma. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as the major chemokine for eosinophil recruitment. Pseudotyped adeno-associated virus (AAV) 2/9, composed by the AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether pseudotyped AAV2/9 virus carrying the small hairpin RNA targeting CCL11 and expressed by CMV/U6 promoter could reduce eosinophilia and asthmatic responses in mite allergen-sensitized mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 allergen (rDp2). AAV2/9 viral vectors were intratracheally injected three days before the first challenge. AAV2/9 sh47 virus significantly reduced airway hyperresponsiveness, airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-10, were also significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. The results suggest that AAV2/9 sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the CMV/U6 promoter with AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma.

Pseudotyped Adeno-Associated Virus 2/9-Delivered CCL11 shRNA Alleviates Lung Inflammation in an Allergen-Sensitized Mouse Model

PubMed Central

Wu, Chia-Jen; Huang, Wen-Chung; Chen, Li-Chen; Shen, Chia-Rui

2012-01-01

Abstract Airway infiltration by eosinophils is a major characteristic of chronic asthma. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as the major chemokine for eosinophil recruitment. Pseudotyped adeno-associated virus (AAV) 2/9, composed by the AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether pseudotyped AAV2/9 virus carrying the small hairpin RNA targeting CCL11 and expressed by CMV/U6 promoter could reduce eosinophilia and asthmatic responses in mite allergen-sensitized mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 allergen (rDp2). AAV2/9 viral vectors were intratracheally injected three days before the first challenge. AAV2/9 sh47 virus significantly reduced airway hyperresponsiveness, airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-10, were also significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. The results suggest that AAV2/9 sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the CMV/U6 promoter with AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma. PMID:22913580

Adeno-associated virus transfer of a gene encoding SNAP-25 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism.

PubMed

Raghunath, Arvind; Perez-Branguli, Francesc; Smith, Leonard; Dolly, J Oliver

2008-04-02

Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

PubMed

Hiramoto, Takafumi; Li, Li B; Funk, Sarah E; Hirata, Roli K; Russell, David W

2018-05-02

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4 + and CD8 + T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

PubMed

Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

1996-07-15

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

PubMed

Li, Hua; Zhang, Feng-Lan; Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

2015-01-01

The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

PubMed

Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

2008-03-01

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

PubMed

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

2017-10-15

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

PubMed Central

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard

2017-01-01

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids

PubMed Central

Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

2016-01-01

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration—at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then—through a variety of mechanisms—result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales

PubMed Central

Negrete, Alejandro; Kotin, Robert M.

2007-01-01

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g.≥1016 particles per production run, utilizes Baculovirus Expression Vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred-tank bioreactors are single-use, disposable bioreactors, e.g. Wave™. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave™ bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave™ bioreactors. Comparable yields of rAAV, ~2e+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration. Keywords: adeno-associated vectors; large-scale production; stirred tank bioreactor; wave bioreactor; gene therapy. PMID:17606302

Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

PubMed

Kia, Azadeh; Yata, Teerapong; Hajji, Nabil; Hajitou, Amin

2013-10-22

Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

A 1-Year Quantitative Survey of Noro-, Adeno-, Human Boca-, and Hepatitis E Viruses in Raw and Secondarily Treated Sewage from Two Plants in Norway.

PubMed

Myrmel, M; Lange, H; Rimstad, E

2015-09-01

A study of enteric viruses in raw and treated sewage from two secondary treatment plants, which received sewage from Oslo city (plant A) and small municipalities in Hedmark county in Norway (plant B), showed high levels of noro-, adeno-, and bocavirus throughout the year. A seasonal variation was observed for adeno- and GII norovirus with higher levels during winter and bocavirus that had more positive samples during winter. The virus concentrations in raw sewage were comparable in the two plants, with medians (log10 genome copies per liter) of 6.1, 6.3, 6.0, and 4.5 for noro GI, noro GII, adeno-, and bocavirus, respectively. The level of hepatitis E virus was not determined as it was below the limit of quantification. The mean log10 virus reduction was 0.55 (plant A) and 1.44 (plant B) with the highest reduction found in the plant with longer hydraulic retention time. The adenoviruses were dominantly serotype 41, while serotype 12 appeared sporadically. Of the 102 raw and treated sewage samples that were tested, eight were positive for hepatitis E virus of which four were from treated sewage. Two of the four obtained gene sequences from hepatitis E virus originated from the rural sewage samples and showed high similarity with a genotype 3 strain of hepatitis E virus detected in local piglets. Two other hepatitis E virus sequences obtained from urban sewage samples showed high similarities with genotype 3 strains isolated from urban sewage in Spain and a human genotype 1 isolate from India. The study gives information on the levels of noroviruses in raw and treated sewage, which is valuable to risk assessment, information indicating that some infections with hepatitis E viruses in Norway have a regional origin and that human bocavirus 2 and 3 are prevalent in the Norwegian population.

Postentry processing of recombinant adeno-associated virus type 1 and transduction of the ferret lung are altered by a factor in airway secretions.

PubMed

Yan, Ziying; Sun, Xingshen; Evans, Idil A; Tyler, Scott R; Song, Yi; Liu, Xiaoming; Sui, Hongshu; Engelhardt, John F

2013-09-01

We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

Adeno Associated Viral-mediated intraosseus labeling of bone marrow derived cells for CNS tracking

PubMed Central

Selenica, Maj-Linda B.; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B.; Nash, Kevin R.; Morgan, Dave; Gordon, Marcia N.; Lee, Daniel C.

2016-01-01

Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseus impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9–GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will

Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

PubMed

Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

2015-10-15

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

PubMed

Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

2009-11-01

Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

PubMed

Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

2016-02-01

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions.

PubMed Central

Oelze, I; Rittner, K; Sczakiel, G

1994-01-01

Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHHH element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHHH also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NF kappa B and SP1 binding sites have no significant influence on the rep-mediated inhibition. Images PMID:8289357

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

PubMed Central

Lock, Martin; McGorray, Susan; Auricchio, Alberto; Ayuso, Eduard; Beecham, E. Jeffrey; Blouin-Tavel, Véronique; Bosch, Fatima; Bose, Mahuya; Byrne, Barry J.; Caton, Tina; Chiorini, John A.; Chtarto, Abdelwahed; Clark, K. Reed; Conlon, Thomas; Darmon, Christophe; Doria, Monica; Douar, Anne; Flotte, Terence R.; Francis, Joyce D.; Francois, Achille; Giacca, Mauro; Korn, Michael T.; Korytov, Irina; Leon, Xavier; Leuchs, Barbara; Lux, Gabriele; Melas, Catherine; Mizukami, Hiroaki; Moullier, Philippe; Müller, Marcus; Ozawa, Keiya; Philipsberg, Tina; Poulard, Karine; Raupp, Christina; Rivière, Christel; Roosendaal, Sigrid D.; Samulski, R. Jude; Soltys, Steven M.; Surosky, Richard; Tenenbaum, Liliane; Thomas, Darby L.; van Montfort, Bart; Veres, Gabor; Wright, J. Fraser; Xu, Yili; Zelenaia, Olga; Zentilin, Lorena

2010-01-01

Abstract A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus–free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 × 1011 particles/ml; 95% confidence interval [CI], 7.89 × 1011 to 1.05 × 1012 particles/ml), vector genomes ({X}, 3.28 × 1010 vector genomes/ml; 95% CI, 2.70 × 1010 to 4.75 × 1010 vector genomes/ml), transducing units ({X}, 5.09 × 108 transducing units/ml; 95% CI, 2.00 × 108 to 9.60 × 108 transducing units/ml), and infectious units ({X}, 4.37 × 109 TCID50 IU/ml; 95% CI, 2.06 × 109 to 9.26 × 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited

Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

PubMed

Earley, Lauriel F; Powers, John M; Adachi, Kei; Baumgart, Joshua T; Meyer, Nancy L; Xie, Qing; Chapman, Michael S; Nakai, Hiroyuki

2017-02-01

Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly. Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities

Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

PubMed Central

Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

2013-01-01

Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer

PubMed Central

Takahashi, Hiroshi; Hirai, Yukihiko; Migita, Makoto; Seino, Yoshihiko; Fukuda, Yuh; Sakuraba, Hitoshi; Kase, Ryoichi; Kobayashi, Toshihide; Hashimoto, Yasuhiro; Shimada, Takashi

2002-01-01

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, α-galactosidase A (α-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the α-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that α-gal A activity in plasma was increased to ≈25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-α-gal A antibodies. The α-gal A activity in various organs of treated Fabry mice remained 5–20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease. PMID:12370426

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

PubMed

van den Brand, Judith M A; Wohlsein, Peter; Herfst, Sander; Bodewes, Rogier; Pfankuche, Vanessa M; van de Bildt, Marco W G; Seehusen, Frauke; Puff, Christina; Richard, Mathilde; Siebert, Ursula; Lehnert, Kristina; Bestebroer, Theo; Lexmond, Pascal; Fouchier, Ron A M; Prenger-Berninghoff, Ellen; Herbst, Werner; Koopmans, Marion; Osterhaus, Albert D M E; Kuiken, Thijs; Baumgärtner, Wolfgang

2016-01-01

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.

Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors.

PubMed

Fraites, Thomas J; Schleissing, Mary R; Shanely, R Andrew; Walter, Glenn A; Cloutier, Denise A; Zolotukhin, Irene; Pauly, Daniel F; Raben, Nina; Plotz, Paul H; Powers, Scott K; Kessler, Paul D; Byrne, Barry J

2002-05-01

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

Prophylaxis and Treatment of Alzheimer's Disease by Delivery of an Adeno-Associated Virus Encoding a Monoclonal Antibody Targeting the Amyloid Beta Protein

PubMed Central

Shimada, Masaru; Abe, Shinya; Takahashi, Toru; Shiozaki, Kazumasa; Okuda, Mitsue; Mizukami, Hiroaki; Klinman, Dennis M.; Ozawa, Keiya; Okuda, Kenji

2013-01-01

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (Aß) protein. Repeated injection of that mAb reduced the accumulation of Aß protein in the brain of human Aß transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0×1010 viral genome of these AAV vectors into C57BL/6 mice generated serum anti-Aß Ab levels up to 0.3 mg/ml. Anti-Aß Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on Aß levels in vivo was examined. A significant decrease in Aß levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-Aß Ab for the prevention and treatment of Alzheimer's disease. PMID:23555563

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

PubMed Central

Adamson-Small, Laura; Potter, Mark; Byrne, Barry J.; Clément, Nathalie

2017-01-01

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 1015 VG in crude cell harvests and an average of 3.5 × 1014 total VG of purified rAAV9 material, resulting in greater than 1 × 105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production. PMID:28117600

Adeno-Associated Virus-Mediated Correction of a Canine Model of Glycogen Storage Disease Type Ia

PubMed Central

Weinstein, David A.; Correia, Catherine E.; Conlon, Thomas; Specht, Andrew; Verstegen, John; Onclin-Verstegen, Karine; Campbell-Thompson, Martha; Dhaliwal, Gurmeet; Mirian, Layla; Cossette, Holly; Falk, Darin J.; Germain, Sean; Clement, Nathalie; Porvasnik, Stacy; Fiske, Laurie; Struck, Maggie; Ramirez, Harvey E.; Jordan, Juan; Andrutis, Karl; Chou, Janice Y.; Byrne, Barry J.

2010-01-01

Abstract Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-α. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver. PMID:20163245

Insulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured Cells.

PubMed

Carrig, Sean; Bijjiga, Enoch; Wopat, Mitchell J; Martino, Ashley T

2016-11-01

Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α 1 -antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates

Transduction of Nonhuman Primate Brain with Adeno-Associated Virus Serotype 1: Vector Trafficking and Immune Response

PubMed Central

Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L.; Bankiewicz, Krystof S.

2009-01-01

Abstract We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohisto-chemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm3 coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4+ lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2. PMID:19292604

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes.

PubMed

Yan, Ziying; Feng, Zehua; Sun, Xingshen; Zhang, Yulong; Zou, Wei; Wang, Zekun; Jensen-Cody, Chandler; Liang, Bo; Park, Soo-Yeun; Qiu, Jianming; Engelhardt, John F

2017-08-01

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients.

PubMed

Leborgne, Christian; Latournerie, Virginie; Boutin, Sylvie; Desgue, Diana; Quéré, Aliénor; Pignot, Elodie; Collaud, Fanny; Charles, Séverine; Simon Sola, Marcelo; Masat, Elisa; Jouen, Fabienne; Boyer, Olivier; Masurier, Carole; Mingozzi, Federico; Veron, Philippe

2018-03-16

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans. Copyright © 2018. Published by Elsevier Inc.

Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

PubMed

Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

2012-03-01

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

PubMed Central

Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny

2012-01-01

Abstract Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

PubMed

Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

2011-06-01

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

PubMed Central

Kilpatrick, Lauren A.; Li, Qian; Yang, John; Goddard, John C; Fekete, Donna M.; Lang, Hainan

2010-01-01

Murine models are ideal for studying cochlear gene transfer as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, due to its small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAV) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear and allows for near-complete preservation of low and middle frequency hearing. In the present study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6, and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus (CMV) promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells (IHCs) were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness. PMID:21209625

Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

PubMed

Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

2002-02-05

Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses ▿ †

PubMed Central

Grimm, Dirk; Lee, Joyce S.; Wang, Lora; Desai, Tushar; Akache, Bassel; Storm, Theresa A.; Kay, Mark A.

2008-01-01

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications. PMID:18400866

Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes

PubMed Central

Halbert, Christine L.; Rutledge, Elizabeth A.; Allen, James M.; Russell, David W.; Miller, A. Dusty

2000-01-01

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors. PMID:10627564

Effective relief of neuropathic pain by adeno-associated virus-mediated expression of a small hairpin RNA against GTP cyclohydrolase 1

PubMed Central

2009-01-01

Background Recent studies show that transcriptional activation of GTP cyclohydrolase I (GCH1) in dorsal root ganglia (DRG) is significantly involved in the development and persistency of pain symptoms. We thus hypothesize that neuropathic pain may be attenuated by down-regulation of GCH1 expression, and propose a gene silencing system for this purpose. Results To interrupt GCH1 synthesis, we designed a bidirectional recombinant adeno-associated virus encoding both a small hairpin RNA against GCH1 and a GFP reporter gene (rAAV-shGCH1). After rAAV-shGCH1 was introduced into the sciatic nerve prior to or following pain-inducing surgery, therapeutic efficacy and the underlying mechanisms were subsequently validated in animal models. The GFP expression data indicates that rAAV effectively delivered transgenes to DRG. Subsequently reduced GCH1 expression was evident from immunohistochemistry and western-blotting analysis. Along with the down-regulation of GCH1, the von Frey test correspondingly indicated a sharp decline in pain symptoms upon both pre- and post-treatment with rAAV-shGCH1. Interestingly, GCH1 down-regulation additionally led to decreased microglial activation in the dorsal horn, implying an association between pain attenuation and reduced inflammation. Conclusion Therefore, the data suggests that GCH1 levels can be reduced by introducing rAAV-shGCH1, leading to pain relief. Based on the results, we propose that GCH1 modulation may be developed as a clinically applicable gene therapy strategy to treat neuropathic pain. PMID:19922668

HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

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

Cho, Mi Ae; Endocrinology, Dong Rae Bong Seng Hospital, Busan; Yashar, Parham

2008-07-04

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the {beta}-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. Themore » expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.« less

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

PubMed Central

Murphy, John E.; Zhou, Shangzhen; Giese, Klaus; Williams, Lewis T.; Escobedo, Jaime A.; Dwarki, Varavani J.

1997-01-01

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity. PMID:9391128

Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.

PubMed

Roche-Molina, Marta; Sanz-Rosa, David; Cruz, Francisco M; García-Prieto, Jaime; López, Sergio; Abia, Rocío; Muriana, Francisco J G; Fuster, Valentín; Ibáñez, Borja; Bernal, Juan A

2015-01-01

Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels. Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses. © 2014 American Heart Association, Inc.

Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport

PubMed Central

Castle, Michael J.; Gershenson, Zachary T.; Giles, April R.; Holzbaur, Erika L.F.

2014-01-01

Abstract Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane

Therapeutic effect of adeno-associated virus (AAV)-mediated ADNF-9 expression on cochlea of kanamycin-deafened guinea pigs.

PubMed

Zheng, Guoxi; Zhu, Zhu; Zhu, Kang; Wei, Junrong; Jing, Yang; Duan, Maoli

2013-10-01

rAAV-NT4-ADNF-9 could ameliorate the damage to auditory function and repair previous impairment of cochlear hair cell loss induced by kanamycin. To investigate the therapeutic effect of ADNF-9 on cochlear hair cells using the recombinant adeno-associated virus (AAV) carrying fusion gene NT4-ADNF-9 and the kanamycin-deafened guinea pig model. Forty white guinea pigs with normal auricle reflex and normal auditory brainstem responses (ABRs) were randomly divided into four groups. Kanamycin was administered to the animals in groups A, B, and C to establish the deafened guinea pig model. rAAV-NT4-ADNF-9, vector only, and artificial perilymph were then delivered to the cochlear tissue of animals in groups A, B, and C, respectively, through the round window membrane. Animals in group D did not receive any treatment and acted as normal controls. The hearing thresholds on the surgery side were recorded before and after the transfection treatment. Fourteen days after treatment, cochleae were removed for paraffin slide preparation and cochlear surface preparation. A phase contrast microscope was used to observe the protective effect of ADNF-9 on hair cells. Significant reduction of the ABR threshold was observed after rAAV-NT4-ADNF-9 treatment (p < 0.05). After 14 days of treatment, the ABR threshold was also significantly different between the rAAV-NT4-ADNF-9-infected group and the non-infected group. Moreover, phase contrast microscopy showed significantly less hair cell damage or hair cell loss in the group treated with rAAV-NT4-ADNF-9 than in the groups treated with vector only or artificial perilymph (p < 0.05).

Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.

PubMed

Matsuzaki, Yasunori; Konno, Ayumu; Mochizuki, Ryuta; Shinohara, Yoichiro; Nitta, Keisuke; Okada, Yukihiro; Hirai, Hirokazu

2018-02-05

Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken β-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0-2%) and astrocytes (0.1-2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

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

Zhao Weihong; Wu Jianqing; Zhong Li

2006-09-30

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency ofmore » conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of

Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm.

PubMed

Lai, Chao-Han; Wang, Kuan-Chieh; Kuo, Cheng-Hsiang; Lee, Fang-Tzu; Cheng, Tsung-Lin; Chang, Bi-Ing; Yang, Yu-Jen; Shi, Guey-Yueh; Wu, Hua-Lin

2017-07-01

Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl 2 -induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl 2 -induced AAA model and angiotensin II-infused AAA model, respectively. In the CaCl 2 -induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (10 11 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

PubMed

Valdor, Markus; Wagner, Anke; Röhrs, Viola; Berg, Johanna; Fechner, Henry; Schröder, Wolfgang; Tzschentke, Thomas M; Bahrenberg, Gregor; Christoph, Thomas; Kurreck, Jens

2018-01-01

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

PubMed

Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

2016-05-01

IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

Photoreceptor protection by adeno-associated virus-mediated LEDGF expression in the RCS rat model of retinal degeneration: probing the mechanism.

PubMed

Raz-Prag, Dorit; Zeng, Yong; Sieving, Paul A; Bush, Ronald A

2009-08-01

Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored. Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control. Retinal function was evaluated 8 weeks later by the electroretinogram and compared with photoreceptor cell layer count. LEDGF mRNA and protein levels and mRNA levels of known stress-related factors were compared in treated and control retinas to explore the mechanism of LEDGF protection. Nine RCS rats were treated with adenovirus-heat shock protein 27 (Ad-HSP27) and examined for protection. Significant photoreceptor protection was evident functionally and morphologically in 65% to 100% of the RCS rats treated at early ages of up to 7 weeks. Cell protection was more prominent in the superior retinal hemisphere which has a slower natural degeneration rate in untreated eyes. Although many of the heat shock proteins and other stress-related genes showed significant elevation in the AAV-Ledgf-treated eyes, all increases were approximately twofold or less. Transduction of retinal cells with Ad-HSP27 also resulted in photoreceptor protection. AAV-Ledgf elicited no photoreceptor functional protection in P23H rhodopsin transgenic rat retina. Chronic LEDGF treatment via AAV-Ledgf administration gave successful protection of photoreceptors in the RCS rat retina and retarded cell death by about 2 weeks. Induction of heat shock proteins also gave photoreceptor protection. However, compelling evidence was not found that LEDGF protection was associated with upregulation of heat shock proteins.

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

PubMed Central

Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

2015-01-01

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

PubMed

Murayama, Asako; Kato, Takanobu; Akazawa, Daisuke; Sugiyama, Nao; Date, Tomoko; Masaki, Takahiro; Nakamoto, Shingo; Tanaka, Yasuhito; Mizokami, Masashi; Yokosuka, Osamu; Nomoto, Akio; Wakita, Takaji

2012-02-01

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.

PubMed

Piedra, Jose; Ontiveros, Maria; Miravet, Susana; Penalva, Cristina; Monfar, Mercè; Chillon, Miguel

2015-02-01

Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

Adeno-associated virus-mediated expression of myostatin propeptide improves the growth of skeletal muscle and attenuates hyperglycemia in db/db mice.

PubMed

Jiang, J G; Shen, G F; Li, J; Qiao, C; Xiao, B; Yan, H; Wang, D W; Xiao, X

2017-03-01

Inhibition of myostatin, a negative growth modulator for muscle, can functionally enhance muscle mass and improve glucose and fat metabolism in myostatin propeptide (MPRO) transgenic mice. This study was to investigate whether myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of MPRO could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in the db/db mice and the mechanisms involved in that process. Eight-week-old male db/db mice were administered saline, AAV-GFP and AAV-MPRO/Fc vectors and monitored random blood glucose levels and body weight for 36 weeks. Body weight gain was not different during follow-up among the groups, but AAV-MPRO/Fc vectors resulted high level of MPRO in the blood companied by an increase in skeletal muscle mass and muscle hypertrophy. In addition, AAV-MPRO/Fc-treated db/db mice showed significantly lower blood glucose and insulin levels and significantly increased glucose tolerance and insulin sensitivity compared with the control groups (P<0.05). Moreover, these mice exhibited lower triglyceride (TG) and free fatty acid (FFA) content in the skeletal muscle, although no difference was observed in fat pad weights and serum TG and FFA levels. Finally, AAV-MPRO/Fc-treated mice had enhanced insulin signaling in the skeletal muscle. These data suggest that AAV-mediated MPRO therapy may provide an important clue for potential clinical applications to prevent type II diabetes, and these studies confirm that MPRO is a therapeutic target for type II diabetes.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

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

Gottlieb, J.; Muzyczka, N.

1988-06-01

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat andmore » in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.« less

Recombinant adeno-associated virus-delivered hypoxia-inducible stanniocalcin-1 expression effectively inhibits hypoxia-induced cell apoptosis in cardiomyocytes.

PubMed

Shi, Xin; Wang, Jianzhong; Qin, Yan

2014-12-01

Ischemia/hypoxia-induced oxidative stress is detrimental for the survival of cardiomyocytes and cardiac function. Stanniocalcin-1 (STC-1), a glycoprotein, has been found to play an inhibitory role in the production of reactive oxygen species (ROS). Here, we speculated that the overexpression of STC-1 might alleviate oxidative damage in cardiomyocytes under conditions of hypoxia. To control the expression of STC-1 in hypoxia, we constructed a recombinant adeno-associated virus (AAV) carrying the hypoxia-responsive element (HRE) to mediate hypoxia induction. Cardiomyocytes were infected with AAV-HRE-STC-1 and cultured in normoxic or hypoxic conditions, and STC-1 overexpression was only detected in hypoxic cultured cardiomyocytes by using quantitative real-time polymerase chain reaction and Western blot analysis. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, AAV-HRE-STC-1 infection was shown to significantly enhance cell survival under hypoxia. Hypoxia-induced cell apoptosis was inhibited by AAV-HRE-STC-1 infection by using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis assay. Moreover, the proapoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2, which were dysregulated by hypoxia, were reversed by AAV-HRE-STC-1 infection. AAV-HRE-STC-1-mediated STC-1 overexpression markedly inhibited ROS production in cardiomyocytes cultured under hypoxic conditions. AAV-HRE-STC-1 infection significantly upregulated uncoupled protein 3 (UCP3), whereas silencing of UCP3 blocked the inhibitory effect of AAV-HRE-STC-1 on ROS production. In contrast, AAV-HRE-STC-1 infection had no effect on UCP2, and knockdown of UCP2 did not block the inhibitory effect of AAV-HRE-STC-1 on ROS production in the cardiomyocytes cultured under hypoxic conditions. Taken together, STC1 activates antioxidant pathway in cardiomyocytes through the induction of UCP3, implying that AAV-HRE-STC-1 has potential in the treatment of ischemic

The effect of surface demineralization of cortical bone allograft on the properties of recombinant adeno-associated virus coatings.

PubMed

Yazici, Cemal; Yanoso, Laura; Xie, Chao; Reynolds, David G; Samulski, R Jude; Samulski, Jade; Yannariello-Brown, Judith; Gertzman, Arthur A; Zhang, Xinping; Awad, Hani A; Schwarz, Edward M

2008-10-01

Freeze-dried recombinant adeno-associated virus (rAAV) coated structural allografts have emerged as an approach to engender necrotic cortical bone with host factors that will persist for weeks following surgery to facilitate revascularization, osteointegration, and remodeling. However, one major limitation is the nonporous cortical surface that prohibits uniform distribution of the rAAV coating prior to freeze-drying. To overcome this we have developed a demineralization method to increase surface absorbance while retaining the structural integrity of the allograft. Demineralized bone wafers (DBW) made from human femoral allograft rings demonstrated a significant 21.1% (73.6+/-3.9% versus 52.5+/-2.6%; p<0.001) increase in percent surface area coating versus mineralized controls. Co-incubation of rAAV-luciferase (rAAV-Luc) coated DBW with a monolayer of C3H10T1/2 cells in culture led to peak luciferase levels that were not significantly different from soluble rAAV-Luc controls (p>0.05), although the peaks occurred at 60h and 12h, respectively. To assess the transduction efficiency of rAAV-Luc coated DBW in vivo, we first performed a dose response with allografts containing 10(7), 10(9) or 10(10) particles that were surgically implanted into the quadriceps of mice, and assayed by in vivo bioluminescence imaging (BLI) on days 1, 3, 5, 7, 10, 14, and 21. The results demonstrated a dose response in which the DBW coated with 10(10) rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 10(7) dose throughout this time period (p<0.01). A direct comparison of mineralized versus DBW coated with 10(10) rAAV-Luc particles failed to demonstrate any significant differences in transduction kinetics or efficiency in vivo. Thus, surface demineralization of human cortical bone allograft increases its absorbance for uniform rAAV coating, without affecting vector transduction efficiency.

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

PubMed

Barbash, I M; Cecchini, S; Faranesh, A Z; Virag, T; Li, L; Yang, Y; Hoyt, R F; Kornegay, J N; Bogan, J R; Garcia, L; Lederman, R J; Kotin, R M

2013-03-01

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Adeno-Associated Viral-Mediated Catalase Expression Suppresses Optic Neuritis in Experimental Allergic Encephalomyelitis

NASA Astrophysics Data System (ADS)

Guy, John; Qi, Xiaoping; Hauswirth, William W.

1998-11-01

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

[Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma].

PubMed

Li, Bai-Ling; Zhang, Guan-Xin; Hou, Xiao-Lei; Tan, Meng-Wei; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Huang, Sheng-Dong

2009-03-01

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. AAV-mediated expression of siRNA could reduce the expression

Adeno-associated virus-RNAi of GlyRα1 and characterization of its synapse-specific inhibition in OFF alpha transient retinal ganglion cells

PubMed Central

Zhang, C.; Rompani, S. B.; Roska, B.

2014-01-01

In the central nervous system, inhibition shapes neuronal excitation. In spinal cord glycinergic inhibition predominates, whereas GABAergic inhibition predominates in the brain. The retina uses GABA and glycine in approximately equal proportions. Glycinergic crossover inhibition, initiated in the On retinal pathway, controls glutamate release from presynaptic OFF cone bipolar cells (CBCs) and directly shapes temporal response properties of OFF retinal ganglion cells (RGCs). In the retina, four glycine receptor (GlyR) α-subunit isoforms are expressed in different sublaminae and their synaptic currents differ in decay kinetics. GlyRα1, expressed in both On and Off sublaminae of the inner plexiform layer, could be the glycinergic isoform that mediates On-to-Off crossover inhibition. However, subunit-selective glycine contributions remain unknown because we lack selective antagonists or cell class-specific subunit knockouts. To examine the role of GlyRα1 in direct inhibition in mature RGCs, we used retrogradely transported adeno-associated virus (AAV) that performed RNAi and eliminated almost all glycinergic spontaneous and visually evoked responses in PV5 (OFFαTransient) RGCs. Comparisons of responses in PV5 RGCs infected with AAV-scrambled-short hairpin RNA (shRNA) or AAV-Glra1-shRNA confirm a role for GlyRα1 in crossover inhibition in cone-driven circuits. Our results also define a role for direct GlyRα1 inhibition in setting the resting membrane potential of PV5 RGCs. The absence of GlyRα1 input unmasked a serial and a direct feedforward GABAAergic modulation in PV5 RGCs, reflecting a complex interaction between glycinergic and GABAAergic inhibition. PMID:25231618

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

PubMed Central

Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

2009-01-01

Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

A Genome-Wide Association Study for the Incidence of Persistent Bovine Viral Diarrhea Virus Infection in Cattle.

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Virus (BVDV) is a diverse group of viruses causing disease in ruminants. The objective was to determine genomic regions harboring single nucleotide polymorphisms (SNP) associated with presence or absence of persistent BVDV infections. A genome wide association approach based on...

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

PubMed

Hashimoto, Haruo; Mizushima, Tomoko; Chijiwa, Tsuyoshi; Nakamura, Masato; Suemizu, Hiroshi

2017-06-15

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction. Copyright © 2017 Elsevier B.V. All rights reserved.

Long-Term Efficacy Following Readministration of an Adeno-Associated Virus Vector in Dogs with Glycogen Storage Disease Type Ia

PubMed Central

Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D.; Landau, Dustin J.; Drake, Elizabeth J.; Kozink, Daniel M.; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R.; Brown, Talmage T.; Kemper, Alex R.

2012-01-01

Abstract Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV

Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia.

PubMed

Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D; Landau, Dustin J; Drake, Elizabeth J; Kozink, Daniel M; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R; Brown, Talmage T; Kemper, Alex R; Koeberl, Dwight D

2012-04-01

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector

Intracellular localization of adeno-associated viral proteins expressed in insect cells.

PubMed

Gallo-Ramírez, Lilí E; Ramírez, Octavio T; Palomares, Laura A

2011-01-01

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Association of an alphasatellite with tomato yellow leaf curl virus and ageratum yellow vein virus in Japan is suggestive of a recent introduction.

PubMed

Shahid, Muhammad Shafiq; Ikegami, Masato; Waheed, Abdul; Briddon, Rob W; Natsuaki, Keiko T

2014-01-14

Samples were collected in 2011 from tomato plants exhibiting typical tomato leaf curl disease symptoms in the vicinity of Komae, Japan. PCR mediated amplification, cloning and sequencing of all begomovirus components from two plants from different fields showed the plants to be infected by Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein virus (AYVV). Both viruses have previously been shown to be present in Japan, although this is the first identification of AYVV on mainland Japan; the virus previously having been shown to be present on the Okinawa Islands. The plant harboring AYVV was also shown to contain the betasatellite Tomato leaf curl Java betasatellite (ToLCJaB), a satellite not previously shown to be present in Japan. No betasatellite was associated with the TYLCV infected tomato plants analyzed here, consistent with earlier findings for this virus in Japan. Surprisingly both plants were also found to harbor an alphasatellite; no alphasatellites having previously been reported from Japan. The alphasatellite associated with both viruses was shown to be Sida yellow vein China alphasatellite which has previously only been identified in the Yunnan Province of China and Nepal. The results suggest that further begomoviruses, and their associated satellites, are being introduced to Japan. The significance of these findings is discussed.

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

PubMed

Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

2007-05-01

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.

PubMed

Lalwani, A K; Walsh, B J; Reilly, P G; Muzyczka, N; Mhatre, A N

1996-07-01

Gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. However, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. The purpose of this study was to assess the feasibility of introducing genetic material directly into the peripheral auditory system using adeno-associated virus (AAV) as the transfection vector and Hartley guinea pigs as the animal model. Approximately 10(5) particles of AAV containing the bacterial beta-galactosidase (beta-gal) sequence with Ad 2 major late promoter were infused into the cochlea of the animal with the aid of an osmotic minipump. Animals were killed after 2 weeks. Two Hartley guinea pigs with intracochlear saline infusion and four unoperated (nonperfused) animals served as negative controls. Both, the infused and the contralateral, non-infused cochleae were harvested from each animal, decalcified, and embedded in paraffin. Sections, 8 microns in width, were cut from the embedded cochleae and assayed for beta-gal expression via immunohistochemistry. Animals perfused with AAV showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti in the perfused cochlea and a much weaker staining but with similar pattern in the contralateral ear. Cochleae from saline-infused and unoperated animals were devoid of the DAB stain. This study demonstrates for the first time in vivo expression of a foreign gene within the mammalian inner ear resulting from its localized, AAV-mediated introduction. The ability to introduce and stably express exogenous genetic material in the peripheral auditory system will have both experimental and therapeutic benefits. These results lay the groundwork for future studies assessing the

Primary sensory neuron-specific interference of TRPV1 signaling by adeno-associated virus-encoded TRPV1 peptide aptamer attenuates neuropathic pain

PubMed Central

Xiang, Hongfei; Liu, Zhen; Wang, Fei; Xu, Hao; Roberts, Christopher; Fischer, Gregory; Stucky, Cheryl L; Dean, Caron; Pan, Bin; Hogan, Quinn H; Yu, Hongwei

2017-01-01

Background TRPV1 (transient receptor potential vanilloid subfamily member 1) is a pain signaling channel highly expressed in primary sensory neurons. Attempts for analgesia by systemic TRPV1 blockade produce undesirable side effects, such as hyperthermia and impaired heat pain sensation. One approach for TRPV1 analgesia is to target TRPV1 along the peripheral sensory pathway. Results For functional blockade of TRPV1 signaling, we constructed an adeno-associated virus (AAV) vector expressing a recombinant TRPV1 interfering peptide aptamer, derived from a 38mer tetrameric assembly domain (TAD), encompassing residues 735 to 772 of rat TRPV1, fused to the C-terminus of enhanced green fluorescent protein (EGFP). AAV-targeted sensory neurons expressing EGFP-TAD after vector injection into the dorsal root ganglia (DRG) revealed decreased inward calcium current and diminished intracellular calcium accumulation in response to capsaicin, compared to neurons of naïve or expressing EGFP alone. To examine the potential for treating neuropathic pain, AAV-EGFP-TAD was injected into fourth and fifth lumbar (L) DRGs of rats subjected to neuropathic pain by tibial nerve injury (TNI). Results showed that AAV-directed selective expression of EGFP-TAD in L4/L5 DRG neuron somata, and their peripheral and central axonal projections can limit TNI-induced neuropathic pain behavior, including hypersensitivity to heat and, to a less extent, mechanical stimulation. Conclusion Selective inhibition of TRPV1 activity in primary sensory neurons by DRG delivery of AAV-encoded analgesic interfering peptide aptamers is efficacious in attenuation of neuropathic pain. With further improvements of vector constructs and in vivo application, this approach might have the potential to develop as an alternative gene therapy strategy to treat chronic pain, especially heat hypersensitivity, without complications due to systemic TRPV1 blockade. PMID:28604222

Adeno-Associated Virus–Mediated Gene Therapy for Metabolic Myopathy

PubMed Central

Mah, Cathryn S.; Soustek, Meghan S.; Todd, A. Gary; McCall, Angela; Smith, Barbara K.; Corti, Manuela; Falk, Darin J.

2013-01-01

Abstract Metabolic myopathies are a diverse group of rare diseases in which impaired breakdown of stored energy leads to profound muscle dysfunction ranging from exercise intolerance to severe muscle wasting. Metabolic myopathies are largely caused by functional deficiency of a single gene and are generally subcategorized into three major types of metabolic disease: mitochondrial, lipid, or glycogen. Treatment varies greatly depending on the biochemical nature of the disease, and unfortunately no definitive treatments exist for metabolic myopathy. Since this group of diseases is inherited, gene therapy is being explored as an approach to personalized medical treatment. Adeno-associated virus–based vectors in particular have shown to be promising in the treatment of several forms of metabolic myopathy. This review will discuss the most recent advances in gene therapy efforts for the treatment of metabolic myopathies. PMID:24164240

Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

ERIC Educational Resources Information Center

Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

PubMed

Kim, Eunmi; Oh, Ji-Seon; Ahn, Ik-Sung; Park, Kook In; Jang, Jae-Hyung

2011-11-01

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Intracranial delivery of Interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway

PubMed Central

Yang, Junling; Kou, Jinghong; Lim, Jeong-Eun; Lalonde, Robert; Fukuchi, Ken-ichiro

2015-01-01

Interleukin-17A (IL-17A) is generally considered as one of the pathogenic factors involved in multiple sclerosis (MS). Indirect evidence for this is that IL-17A-producing T helper 17 (Th17) cells preferentially accumulate in lesions of MS and experimental autoimmune encephalomyelitis (EAE). However, a direct involvement of IL-17A in MS pathogenesis is still an open question. In this study, we overexpressed IL-17A in the brains of mice (IL-17A-in-Brain mice) via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated gene delivery. In spite of high levels of IL-17A expression in the brain and blood, IL-17A-in-Brain mice exhibit no inflammatory responses and no abnormalities in motor coordination and spatial orientation. Unexpectedly, IL-17A-in-Brain mice show decreases in body weight and adipose tissue mass and an improvement in glucose tolerance and insulin sensitivity. IL-17A enhances glucose uptake in PC12 cells by activation of AKT. Our results provide direct evidence for the first time that IL-17A overexpression in the central nervous system does not cause physical and learning disabilities and neuroinflammation and suggest that IL-17A may regulate glucose metabolism through the AKT signaling pathway. PMID:26562537

Seroprevalence of Antibodies against Seal Influenza A(H10N7) Virus in Harbor Seals and Gray Seals from the Netherlands.

PubMed

Bodewes, Rogier; Rubio García, Ana; Brasseur, Sophie M; Sanchez Conteras, Guillermo J; van de Bildt, Marco W G; Koopmans, Marion P G; Osterhaus, Albert D M E; Kuiken, Thijs

2015-01-01

In the spring and summer 2014, an outbreak of seal influenza A(H10N7) virus infection occurred among harbor seals (Phoca vitulina) off the coasts of Sweden and Denmark. This virus subsequently spread to harbor seals off the coasts of Germany and the Netherlands. While thousands of seals were reported dead in Sweden, Denmark and Germany, only a limited number of seals were found dead in the Netherlands. To determine the extent of exposure of seals in the Netherlands to influenza A/H10N7 virus, we measured specific antibody titers in serum samples from live-captured seals and seals admitted for rehabilitation in the Netherlands by use of a hemagglutination inhibition assay and an ELISA. In harbor seals in 2015, antibodies against seal influenza A(H10N7) virus were detected in 41% (32 out of 78) pups, 10% (5 out of 52) weaners, and 58% (7 out of 12) subadults or adults. In gray seals (Halichoerus grypus) in 2015, specific antibodies were not found in the pups (n = 26), but in 26% (5 out of 19) of the older animals. These findings indicate that, despite apparent low mortality, infection with seal influenza A(H10N7) virus was geographically widespread and also occurred in grey seals.

Targeted Gene Addition to a Safe Harbor locus in human CD34+ Hematopoietic Stem Cells for Correction of X-linked Chronic Granulomatous Disease

PubMed Central

De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi; Li, Linhong; Wu, Xiaolin; Su, Ling; Raley, Castle; Theobald, Narda; Choi, Uimook; Song, Alexander H.; Chan, Andy; Pearl, Jocelynn R.; Paschon, David E.; Lee, Janet; Newcombe, Hannah; Koontz, Sherry; Sweeney, Colin; Shivak, David A.; Zarember, Kol A.; Peshwa, Madhusudan V.; Gregory, Philip D.; Urnov, Fyodor D.; Malech, Harry L.

2016-01-01

Gene therapy with genetically modified human CD34+ hematopoietic stem cells (HSCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno associated virus (AAV) 6 delivery of donor constructs in human HSCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus-positive HSCs with 6–16% human cell marking were observed following engraftment into mice. In HSCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 in resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. PMID:26950749

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus (AAV) Types 2, 5, and 6 in Cystic Fibrosis and Normal Populations: Implications for Gene Therapy using AAV Vectors

PubMed Central

Halbert, Christine L.; Miller, A. Dusty; McNamara, Sharon; Emerson, Julia; Gibson, Ronald L.; Ramsey, Bonnie; Aitken, Moira L.

2014-01-01

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared to that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20–30% were seropositive for AAV6, and 10–20% were seropositive for AAV5. CF children were seropositive for AAV types 2, 5, or 6 at rates of 4–15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared to the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children. PMID:16610931

Variation in Tomato spotted wilt virus titer in Frankliniella occidentalis and its association with frequency of transmission.

PubMed

Rotenberg, Dorith; Krishna Kumar, Nallur K; Ullman, Diane E; Montero-Astúa, Mauricio; Willis, David K; German, Thomas L; Whitfield, Anna E

2009-04-01

Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by Frankliniella occidentalis, the western flower thrips. While it is well established that vector competence depends on TSWV acquisition by young larvae and virus replication within the insect, the biological factors associated with frequency of transmission have not been well characterized. We hypothesized that the number of transmission events by a single adult thrips is determined, in part, by the amount of virus harbored (titer) by the insect. Transmission time-course experiments were conducted using a leaf disk assay to determine the efficiency and frequency of TSWV transmission following 2-day inoculation access periods (IAPs). Virus titer in individual adult thrips was determined by real-time quantitative reverse transcriptase-PCR (qRT-PCR) at the end of the experiments. On average, 59% of adults transmitted the virus during the first IAP (2 to 3 days post adult-eclosion). Male thrips were more efficient at transmitting TSWV multiple times compared with female thrips of the same cohort. However, females harbored two to three times more copies of TSWV-N RNA per insect, indicating that factors other than absolute virus titer in the insect contribute to a successful transmission event. Examination of virus titer in individual insects at the end of the third IAP (7 days post adult-eclosion) revealed significant and consistent positive associations between frequency of transmission and virus titer. Our data support the hypothesis that a viruliferous thrips is more likely to transmit multiple times if it harbors a high titer of virus. This quantitative relationship provides new insights into the biological parameters that may influence the spread of TSWV by thrips.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

PubMed Central

Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V.; Nair, Sukesh C.; Srinivasan, Narayanaswamy; Srivastava, Alok

2013-01-01

Abstract Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (∼9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

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

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

PubMed

Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

2014-09-01

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

Myocardial Adeno-Associated Virus Serotype 6–βARKct Gene Therapy Improves Cardiac Function and Normalizes the Neurohormonal Axis in Chronic Heart Failure

PubMed Central

Rengo, Giuseppe; Lymperopoulos, Anastasios; Zincarelli, Carmela; Donniacuo, Maria; Soltys, Stephen; Rabinowitz, Joseph E.; Koch, Walter J.

2009-01-01

Background The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function. The peptide βARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve βAR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term βARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results In HF rats, we delivered βARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the β-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. βARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac βAR signaling. Addition of metoprolol neither enhanced nor decreased βARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions Long-term cardiac AAV6-βARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, βARKct alone improves outcomes more than a β-blocker alone, whereas both treatments are compatible. These findings show that βARKct gene therapy can be of long-term therapeutic value in HF. PMID:19103992

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

PubMed

Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

2012-03-01

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

PubMed

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

PubMed Central

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G.; Corydon, Thomas J.; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-01-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

Novel Adeno-Associated Viral Vector Delivering the Utrophin Gene Regulator Jazz Counteracts Dystrophic Pathology in mdx Mice

PubMed Central

Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

2014-01-01

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor “Jazz” that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. J. Cell. Physiol. 229: 1283–1291, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:24469912

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

PubMed Central

Halbert, Christine L.; Allen, James M.; Miller, A. Dusty

2001-01-01

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis. PMID:11413329

Adeno-associated virus type 8 vector–mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model

PubMed Central

Igarashi, Tsutomu; Miyake, Noriko; Fujimoto, Chiaki; Yaguchi, Chiemi; Iijima, Osamu; Shimada, Takashi; Takahashi, Hiroshi

2014-01-01

Purpose To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated an adeno-associated virus type 8 vector (AAV2/8) encoding an siRNA targeting vascular endothelial growth factor (VEGF), and determined the AAV2/8 vector’s ability to inhibit angiogenesis. Methods We initially transfected 3T3 cells expressing VEGF with the AAV2/8 plasmid vector psiRNA-VEGF using the H1 promoter and found that VEGF expression was significantly diminished in the transfectants. We next injected 1 μl (3 × 1014 vg/ml) of AAV2/8 vector encoding siRNA targeting VEGF (AAV2/8/SmVEGF-2; n = 12) or control vector encoding green fluorescent protein (GFP) (AAV2/8/GFP; n = 14) into the subretinal space in C57BL/6 mice. One week later, CNV was induced by using a diode laser to make four separate choroidal burns around the optic nerve in each eye. After an additional 2 weeks, the eyes were removed for flat mount analysis of the CNV surface area. Results Subretinal delivery of AAV2/8/SmVEGF-2 significantly diminished CNV at the laser lesions, compared to AAV8/GFP (1597.3±2077.2 versus 5039.5±4055.9 µm2; p<0.05). Using an enzyme-linked immunosorbent assay, we found that VEGF levels were reduced by approximately half in the AAV2/8/SmVEGF-2 treated eyes. Conclusions These results suggest that siRNA-VEGF can be expressed across the retina and that long-term suppression of CNV is possible through the use of stable AAV2/8-mediated siRNA-VEGF expression. In vivo gene therapy may thus be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration. PMID:24744609

The conundrum of the Epstein-Barr virus-associated gastric carcinoma in the Americas

PubMed Central

Carrasco-Avino, Gonzalo; Riquelme, Ismael; Padilla, Oslando; Villaseca, Miguel; Aguayo, Francisco R.; Corvalan, Alejandro H.

2017-01-01

Epstein-Barr virus-associated gastric carcinoma shows a higher prevalence in the Americas than Asia. We summarize all studies of Epstein Barr virus-associated gastric carcinoma in the Americas, focusing on host characteristics, environmental associations and phylogeographic diversity of Epstein-Barr virus strains. In the Americas, the prevalence of Epstein Barr virus-associated gastric carcinoma is 11.4%, more frequent in males and portray predominantly diffuse-type histology. EBERs, EBNAs, BARTs and LMP are the highest expressed genes; their variations in healthy individuals may explain the phylogeographic diversity of Epstein-Barr virus across the region. Gastric cancer cases harbor exclusively the western genotype (subtype D and kept Xho I site), suggesting a disrupted co-evolution between the pathogen and its host. Epstein-Barr virus-associated gastric carcinoma molecular subtype cases from The Cancer Genome Atlas display PIK3CA gene mutations, amplification of JAK2, PD-L1 and PD-L2 and CpG island methylator phenotype, leading to more extensive methylation of host and viral genomes than any other subtypes from the study. Environmental conditions include negative- and positive- associations with being firstborn child and smoking, respectively. A marginal association with H. pylori has also been reported. Lymphoepithelioma-like carcinoma is associated with Epstein Barr virus in 80%–86% of cases, most of which have been included as part of Epstein Barr virus-associated gastric carcinoma series (prevalence 1.1%–7.6%). Whether these cases represent a variant of Epstein-Barr virus-associated gastric carcinoma is discussed. We propose novel research strategies to solve the conundrum of the high prevalence of Epstein-Barr virus-associated gastric carcinoma in the Americas. PMID:29088902

ADenoVirus Initiative Study in Epidemiology (ADVISE): Results of a multicentric epidemiology study in Spain.

PubMed

Muñoz-Hernández, A M; Duquesroix, B; Benítez-Del-Castillo, J M

2018-03-01

Non-interventional, observational, epidemiology study to assess clinical characteristics and frequency of adenovirus conjunctivitis diagnosed by AdenoPlus ® in patients who presented with signs and symptoms of acute conjunctivitis. Safety aspects during use of the test were analyzed as well. This analysis presents the data obtained from the 386 patients enrolled in Spain. Patients had to present with acute signs and symptoms of conjunctivitis ≤7days. The minimum age was 1year old. Patients who had already used local antiviral therapies, topical steroids or immuno-modulators were not allowed to enter the study. A standardized questionnaire was used to collect patient's ocular history and test results. A total of 386 patients were recruited in 22 sites, being analyzed 329 patients. Among them, the percentage of "AdenoPlus ® positive" was 36,2% (119/329). Before the test was performed, in 84,1% of the cases, investigators believed that the conjunctivitis was of viral origin but only 50,3% of the investigators had their clinical assessment confirmed by the test. Patients who tested positive for adenoviral conjunctivitis presented higher percentages of signs and symptoms than the rest of the patients. None of the signs or symptoms could be qualified as pathognomonic of the disease, being difficult for the clinicians to perform an accurate diagnosis. AdenoPlus ® test is an antigen based immunoassay test that detects the presence of adenovirus directly from tears, and it can be an useful tool to help early differential diagnosis in patients with conjunctivitis signs and symptoms lasting for less than or equal to 7days. Copyright © 2017 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

PubMed Central

Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W.; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A.; Balogh, Karla K.; Tossi, Kerstin Pino; Roden, Richard B.S.; Christensen, Neil D.

2016-01-01

Vaccination with the minor capsid protein L2, notably the 17–36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17–36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum ± MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17–36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant. PMID:26382603

Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

PubMed Central

Xia, Li; Yin, Shankai; Wang, Jian

2012-01-01

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

Identification of Multiple Novel Viruses, Including a Parvovirus and a Hepevirus, in Feces of Red Foxes

PubMed Central

van der Giessen, Joke; Haagmans, Bart L.; Osterhaus, Albert D. M. E.; Smits, Saskia L.

2013-01-01

Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified. PMID:23616657

Muju Virus, Harbored by Myodes regulus in Korea, Might Represent a Genetic Variant of Puumala Virus, the Prototype Arvicolid Rodent-Borne Hantavirus

PubMed Central

Lee, Jin Goo; Gu, Se Hun; Baek, Luck Ju; Shin, Ok Sarah; Park, Kwang Sook; Kim, Heung-Chul; Klein, Terry A.; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture. PMID:24736214

Tunable protease-activatable virus nanonodes.

PubMed

Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

2014-05-27

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

NLX-P101, an adeno-associated virus gene therapy encoding glutamic acid decarboxylase, for the potential treatment of Parkinson's disease.

PubMed

Diaz-Nido, Javier

2010-07-01

Parkinson's disease (PD) is a neurodegenerative disease affecting nigrostriatal dopaminergic neurons. Dopamine depletion in the striatum leads to functional changes in several deep brain nuclei, including the subthalamic nucleus (STN), which becomes disinhibited and perturbs the control of body movement. Although there is no cure for PD, some pharmacological and surgical treatments can significantly improve the functional ability of patients, particularly in the early stages of the disease. Among neurodegenerative diseases, PD is a particularly suitable target for gene therapy because the neuropathology is largely confined to a relatively small region of the brain. Neurologix Inc is developing NLX-P101 (AAV2-GAD), an adeno-associated viral vector encoding glutamic acid decarboxylase (GAD), for the potential therapy of PD. As GAD potentiates inhibitory neurotransmission from the STN, sustained expression of GAD in the STN by direct delivery of NLX-P101 decreases STN overactivation. This procedure was demonstrated to be a safe and efficient method of reducing motor deficits in animal models of PD. A phase I clinical trial has demonstrated that NLX-P101 was safe and indicated the efficacy of this approach in patients with PD. Results from an ongoing phase II clinical trial of NLX-P101 are awaited to establish the clinical efficacy of this gene therapy.

Tunable Protease-Activatable Virus Nanonodes

PubMed Central

2015-01-01

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery. PMID:24796495

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

PubMed Central

Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

2016-01-01

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

In vitro evaluation of mitochondrial dysfunction and treatment with adeno-associated virus vector in fibroblasts from Doberman Pinschers with dilated cardiomyopathy and a pyruvate dehydrogenase kinase 4 mutation.

PubMed

Sosa, Ivan; Estrada, Amara H; Winter, Brandy D; Erger, Kirsten E; Conlon, Thomas J

2016-02-01

To compare mitochondrial oxygen consumption rate (OCR) of fibroblasts from Doberman Pinschers with and without dilated cardiomyopathy (DCM) and mutation of the gene for pyruvate dehydrogenase kinase isozyme 4 (PDK4) and to evaluate in vitro whether treatment with adeno-associated virus (AAV) vector (i.e., gene therapy) would alter metabolic efficiency. 10 Doberman Pinschers screened for DCM and PDK4 mutation. PROCEDURES Fibroblasts were harvested from skin biopsy specimens obtained from Doberman Pinschers, and dogs were classified as without DCM or PDK4 mutation (n = 3) or with occult DCM and heterozygous (4) or homozygous (3) for PDK4 mutation. Fibroblasts were or were not treated with tyrosine mutant AAV type 2 vector containing PDK4 at multiplicities of infection of 1,000. Mitochondrial OCR was measured to evaluate mitochondrial metabolism. The OCR was compared among dog groups and between untreated and treated fibroblasts within groups. Mean ± SD basal OCR of fibroblasts from heterozygous (74 ± 8 pmol of O2/min) and homozygous (58 ± 12 pmol of O2/min) dogs was significantly lower than that for dogs without PDK4 mutation (115 ± 9 pmol of O2/min). After AAV transduction, OCR did not increase significantly in any group (mutation-free group, 121 ± 26 pmol of O2/min; heterozygous group, 88 ± 6 pmol of O2/min; homozygous group, 59 ± 3 pmol of O2/min). Mitochondrial function was altered in skin fibroblasts of Doberman Pinschers with DCM and PDK4 mutation. Change in mitochondrial function after in vitro gene therapy at the multiplicities of infection used in this study was not significant.

Successful gene therapy in older Rpe65-deficient dogs following subretinal injection of an adeno-associated vector expressing RPE65.

PubMed

Annear, Matthew J; Mowat, Freya M; Bartoe, Joshua T; Querubin, Janice; Azam, Selina A; Basche, Mark; Curran, Paul G; Smith, Alexander J; Bainbridge, James W B; Ali, Robin R; Petersen-Jones, Simon M

2013-10-01

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors.

PubMed

Gray, Steven J; Foti, Stacey B; Schwartz, Joel W; Bachaboina, Lavanya; Taylor-Blake, Bonnie; Coleman, Jennifer; Ehlers, Michael D; Zylka, Mark J; McCown, Thomas J; Samulski, R Jude

2011-09-01

With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

Humanized Mouse Models of Epstein-Barr Virus Infection and Associated Diseases

PubMed Central

Fujiwara, Shigeyoshi; Matsuda, Go; Imadome, Ken-Ichi

2013-01-01

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus infecting more than 90% of the adult population of the world. EBV is associated with a variety of diseases including infectious mononucleosis, lymphoproliferative diseases, malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma, and autoimmune diseases including rheumatoid arthritis (RA). EBV in nature infects only humans, but in an experimental setting, a limited species of new-world monkeys can be infected with the virus. Small animal models, suitable for evaluation of novel therapeutics and vaccines, have not been available. Humanized mice, defined here as mice harboring functioning human immune system components, are easily infected with EBV that targets cells of the hematoimmune system. Furthermore, humanized mice can mount both cellular and humoral immune responses to EBV. Thus, many aspects of human EBV infection, including associated diseases (e.g., lymphoproliferative disease, hemophagocytic lymphohistiocytosis and erosive arthritis resembling RA), latent infection, and T-cell-mediated and humoral immune responses have been successfully reproduced in humanized mice. Here we summarize recent achievements in the field of humanized mouse models of EBV infection and show how they have been utilized to analyze EBV pathogenesis and normal and aberrant human immune responses to the virus. PMID:25436886

Emergence of fatal avian influenza in New England harbor seals

USGS Publications Warehouse

Anthony, S.J.; St. Leger, J. A.; Pugliares, K.; Ip, Hon S.; Chan, J.M.; Carpenter, Z.W.; Navarrete-Macias, I.; Sanchez-Leon, M.; Saliki, J.T.; Pedersen, J.; Karesh, W.; Daszak, P.; Rabadan, R.; Rowles, T.; Lipkin, W.I.

2012-01-01

From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission.

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

PubMed Central

Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

2016-01-01

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

Intranasal Adeno-Associated Virus Mediated Gene Delivery and Expression of Human Iduronidase in the Central Nervous System: A Noninvasive and Effective Approach for Prevention of Neurologic Disease in Mucopolysaccharidosis Type I.

PubMed

Belur, Lalitha R; Temme, Alexa; Podetz-Pedersen, Kelly M; Riedl, Maureen; Vulchanova, Lucy; Robinson, Nicholas; Hanson, Leah R; Kozarsky, Karen F; Orchard, Paul J; Frey, William H; Low, Walter C; McIvor, R Scott

2017-07-01

Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.

Human beta defensin-1 is involved in the susceptibility to adeno-tonsillar hypertrophy.

PubMed

Zupin, Luisa; Celsi, Fulvio; Bresciani, Martina; Orzan, Eva; Grasso, Domenico Leonardo; Crovella, Sergio

2018-04-01

Innate immunity molecules are known to play a pivotal role in the homeostasis of the oral mucosa, permitting the presence of commensal microflora and, at the same time, providing a first line of defense against pathogens attempting to invade the oral cavity. Tonsils represent the local immune tissue in oral cavity, being able to provide a non-specific response to pathogens; however, in the presence of microbes or foreign materials present in the mouth tonsils could became infected and develop chronic inflammation, thus leading to hypertrophy. The etiology of the disease is multifactorial depending upon environmental and host factors, the latter including molecules of mucosal innate immunity. Ninety-five children with adeno-tonsillar hypertrophy subjected to adeno-tonsillectomy were recruited at the pediatric otorhinolaryngology service of the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste (Italy). The specimen discarded from the surgery were used for genomic DNA extraction and genotyping, for mRNA extraction and gene expression analysis, finally the samples were cut and used to prepare slides to perform immunohistochemistry. Functional polymorphisms within DEFB1 gene, encoding the human beta defensin-1 (hBD-1), were analyzed finding association between DEFB1 rare haplotypes and susceptibility to adeno-tonsillar hypertrophy. DEFB1 mRNA expression was detected in the tonsils and the hBD-1 protein was localized at the epithelia of tonsils mainly in the proximity of the basal lamina. Our findings lead us to hypothesize an involvement of hBD-1 mediated innate immunity in the modulation of the susceptibility towards adeno-tonsillar hypertrophy development. Copyright © 2018 Elsevier B.V. All rights reserved.

Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

PubMed Central

Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

2008-01-01

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes.

PubMed

Dutra, Heverton Leandro Carneiro; Rocha, Marcele Neves; Dias, Fernando Braga Stehling; Mansur, Simone Brutman; Caragata, Eric Pearce; Moreira, Luciano Andrade

2016-06-08

The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epidemic. Wolbachia-harboring mosquitoes displayed lower viral prevalence and intensity and decreased disseminated infection and, critically, did not carry infectious virus in the saliva, suggesting that viral transmission was blocked. Our data indicate that the use of Wolbachia-harboring mosquitoes could represent an effective mechanism to reduce Zika virus transmission and should be included as part of Zika control strategies. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

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

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

Comparative Pathology of Hepatitis A Virus and Hepatitis E Virus Infection.

PubMed

Cullen, John M; Lemon, Stanley M

2018-04-30

Hepatitis A virus (HAV) and hepatitis E virus (HEV) cause acute, self-limiting hepatic infections that are usually spread by the fecal-oral route in humans. Naturally occurring and experimental infections are possible in a variety of nonhuman primates and, in the case of HEV, a number of other species. Many advances in understanding the pathogenesis of these viruses have come from studies in experimental animals. In general, animals infected with these viruses recapitulate the histologic lesions seen in infected humans, but typically with less severe clinical and histopathological manifestations. This review describes the histopathologic changes associated with HAV and HEV infection in humans and experimental animals. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease

PubMed Central

Karumuthil-Melethil, Subha; Kalburgi, Sahana Nagabhushan; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D.; Keimel, John G.; Mark, Brian L.; Mahuran, Don; Walia, Jagdeep S.; Gray, Steven J.

2016-01-01

GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α–β), “A” isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP– GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter–intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the

Phocine distemper virus: current knowledge and future directions.

PubMed

Duignan, Pádraig J; Van Bressem, Marie-Françoise; Baker, Jason D; Barbieri, Michelle; Colegrove, Kathleen M; De Guise, Sylvain; de Swart, Rik L; Di Guardo, Giovanni; Dobson, Andrew; Duprex, W Paul; Early, Greg; Fauquier, Deborah; Goldstein, Tracey; Goodman, Simon J; Grenfell, Bryan; Groch, Kátia R; Gulland, Frances; Hall, Ailsa; Jensen, Brenda A; Lamy, Karina; Matassa, Keith; Mazzariol, Sandro; Morris, Sinead E; Nielsen, Ole; Rotstein, David; Rowles, Teresa K; Saliki, Jeremy T; Siebert, Ursula; Waltzek, Thomas; Wellehan, James F X

2014-12-22

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.

Phocine Distemper Virus: Current Knowledge and Future Directions

PubMed Central

Duignan, Pádraig J.; Van Bressem, Marie-Françoise; Baker, Jason D.; Barbieri, Michelle; Colegrove, Kathleen M.; De Guise, Sylvain; de Swart, Rik L.; Di Guardo, Giovanni; Dobson, Andrew; Duprex, W. Paul; Early, Greg; Fauquier, Deborah; Goldstein, Tracey; Goodman, Simon J.; Grenfell, Bryan; Groch, Kátia R.; Gulland, Frances; Hall, Ailsa; Jensen, Brenda A.; Lamy, Karina; Matassa, Keith; Mazzariol, Sandro; Morris, Sinead E.; Nielsen, Ole; Rotstein, David; Rowles, Teresa K.; Saliki, Jeremy T.; Siebert, Ursula; Waltzek, Thomas; Wellehan, James F.X.

2014-01-01

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years. PMID:25533658

Gene therapy for inherited muscle diseases: where genetics meets rehabilitation medicine.

PubMed

Braun, Robynne; Wang, Zejing; Mack, David L; Childers, Martin K

2014-11-01

The development of clinical vectors to correct genetic mutations that cause inherited myopathies and related disorders of skeletal muscle is advancing at an impressive rate. Adeno-associated virus vectors are attractive for clinical use because (1) adeno-associated viruses do not cause human disease and (2) these vectors are able to persist for years. New vectors are now becoming available as gene therapy delivery tools, and recent preclinical experiments have demonstrated the feasibility, safety, and efficacy of gene therapy with adeno-associated virus for long-term correction of muscle pathology and weakness in myotubularin-deficient canine and murine disease models. In this review, recent advances in the application of gene therapies to treat inherited muscle disorders are presented, including Duchenne muscular dystrophy and x-linked myotubular myopathy. Potential areas for therapeutic synergies between rehabilitation medicine and genetics are also discussed.

A genome-wide association study for the incidence of persistent bovine viral diarrhea virus infection in cattle

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Viruses (BVDV) comprises a diverse group of viruses that causes disease in cattle. BVDV may establish both, transient and persistent infections depending on the developmental stage of the animal at exposure. The objective was to determine if genomic regions harboring single nuc...

A genome-wide association study for the incidence of persistent bovine viral diarrhea virus infection in cattle

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Viruses (BVDV) comprise a diverse group of viruses that cause disease in cattle. BVDV may establish both, transient and persistent infections depending on the developmental stage of the animal at exposure. The objective was to determine if genomic regions harboring single nucle...

Generation of influenza A viruses as live but replication-incompetent virus vaccines.

PubMed

Si, Longlong; Xu, Huan; Zhou, Xueying; Zhang, Ziwei; Tian, Zhenyu; Wang, Yan; Wu, Yiming; Zhang, Bo; Niu, Zhenlan; Zhang, Chuanling; Fu, Ge; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Zhou, Demin

2016-12-02

The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus. Copyright © 2016, American Association for the Advancement of Science.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

PubMed

Monahan, Paul E; Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B; Wichlan, David G; Wu, Zhijian; Grieger, Joshua C; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W; Booth, Carmen J; Samulski, Jade J; Kafri, Tal; McPhee, Scott W J; Samulski, R Jude

2015-02-01

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity

Floating-Harbor syndrome associated with middle ear abnormalities.

PubMed

Hendrickx, Jan-Jaap; Keymolen, Kathelijn; Desprechins, Brigitte; Casselman, Jan; Gordts, Frans

2010-01-01

Floating-Harbor syndrome is a rare syndrome of unknown etiology, which was first described in 1973. A triad of main features characterizes Floating-Harbor syndrome: short stature, characteristic face, and an expressive speech delay. We present a patient in whom the hearing thresholds improved insufficiently after placement of grommets. High-resolution CT scan of the temporal bone showed a prominent soft-tissue thickening suspected of causing fixation of the malleus, and fusion of the malleus head with the body of the incus. To our knowledge this is the first reported abnormal middle ear anatomy in a patient with Floating-Harbor syndrome. A conservative treatment with hearing aids was preferred as an initial treatment in favor of a surgical exploration.

Real-time MR imaging of adeno-associated viral vector delivery to the primate brain

PubMed Central

Fiandaca, Massimo S.; Varenika, Vanja; Eberling, Jamie; McKnight, Tracy; Bringas, John; Pivirotto, Phillip; Beyer, Janine; Hadaczek, Piotr; Bowers, William; Park, John; Federoff, Howard; Forsayeth, John; Bankiewicz, Krystof S.

2009-01-01

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes. PMID:19095069

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

PubMed Central

Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

2017-01-01

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

BACTERIAL MICROBIOTA IN HARBOR SEALS (PHOCA VITULINA) FROM THE NORTH SEA OF SCHLESWIG-HOLSTEIN, GERMANY, AROUND THE TIME OF MORBILLIVIRUS AND INFLUENZA EPIDEMICS.

PubMed

Siebert, Ursula; Rademaker, Marion; Ulrich, Sophie A; Wohlsein, Peter; Ronnenberg, Katrin; Prenger-Berninghoff, Ellen

2017-04-01

We present microbiologic findings in harbor seal (phoca; Phoca vitulina ) carcasses collected from the North Sea of Schleswig-Holstein, Germany, 1996-2014, and interpret results in relation to potential variations caused by phocine distemper virus and influenza A virus mass mortalities. We conducted microbiologic investigations on 2,124 tissue samples from lung, liver, kidney, spleen, intestine, and mesenteric lymph nodes from 549 dead harbor seals of the German North Sea. A large variety of bacteria, including potentially pathogenic species such as Bordetella bronchiseptica , Brucella spp., Clostridium perfringens , Escherichia coli , Erysipelothrix rhusiopathiae , β-hemolytic streptococci, and Staphylococcus aureus , were isolated. These bacteria were associated with bronchopneumonia, gastroenteritis, hepatitis, polyarthritis, nephritis, myositis, myocarditis, and septicemia. Bordetella bronchiseptica and Streptococcus equi subsp. zooepidemicus were significantly associated with the seal die-offs from phocine distemper in 2002 and influenza in 2014. Many bacteria were detected in tissues of dead harbor seals, of which E. coli , β-hemolytic streptococci, and Brucella spp. might be responsible for pathologic changes. Zoonotic bacteria such as Brucella spp. and E. rhusiopathiae are frequently isolated from harbor seals. Brucella spp. was less and Vibrio spp. more frequently found in summer. Erysipelothrix rhusiopathiae showed an almost regular 4-yr oscillating trend. We found C. perfringens less frequently and E. coli more frequently in harbor seals from St. Peter-Ording. Because zoonotic bacteria are regularly found, handling of dead and live harbor seal specimens should be conducted carefully to prevent transmission to humans. Further investigations are needed to understand microbiota changes in relation to increasing seal populations, reintroduction of rehabilitated seals to the wild, and increasing pressure from anthropogenic activities.

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice

PubMed Central

Mak, Kai Y; Chin, Ruth; Cunningham, Sharon C; Habib, Miriam R; Torresi, Joseph; Sharland, Alexandra F; Alexander, Ian E; Angus, Peter W; Herath, Chandana B

2015-01-01

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1–7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1–7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects. PMID:25997428

Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

PubMed Central

Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

2016-01-01

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal

Initial characterization of 17 viruses harboring mutant forms of the immediate-early gene of equine herpesvirus 1.

PubMed

Buczynski, Kimberly A; Kim, Seong K; O'Callaghan, Dennis J

2005-10-01

The sole immediate-early (IE) gene of equine herpesvirus 1 (EHV-1) encodes a major regulatory protein of 1487 amino acids (aa) capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Using a specially designed recombination system and a library of IE linker-insertion, deletion, point, and nonsense mutant constructs that encode forms of the IE protein (IEP) harboring mutations within all five regions, 17 mutant viruses were generated and characterized. Ribonuclease protection analyses revealed that all 17 mutants synthesize the IE mRNA in RK-13 cells, whereas those that failed to replicate on non-complementing RK-13 cells displayed a defect in the transcription of either an important early gene (EICP0) and/or an essential late gene (glycoprotein D). Western blot analyses showed that the IEP was synthesized and detectable in cells infected with each mutant virus, including those mutants that failed to replicate on non-complementing RK-13 cells. Eleven of the 17 mutants were capable of growth on non-complementing RK-13 cells, whereas mutant viruses with deletions within the serine-rich tract (SRT), nucleus localization signal (NLS), or DNA-binding domain (DBD) were capable of growth only on the IEP-producing cell line (IE13.1). Lastly, temperature shift experiments revealed that mutant viruses containing deletions within the C-terminus (KyAn1029 and KyAn1411) or within the SRT (KyADeltaSRT2) of the IEP exhibited a temperature-sensitive phenotype in that these viruses, in contrast to the parent KyA, failed to replicate at 39 degrees C. Overall, these results indicate that the C-terminus of the IEP is not essential for IEP function in cell culture, but this region contains elements that enhance the function(s) of the IEP. The initial characterization of these 17 EHV-1 mutants has shown that sequences totaling at least 43% of the IEP are not essential for virus replication in cell culture.

Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction.

PubMed

Furtak, Vyacheslav; Roivainen, Merja; Mirochnichenko, Olga; Zagorodnyaya, Tatiana; Laassri, Majid; Zaidi, Sohail Z; Rehman, Lubna; Alam, Muhammad M; Chizhikov, Vladimir; Chumakov, Konstantin

2016-04-14

An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.

Landscape of DNA Virus Associations across Human Malignant Cancers: Analysis of 3,775 Cases Using RNA-Seq

PubMed Central

Tannir, Nizar M.; Williams, Michelle D.; Chen, Yunxin; Yao, Hui; Zhang, Jianping; Thompson, Erika J.; Meric-Bernstam, Funda; Medeiros, L. Jeffrey; Weinstein, John N.

2013-01-01

Elucidation of tumor-DNA virus associations in many cancer types has enhanced our knowledge of fundamental oncogenesis mechanisms and provided a basis for cancer prevention initiatives. RNA-Seq is a novel tool to comprehensively assess such associations. We interrogated RNA-Seq data from 3,775 malignant neoplasms in The Cancer Genome Atlas database for the presence of viral sequences. Viral integration sites were also detected in expressed transcripts using a novel approach. The detection capacity of RNA-Seq was compared to available clinical laboratory data. Human papillomavirus (HPV) transcripts were detected using RNA-Seq analysis in head-and-neck squamous cell carcinoma, uterine endometrioid carcinoma, and squamous cell carcinoma of the lung. Detection of HPV by RNA-Seq correlated with detection by in situ hybridization and immunohistochemistry in squamous cell carcinoma tumors of the head and neck. Hepatitis B virus and Epstein-Barr virus (EBV) were detected using RNA-Seq in hepatocellular carcinoma and gastric carcinoma tumors, respectively. Integration sites of viral genes and oncogenes were detected in cancers harboring HPV or hepatitis B virus but not in EBV-positive gastric carcinoma. Integration sites of expressed viral transcripts frequently involved known coding areas of the host genome. No DNA virus transcripts were detected in acute myeloid leukemia, cutaneous melanoma, low- and high-grade gliomas of the brain, and adenocarcinomas of the breast, colon and rectum, lung, prostate, ovary, kidney, and thyroid. In conclusion, this study provides a large-scale overview of the landscape of DNA viruses in human malignant cancers. While further validation is necessary for specific cancer types, our findings highlight the utility of RNA-Seq in detecting tumor-associated DNA viruses and identifying viral integration sites that may unravel novel mechanisms of cancer pathogenesis. PMID:23740984

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

PubMed

Cunningham, Sharon C; Siew, Susan M; Hallwirth, Claus V; Bolitho, Christine; Sasaki, Natsuki; Garg, Gagan; Michael, Iacovos P; Hetherington, Nicola A; Carpenter, Kevin; de Alencastro, Gustavo; Nagy, Andras; Alexander, Ian E

2015-08-01

Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. © 2015 by

History of the Discovery of Hepatitis A Virus.

PubMed

Feinstone, Stephen M

2018-04-30

Disease outbreaks resembling hepatitis A have been known since antiquity. However, it was not until World War II when two forms of viral hepatitis were clearly differentiated. After the discovery of Australia antigen and its association with hepatitis B, similar methodologies were used to find the hepatitis A virus. The virus was ultimately identified when investigators changed the focus of their search from serum to feces and applied appropriate technology. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

High-Resolution Metatranscriptomics Reveals the Ecological Dynamics of Mosquito-Associated RNA Viruses in Western Australia

PubMed Central

Shi, Mang; Neville, Peter; Nicholson, Jay; Eden, John-Sebastian

2017-01-01

ABSTRACT Mosquitoes harbor a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a metatranscriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes of the Aedes genus exhibiting substantially less diversity and lower abundances than those of mosquitoes of the Culex genus, within which the viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that the host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95 to 98% nucleotide identity) to those from Indonesia and China. Finally, using abundance-level and phylogenetic relationships, we were able to distinguish potential mosquito viruses from those present in coinfecting bacteria, fungi, and protists. In sum, our metatranscriptomics approach provides important insights into the ecology of mosquito RNA viruses. IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize metatranscriptomic approaches to reveal both complete virus diversity and the relative abundance of these viruses. We used such a

Epstein-Barr virus-associated lymphomas.

PubMed

Shannon-Lowe, Claire; Rickinson, Alan B; Bell, Andrew I

2017-10-19

Epstein-Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Authors.

Hydroxyurea enhances the activity of acyclovir and cidofovir against herpes simplex virus type 1 resistant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes.

PubMed

Sergerie, Yan; Boivin, Guy

2008-01-01

Drug-resistant herpes simplex virus type 1 (HSV-1) recombinant strains harboring mutations in the thymidine kinase and/or the DNA polymerase genes were evaluated for their susceptibility to various antivirals in the presence of 25 microg/ml of hydroxyurea (HyU). The latter compound decreased the 50% inhibitory concentrations of acyclovir by 1.5-3.8-fold and that of cidofovir by 2.7-14.4-fold. However, HyU did not affect the susceptibilities of the various recombinant mutants to foscarnet. Hydroxyurea, a ribonucleotide reductase inhibitor, can increase the activity of nucleoside/nucleotide analogues against drug-resistant viruses.

Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.

PubMed

De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi; Li, Linhong; Wu, Xiaolin; Su, Ling; Raley, Castle; Theobald, Narda; Choi, Uimook; Song, Alexander H; Chan, Andy; Pearl, Jocelynn R; Paschon, David E; Lee, Janet; Newcombe, Hannah; Koontz, Sherry; Sweeney, Colin; Shivak, David A; Zarember, Kol A; Peshwa, Madhusudan V; Gregory, Philip D; Urnov, Fyodor D; Malech, Harry L

2016-04-01

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

An infectious bat chimeric influenza virus harboring the entry machinery of a influenza A virus

PubMed Central

Juozapaitis, Mindaugas; Moreira, Étori Aguiar; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

2017-01-01

In 2012 the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the HA and NA proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event. PMID:25055345

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

Engineered Viruses as Genome Editing Devices

PubMed Central

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-01-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

Engineered Viruses as Genome Editing Devices.

PubMed

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-03-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR-Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole.

BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

PubMed

McClung, Joseph M; McCord, Timothy J; Ryan, Terence E; Schmidt, Cameron A; Green, Tom D; Southerland, Kevin W; Reinardy, Jessica L; Mueller, Sarah B; Venkatraman, Talaignair N; Lascola, Christopher D; Keum, Sehoon; Marchuk, Douglas A; Spangenburg, Espen E; Dokun, Ayotunde; Annex, Brian H; Kontos, Christopher D

2017-07-18

Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1 , that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6- Lsq1-3 ). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3 Ile81 , but not BAG3 Met81 , improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3 Ile81 (n=9), but not BAG3 Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3 Met81 , BAG3 Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle

The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA.

PubMed

Riegger, David; Hai, Rong; Dornfeld, Dominik; Mänz, Benjamin; Leyva-Grado, Victor; Sánchez-Aparicio, Maria T; Albrecht, Randy A; Palese, Peter; Haller, Otto; Schwemmle, Martin; García-Sastre, Adolfo; Kochs, Georg; Schmolke, Mirco

2015-02-01

Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans. The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts

Oseltamivir-resistant influenza A(H1N1)pdm09 virus associated with high case fatality, India 2015.

PubMed

Tandel, Kundan; Sharma, Shashi; Dash, Paban Kumar; Parida, ManMohan

2018-05-01

Influenza A viruses has been associated with severe global pandemics of high morbidity and mortality with devastating impact on human health and global economy. India witnessed a major outbreak of influenza A(H1N1)pdm09 in 2015. This study comprises detailed investigation of cases died of influenza A(H1N1)pdm09 virus infection during explosive outbreak of 2015, in central part of India. To find out presence of drug resistant virus among patients who died of influenza A(H1N1)pdm09 virus infection and to find out presence of other mutations contributing to the morbidity and mortality. Twenty-two patients having confirmed influenza A(H1N1)pdm09 infection and subsequently died of this infection along with 20 non fatal cases with influenza A(H1N1)pdm09 infection were included in the study. Samples were investigated through RT-PCR/RFLP analysis, followed by nucleotide cycle sequencing of whole NA gene for detection of H275Y amino acid substitution in NA gene responsible for oseltamivir drug resistance. Out of 22 fatal cases, 6 (27.27%) were found to harbor oseltamivir resistant virus strains, whereas the H275Y mutation was not observed among the 20 non fatal cases. Amino acid substitution analysis of complete NA gene revealed V241I, N369K, N386K substitution in all strains playing synergistic role in oseltamivir drug resistance. High morbidity and mortality associated with influenza A(H1N1)pdm09 viruses can be explained by presence of drug resistant strains circulating in this outbreak. Presence of Oseltamivir resistant influenza A(H1N1)pdm09 viruses is a cause of great concern and warrants continuous screening for the circulation of drug resistant strains. © 2017 Wiley Periodicals, Inc.

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

Epstein–Barr virus-associated lymphomas

PubMed Central

Shannon-Lowe, Claire; Rickinson, Alan B.

2017-01-01

Epstein–Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host. This article is part of the themed issue ‘Human oncogenic viruses’. PMID:28893938

Human papilloma virus vaccine associated uveitis.

PubMed

Holt, Henry D; Hinkle, David M; Falk, Naomi S; Fraunfelder, Frederick T; Fraunfelder, Frederick W

2014-03-01

To report a possible association between human papilloma virus (HPV) vaccination and uveitis. Spontaneous reports from the National Registry of Drug-Induced Ocular Side effects, World Health Organization and Food and Drug Administration were collected on uveitis associated with human papilloma virus vaccination. A MEDLINE search was performed using keywords "uveitis," "iritis," "iridocyclitis," "human papilloma virus," "Cervarix", and "Gardasil." Data garnered from spontaneous reports included the age, gender, adverse drug reaction (ADR), date of administration, concomitant administration of other vaccinations, time until onset of ADR, other systemic reactions, and dechallenge and rechallenge data. A total of 24 case reports of uveitis associated with human papilloma virus vaccination were identified, all cases were female, and the median age was 17. Median time from HPV vaccination to reported ADR was 30 days (range 0-476 days). According to World Health Organization criteria, the relationship between human papilloma virus vaccination and uveitis is "possible." Causality assessments are based on the time relationship of drug administration, uveitis development and re-challenge data. Clinicians should be aware of a possible bilateral uveitis and papillitis following HPV vaccination.

Detection of canine distemper virus serum neutralizing antibodies in captive U.S. phocids.

PubMed

Clancy, Meredith M; Gamble, Kathryn C; Travis, Dominic A

2013-03-01

Antibodies to morbilliviruses have been documented in free-ranging pinnipeds throughout populations in the Atlantic and Arctic Oceans, but not from the Pacific Ocean. As a symbolic geographic barrier between the exposed Atlantic and naive Pacific populations, the captive phocid population in North America had undocumented serologic status. In this study, canine distemper virus (CDV) serum neutralization assays were used to assess the prevalence of antibodies in this population with participation of 25 U.S. institutions from grey seals (Halichoerus grypus, n = 6) and harbor seals (Phoca vitulina, n = 108). Historic and environmental risk factors associated with the epidemiology of distemper virus were collected by survey. Based on antibodies to canine distemper virus, the prevalence of exposure in this population was 25.5%, with 28 seals (grey, n = 2; harbor, n = 26) demonstrating antibody titers > or = 1:16, and positive titers ranged from 1:4 to 1:1,536. By survey analysis, strong associations with seropositive status were identified for captive origin (P = 0.013) and movement among institutions (P = 0.024). Size of population has positive correlation with likelihood of seropositive seals at an institution (P = 0.020). However, no major husbandry or enclosure-based risk factors were identified in institutions with seropositive seals, and no interaction between individual or institutional risk factors was identified. Previously undocumented prior to this study, CDV antibodies were measured in harbor seals (n = 2) recently stranded from the Pacific coast.

Population dynamics of rhesus macaques and associated foamy virus in Bangladesh

PubMed Central

Feeroz, Mostafa M; Soliven, Khanh; Small, Christopher T; Engel, Gregory A; Andreina Pacheco, M; Yee, JoAnn L; Wang, Xiaoxing; Kamrul Hasan, M; Oh, Gunwha; Levine, Kathryn L; Rabiul Alam, SM; Craig, Karen L; Jackson, Dana L; Lee, Eun-Gyung; Barry, Peter A; Lerche, Nicholas W; Escalante, Ananias A; Matsen IV, Frederick A; Linial, Maxine L; Jones-Engel, Lisa

2013-01-01

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses. PMID:26038465

Bacteriophage P23-77 Capsid Protein Structures Reveal the Archetype of an Ancient Branch from a Major Virus Lineage

PubMed Central

Rissanen, Ilona; Grimes, Jonathan M.; Pawlowski, Alice; Mäntynen, Sari; Harlos, Karl; Bamford, Jaana K.H.; Stuart, David I.

2013-01-01

Summary It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor. PMID:23623731

Pulmonary inflammatory myofibroblastic tumor harboring EML4-ALK fusion gene.

PubMed

Sokai, Akihiko; Enaka, Makiko; Sokai, Risa; Mori, Shoichi; Mori, Shunsuke; Gunji, Masaharu; Fujino, Masahiko; Ito, Masafumi

2014-01-01

Inflammatory myofibroblastic tumor is a rare tumor deriving from mesenchymal tissue. Approximately 50% of inflammatory myofibroblastic tumors harbor an anaplastic lymphoma kinase fusion gene. Pulmonary inflammatory myofibroblastic tumors harboring tropomyosin3-anaplastic lymphoma kinase or protein tyrosine phosphatase receptor-type F polypeptide-interacting protein-binding protein 1-anaplastic lymphoma kinase have been reported previously. However, it has not been reported that inflammatory myofibroblastic tumors harbor echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene which is considered to be very specific to lung cancers. A few tumors harboring echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene other than lung cancers have been reported and the tumors were all carcinomas. A 67-year-old man had been followed up for a benign tumor for approximately 3 years before the tumor demonstrated malignant transformation. Lobectomy and autopsy revealed that an inflammatory myofibroblastic tumor harboring echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene had transformed into an undifferentiated sarcoma. This case suggests that echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion is an oncogenic event in not only carcinomas but also sarcomas originating from stromal cells.

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

PubMed

Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

1999-01-01

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Oxybuprocaine induces a false-positive response in immunochromatographic SAS Adeno Test.

PubMed

Hoshino, Takeshi; Takanashi, Taiji; Okada, Morio; Uchida, Sunao

2002-04-01

To investigate whether a solution of oxybuprocaine hydrochloride, 0.4%, results in a false-positive response in an immunochromatographic SAS Adeno Test. Experimental study. Physiologic saline and 2% lidocaine. Each chemical (100 microl) was diluted in a transport medium. Five drops (200 microl) of the resultant solution were dispensed into the round sample well of a test device. Fifteen samples were tested in each group. Ten minutes after the start of the test, a colored line in the "specimen" portion of the test membrane was visually read as positive or negative by a masked technician. No positive reaction was observed in the control groups (physiologic saline and lidocaine). A false-positive reaction was observed in six samples (33.3%) in the oxybuprocaine group. The positive rate was significantly higher in the oxybuprocaine group compared with those in control groups (P = 0.0062, Fisher's extract probability test). Oxybuprocaine may induce a false-positive reaction in an immunochromatographic SAS Adeno Test. We recommend the use of lidocaine, instead of oxybuprocaine, for local anesthesia in taking eye swabs from patients with suspected adenovirus infection.

Surveillance for zoonotic and selected pathogens in harbor seals Phoca vitulina from central California

USGS Publications Warehouse

Greig, Denise J.; Ip, Hon S.; Gulland, Frances M. D.; Miller, Woutrina A.; Conrad, Patricia A.; Field, Cara L.; Fleetwood, Michelle; Harvey, James T.; Jang, Spencer; Packham, Andrea; Wheeler, Elizabeth; Hall, Ailsa J.

2014-01-01

The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Polycyclic aromatic hydrocarbons and trace elements bounded to airborne PM10 in the harbor of Volos, Greece: Implications for the impact of harbor activities

NASA Astrophysics Data System (ADS)

Manoli, E.; Chelioti-Chatzidimitriou, A.; Karageorgou, K.; Kouras, A.; Voutsa, D.; Samara, C.; Kampanos, I.

2017-10-01

Harbors are often characterized by high levels of air pollutants that are emitted from ship traffic and other harbor activities. In the present study, the concentrations of Polycyclic Aromatic Hydrocarbons (PAHs) and trace elements (As, Cd, Ni, Pb, Cr, Mn, Zn, and Fe) bounded to the inhalable particulate matter PM10 were studied in the harbor of Volos, central Greece, during a 2-year period (2014-2015). Seasonal and daily variations were investigated. Moreover, total carcinogenic and mutagenic activities of PAHs were calculated. The effect of major wind sectors (sea, city, industrial, harbor) was estimated to assess the potential contribution of ship traffic and harbor activities, such as scrap metal handling operations. Results showed that the harbor sector (calm winds ≤ 0.5 m s-1) was associated with the highest concentrations of PM10. The harbor sector was also associated with relatively increased levels of trace elements (As, Fe, Cr, Mn, Ni), however the effect of this sector was lower than the corresponding effect of the industrial wind sector. The sea sector showed only a slight increase in B[a]Py and Σ12PAHs, whereas the highest increasing effect for PAHs and traffic-related elements, such as Pb and Zn, was evidenced for the city sector.

Nonhuman Primate Models of Hepatitis A Virus and Hepatitis E Virus Infections.

PubMed

Lanford, Robert E; Walker, Christopher M; Lemon, Stanley M

2018-04-23

Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

West Nile Flavivirus Polioencephalomyelitis in a harbor seal (Phoca vitulina).

PubMed

Del Piero, F; Stremme, D W; Habecker, P L; Cantile, C

2006-01-01

A 12-year-old male harbor seal presented with progressive signs of neurologic dysfunction including head tremors, muzzle twitching, clonic spasms, and weakness. Lesions included polioencephalomyelitis with glial nodules, spheroids, neuronophagia, ring hemorrhages, and a few neutrophils. Neurons, fibers, and glial nodules were multifocally colonized with intracytoplasmic West Nile flavivirus antigens that were demonstrated using indirect immunohistochemical analysis. Flavivirus on cultured cells also was isolated and was identified by use of monoclonal antibodies and reverse transcriptase-polymerase chain reaction analysis. Clinical signs of disease and lesion morphology and distribution were similar to those of equine West Nile virus infection. Similar to horses, alpacas, humans, dogs, and reptiles, seals can be dead-end hosts of West Nile virus.

Secreted Klotho Attenuates Inflammation-Associated Aortic Valve Fibrosis in Senescence-Accelerated Mice P1.

PubMed

Chen, Jianglei; Fan, Jun; Wang, Shirley; Sun, Zhongjie

2018-05-01

Senescence-accelerated mice P1 (SAMP1) is an aging model characterized by shortened lifespan and early signs of senescence. Klotho is an aging-suppressor gene. The purpose of this study is to investigate whether in vivo expression of secreted klotho ( Skl ) gene attenuates aortic valve fibrosis in SAMP1 mice. SAMP1 mice and age-matched (AKR/J) control mice were used. SAMP1 mice developed obvious fibrosis in aortic valves, namely fibrotic aortic valve disease. Serum level of Skl was decreased drastically in SAMP1 mice. Expression of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule 1), F4/80, and CD68 was increased in aortic valves of SAMP1 mice, indicating inflammation. An increase in expression of α-smooth muscle actin (myofibroblast marker), transforming growth factorβ-1, and scleraxis (a transcription factor of collagen synthesis) was also found in aortic valves of SAMP1 mice, suggesting that accelerated aging is associated with myofibroblast transition and collagen gene activation. We constructed adeno-associated virus 2 carrying mouse Skl cDNA for in vivo expression of Skl. Skl gene delivery effectively increased serum Skl of SAMP1 mice to the control level. Skl gene delivery inhibited inflammation and myofibroblastic transition in aortic valves and attenuated fibrotic aortic valve disease in SAMP1 mice. It is concluded that senescence-related fibrotic aortic valve disease in SAMP1 mice is associated with a decrease in serum klotho leading to inflammation, including macrophage infiltration and transforming growth factorβ-1/scleraxis-driven myofibroblast differentiation in aortic valves. Restoration of serum Skl levels by adeno-associated virus 2 carrying mouse Skl cDNA effectively suppresses inflammation and myofibroblastic transition and attenuates aortic valve fibrosis. Skl may be a potential therapeutic target for fibrotic aortic valve disease. © 2018 American Heart Association, Inc.

Antiviral activity in vitro of two preparations of the herbal medicinal product Sinupret® against viruses causing respiratory infections.

PubMed

Glatthaar-Saalmüller, B; Rauchhaus, U; Rode, S; Haunschild, J; Saalmüller, A

2011-12-15

Sinupret(®), a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret(®) oral drops (hereinafter referred to as "oral drops") and Sinupret(®) dry extract (hereinafter referred to as "dry extract"), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC(50) between 13.8 and 124.8 μg/ml) of Sinupret(®) was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret(®) shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections. Copyright © 2011 Elsevier GmbH. All rights reserved.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Sensitivity and specificity of the AdenoPlus point-of-care system in detecting adenovirus in conjunctivitis patients at an ophthalmic emergency department: a diagnostic accuracy study.

PubMed

Kam, K Y Ronald; Ong, Hon Shing; Bunce, Catey; Ogunbowale, Lola; Verma, Seema

2015-09-01

To estimate the diagnostic accuracy (sensitivity and specificity) of the AdenoPlus point-of-care adenoviral test compared to PCR in an ophthalmic accident and emergency service. These findings were compared with those of a previous study. This was a prospective diagnostic accuracy study on 121 patients presenting to an emergency eye unit with a clinical picture of acute adenoviral conjunctivitis. AdenoPlus testing was carried out on one eye of each patient and a PCR analysis was also performed on a swab taken from the same eye. AdenoPlus and PCR results were interpreted by masked personnel. Sensitivity and specificity for the AdenoPlus test were calculated using PCR results as the reference standard. 121 patients were enrolled and 109 met the inclusion criteria. 43 patients (39.4%) tested positive for adenovirus by PCR analysis. The sensitivity of the AdenoPlus swab in detecting adenovirus was 39.5% (17/43, 95% CI 26% to 54%) and specificity was 95.5% (63/66, 95% CI 87% to 98%) compared to PCR. The AdenoPlus test has a high specificity for diagnosing adenoviral conjunctivitis, but in this clinical setting, we could not reproduce the high sensitivity that has been previously published. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Nerve Growth Factor Gene Therapy Using Adeno-Associated Viral Vectors Prevents Cardiomyopathy in Type 1 Diabetic Mice

PubMed Central

Meloni, Marco; Descamps, Betty; Caporali, Andrea; Zentilin, Lorena; Floris, Ilaria; Giacca, Mauro; Emanueli, Costanza

2012-01-01

Diabetes is a cause of cardiac dysfunction, reduced myocardial perfusion, and ultimately heart failure. Nerve growth factor (NGF) exerts protective effects on the cardiovascular system. This study investigated whether NGF gene transfer can prevent diabetic cardiomyopathy in mice. We worked with mice with streptozotocin-induced type 1 diabetes and with nondiabetic control mice. After having established that diabetes reduces cardiac NGF mRNA expression, we tested NGF gene therapies with adeno-associated viral vectors (AAVs) for the capacity to protect the diabetic mouse heart. To this aim, after 2 weeks of diabetes, cardiac expression of human NGF or β-Gal (control) genes was induced by either intramyocardial injection of AAV serotype 2 (AAV2) or systemic delivery of AAV serotype 9 (AAV9). Nondiabetic mice were given AAV2–β-Gal or AAV9–β-Gal. We found that the diabetic mice receiving NGF gene transfer via either AAV2 or AAV9 were spared the progressive deterioration of cardiac function and left ventricular chamber dilatation observed in β-Gal–injected diabetic mice. Moreover, they were additionally protected from myocardial microvascular rarefaction, hypoperfusion, increased deposition of interstitial fibrosis, and increased apoptosis of endothelial cells and cardiomyocytes, which afflicted the β-Gal–injected diabetic control mice. Our data suggest therapeutic potential of NGF for the prevention of cardiomyopathy in diabetic subjects. PMID:22187379

Partial Correction of the CNS Lysosomal Storage Defect in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis by Neonatal CNS Administration of an Adeno-Associated Virus Serotype rh.10 Vector Expressing the Human CLN3 Gene

PubMed Central

Sondhi, Dolan; Scott, Emma C.; Chen, Alvin; Hackett, Neil R.; Wong, Andrew M.S.; Kubiak, Agnieszka; Nelvagal, Hemanth R.; Pearse, Yewande; Cotman, Susan L.; Cooper, Jonathan D.

2014-01-01

Abstract Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3Δex7/8 knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3Δex7/8 mice were administered 3×1010 genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3Δex7/8 mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3Δex7/8 mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3Δex7/8 mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions.

PubMed

Link, Nils; Brunner, Tobias J; Dreesen, Imke A J; Stark, Wendelin J; Fussenegger, Martin

2007-12-01

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus.

PubMed

O'Donnell, Vivian; Holinka, Lauren G; Gladue, Douglas P; Sanford, Brenton; Krug, Peter W; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R; Borca, Manuel V

2015-06-01

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus

PubMed Central

O'Donnell, Vivian; Holinka, Lauren G.; Gladue, Douglas P.; Sanford, Brenton; Krug, Peter W.; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R.

2015-01-01

ABSTRACT African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 102 or 104 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer

Memantine Attenuates Alzheimer’s Disease-Like Pathology and Cognitive Impairment

PubMed Central

Wang, Xiaochuan; Blanchard, Julie; Iqbal, Khalid

2015-01-01

Deficiency of protein phosphatase-2A is a key event in Alzheimer’s disease. An endogenous inhibitor of protein phosphatase-2A, inhibitor-1, I1 PP2A, which inhibits the phosphatase activity by interacting with its catalytic subunit protein phosphatase-2Ac, is known to be upregulated in Alzheimer’s disease brain. In the present study, we overexpressed I1 PP2A by intracerebroventricular injection with adeno-associated virus vector-1-I1 PP2A in Wistar rats. The I1 PP2A rats showed a decrease in brain protein phosphatase-2A activity, abnormal hyperphosphorylation of tau, neurodegeneration, an increase in the level of activated glycogen synthase kinase-3beta, enhanced expression of intraneuronal amyloid-beta and spatial reference memory deficit; littermates treated identically but with vector only, i.e., adeno-associated virus vector-1-enhanced GFP, served as a control. Treatment with memantine, a noncompetitive NMDA receptor antagonist which is an approved drug for treatment of Alzheimer’s disease, rescued protein phosphatase-2A activity by decreasing its demethylation at Leu309 selectively and attenuated Alzheimer’s disease-like pathology and cognitive impairment in adeno-associated virus vector-1-I1 PP2A rats. These findings provide new clues into the possible mechanism of the beneficial therapeutic effect of memantine in Alzheimer’s disease patients. PMID:26697860

Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody Gene Transfer Protects Nonhuman Primates from Mucosal Simian-Human Immunodeficiency Virus Infection

PubMed Central

Saunders, Kevin O.; Wang, Lingshu; Joyce, M. Gordon; Yang, Zhi-Yong; Balazs, Alejandro B.; Cheng, Cheng; Ko, Sung-Youl; Kong, Wing-Pui; Rudicell, Rebecca S.; Georgiev, Ivelin S.; Duan, Lijie; Foulds, Kathryn E.; Donaldson, Mitzi; Xu, Ling; Schmidt, Stephen D.; Todd, John-Paul; Baltimore, David; Roederer, Mario; Haase, Ashley T.; Kwong, Peter D.; Rao, Srinivas S.

2015-01-01

ABSTRACT Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 μg/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled. IMPORTANCE Sustained interventions that can prevent HIV-1 infection are needed to halt the spread of the HIV-1 pandemic. The protective capacity of anti-HIV antibody gene therapy has been established in mouse models of HIV-1 infection but has not been established for primates. We show here a proof-of-concept that gene transfer of anti-HIV antibody genes can protect against infection by viruses that cause AIDS in primates when host immune responses are controlled. PMID:26041300

Brain Meta-Transcriptomics from Harbor Seals to Infer the Role of the Microbiome and Virome in a Stranding Event.

PubMed

Rosales, Stephanie M; Thurber, Rebecca Vega

2015-01-01

Marine diseases are becoming more frequent, and tools for identifying pathogens and disease reservoirs are needed to help prevent and mitigate epizootics. Meta-transcriptomics provides insights into disease etiology by cataloguing and comparing sequences from suspected pathogens. This method is a powerful approach to simultaneously evaluate both the viral and bacterial communities, but few studies have applied this technique in marine systems. In 2009 seven harbor seals, Phoca vitulina, stranded along the California coast from a similar brain disease of unknown cause of death (UCD). We evaluated the differences between the virome and microbiome of UCDs and harbor seals with known causes of death. Here we determined that UCD stranded animals had no viruses in their brain tissue. However, in the bacterial community, we identified Burkholderia and Coxiella burnetii as important pathogens associated with this stranding event. Burkholderia were 100% prevalent and ~2.8 log2 fold more abundant in the UCD animals. Further, while C. burnetii was found in only 35.7% of all samples, it was highly abundant (~94% of the total microbial community) in a single individual. In this harbor seal, C. burnetii showed high transcription rates of invading and translation genes, implicating it in the pathogenesis of this animal. Based on these data we propose that Burkholderia taxa and C. burnetii are potentially important opportunistic neurotropic pathogens in UCD stranded harbor seals.

Twenty-Five New Viruses Associated with the Drosophilidae (Diptera)

PubMed Central

Webster, Claire L.; Longdon, Ben; Lewis, Samuel H.; Obbard, Darren J.

2016-01-01

Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here, we use metagenomic RNA sequencing of about 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses, Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not been previously reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and Drosophila obscura, we identify sequences very closely related to Armadillidium vulgare iridescent virus (Invertebrate iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three. PMID:27375356

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors.

PubMed

Vande Velde, G; Rangarajan, J R; Toelen, J; Dresselaers, T; Ibrahimi, A; Krylychkina, O; Vreys, R; Van der Linden, A; Maes, F; Debyser, Z; Himmelreich, U; Baekelandt, V

2011-06-01

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential

PubMed Central

Watanabe, Tokiko; Zhong, Gongxun; Russell, Colin A.; Nakajima, Noriko; Hatta, Masato; Hanson, Anthony; McBride, Ryan; Burke, David F.; Takahashi, Kenta; Fukuyama, Satoshi; Tomita, Yuriko; Maher, Eileen A.; Watanabe, Shinji; Imai, Masaki; Neumann, Gabriele; Hasegawa, Hideki; Paulson, James C.; Smith, Derek J.; Kawaoka, Yoshihiro

2014-01-01

Summary Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited higher pathogenicity in mice and ferrets than an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential. PMID:24922572

[Skin symptoms associated with human immunodeficiency virus infection].

PubMed

Tamási, Béla; Marschalkó, Márta; Kárpáti, Sarolta

2015-01-04

The recently observed accelerated increase of human immunodeficiency virus infection in Hungary poses a major public concern for the healthcare system. Given the effective only but not the curative therapy, prevention should be emphasized. Current statistics estimate that about 50% of the infected persons are not aware of their human immunodeficiency virus-positivity. Thus, early diagnosis of the infection by serological screening and timely recognition of the disease-associated symptoms are crucial. The authors' intention is to facilitate early infection detection with this review on human immunodeficiency virus-associated skin symptoms, and highlight the significance of human immunodeficiency virus care in the everyday medical practice.

Discovery of Culex pipiens associated tunisia virus: a new ssRNA(+) virus representing a new insect associated virus family

PubMed Central

Bigot, Diane; Atyame, Célestine M; Weill, Mylène; Justy, Fabienne

2018-01-01

Abstract In the global context of arboviral emergence, deep sequencing unlocks the discovery of new mosquito-borne viruses. Mosquitoes of the species Culex pipiens, C. torrentium, and C. hortensis were sampled from 22 locations worldwide for transcriptomic analyses. A virus discovery pipeline was used to analyze the dataset of 0.7 billion reads comprising 22 individual transcriptomes. Two closely related 6.8 kb viral genomes were identified in C. pipiens and named as Culex pipiens associated tunisia virus (CpATV) strains Ayed and Jedaida. The CpATV genome contained four ORFs. ORF1 possessed helicase and RNA-dependent RNA polymerase (RdRp) domains related to new viral sequences recently found mainly in dipterans. ORF2 and 4 contained a capsid protein domain showing strong homology with Virgaviridae plant viruses. ORF3 displayed similarities with eukaryotic Rhoptry domain and a merozoite surface protein (MSP7) domain only found in mosquito-transmitted Plasmodium, suggesting possible interactions between CpATV and vertebrate cells. Estimation of a strong purifying selection exerted on each ORFs and the presence of a polymorphism maintained in the coding region of ORF3 suggested that both CpATV sequences are genuine functional viruses. CpATV is part of an entirely new and highly diversified group of viruses recently found in insects, and that bears the genomic hallmarks of a new viral family. PMID:29340209

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

PubMed

Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M; Borel, Florie; Reeves, Emer P; Calcedo, Roberto; Rouhani, Farshid N; Yachnis, Anthony; Humphries, Margaret; Campbell-Thompson, Martha; Messina, Louis; Chulay, Jeffrey D; Trapnell, Bruce; Wilson, James M; McElvaney, Noel G; Flotte, Terence R

2017-06-07

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Gene repertoire of amoeba-associated giant viruses.

PubMed

Colson, Philippe; Raoult, Didier

2010-01-01

Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. Copyright 2010 S. Karger AG, Basel.

Genetic Mutation and Exosome Signature of Human Papilloma Virus Associated Oropharyngeal Cancer

PubMed Central

Kannan, Anbarasu; Hertweck, Kate L.; Philley, Julie V.; Wells, Robert B.; Dasgupta, Santanu

2017-01-01

Human papilloma virus-16 (HPV-16) associated oropharyngeal cancer (HPVOPC) is increasing alarmingly in the United States. We performed whole genome sequencing of a 44 year old, male HPVOPC subject diagnosed with moderately differentiated tonsillar carcinoma. We identified new somatic mutation in MUC16 (A.k.a. CA-125), MUC12, MUC4, MUC6, MUC2, SIRPA, HLA-DRB1, HLA-A and HLA-B molecules. Increased protein expression of MUC16, SIRPA and decreased expression of HLA-DRB1 was further demonstrated in this HPVOPC subject and an additional set of 15 HPVOPC cases. Copy number gain (3 copies) was also observed for MUC2, MUC4, MUC6 and SIRPA. Enhanced expression of MUC16, SIRPA and HPV-16-E7 protein was detectable in the circulating exosomes of numerous HPVOPC subjects. Treatment of non-tumorigenic mammary epithelial cells with exosomes derived from aggressive HPVOPC cells harboring MUC16, SIRPA and HPV-16-E7 proteins augmented invasion and induced epithelial to mesenchymal transition (EMT) accompanied by an increased expression ratio of the EMT markers Vimentin/E-cadherin. Exosome based screening of key HPVOPC associated molecules could be beneficial for early cancer diagnosis, monitoring and surveillance. PMID:28383029

Transgene expression in target-defined neuron populations mediated by retrograde infection with adeno-associated viral vectors.

PubMed

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta; Wachowiak, Matt

2013-09-18

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

PubMed Central

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta

2013-01-01

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors—in particular, recombinant adeno-associated viral vectors (rAAVs)—have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested—in particular, though not exclusively, Cre-dependent vectors—showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal. PMID:24048849

Carrot yellow leaf virus is associated with carrot internal necrosis.

PubMed

Adams, Ian P; Skelton, Anna; Macarthur, Roy; Hodges, Tobias; Hinds, Howard; Flint, Laura; Nath, Palash Deb; Boonham, Neil; Fox, Adrian

2014-01-01

Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots.

Carrot yellow leaf virus Is Associated with Carrot Internal Necrosis

PubMed Central

Adams, Ian P.; Skelton, Anna; Macarthur, Roy; Hodges, Tobias; Hinds, Howard; Flint, Laura; Nath, Palash Deb; Boonham, Neil; Fox, Adrian

2014-01-01

Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots. PMID:25365290

A parvovirus isolated from royal python (Python regius) is a member of the genus Dependovirus.

PubMed

Farkas, Szilvia L; Zádori, Zoltán; Benko, Mária; Essbauer, Sandra; Harrach, Balázs; Tijssen, Peter

2004-03-01

Parvoviruses were isolated from Python regius and Boa constrictor snakes and propagated in viper heart (VH-2) and iguana heart (IgH-2) cells. The full-length genome of a snake parvovirus was cloned and both strands were sequenced. The organization of the 4432-nt-long genome was found to be typical of parvoviruses. This genome was flanked by inverted terminal repeats (ITRs) of 154 nt, containing 122 nt terminal hairpins and contained two large open reading frames, encoding the non-structural and structural proteins. Genes of this new parvovirus were most similar to those from waterfowl parvoviruses and from adeno-associated viruses (AAVs), albeit to a relatively low degree and with some organizational differences. The structure of its ITRs also closely resembled those of AAVs. Based on these data, we propose to classify this virus, the first serpentine parvovirus to be identified, as serpentine adeno-associated virus (SAAV) in the genus Dependovirus.

33 CFR 100.109 - Winter Harbor Lobster Boat Race, Winter Harbor, ME.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Winter Harbor Lobster Boat Race, Winter Harbor, ME. 100.109 Section 100.109 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF... Lobster Boat Race, Winter Harbor, ME. (a) Regulated area. The regulated area includes all waters of Winter...

Zika Virus-Associated Cognitive Impairment in Adolescent, 2016.

PubMed

Zucker, Jason; Neu, Natalie; Chiriboga, Claudia A; Hinton, Veronica J; Leonardo, Marc; Sheikh, Arif; Thakur, Kiran

2017-06-01

Incidence of neurologic manifestations associated with Zika virus infection has been increasing. In 2016, neuropsychological and cognitive changes developed in an adolescent after travel to a Zika virus-endemic area. Single-photon emission computed tomography and neuropsychological testing raised the possibility that Zika virus infection may lead to neuropsychiatric and cognitive symptoms.

Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

PubMed

Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

2010-06-01

Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

Comparison of variable region 3 sequences of human immunodeficiency virus type 1 from infected children with the RNA and DNA sequences of the virus populations of their mothers.

PubMed Central

Scarlatti, G; Leitner, T; Halapi, E; Wahlberg, J; Marchisio, P; Clerici-Schoeller, M A; Wigzell, H; Fenyö, E M; Albert, J; Uhlén, M

1993-01-01

We have compared the variable region 3 sequences from 10 human immunodeficiency virus type 1 (HIV-1)-infected infants to virus sequences from the corresponding mothers. The sequences were derived from DNA of uncultured peripheral blood mononuclear cells (PBMC), DNA of cultured PBMC, and RNA from serum collected at or shortly after delivery. The infected infants, in contrast to the mothers, harbored homogeneous virus populations. Comparison of sequences from the children and clones derived from DNA of the corresponding mothers showed that the transmitted virus represented either a minor or a major virus population of the mother. In contrast to an earlier study, we found no evidence of selection of minor virus variants during transmission. Furthermore, the transmitted virus variant did not show any characteristic molecular features. In some cases the transmitted virus was more related to the virus RNA population of the mother and in other cases it was more related to the virus DNA population. This suggests that either cell-free or cell-associated virus may be transmitted. These data will help AIDS researchers to understand the mechanism of transmission and to plan strategies for prevention of transmission. PMID:8446584

In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

PubMed Central

Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

2016-01-01

Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia. PMID:26865405

Viruses Associated with Human Cancer

PubMed Central

McLaughlin-Drubin, Margaret E.; Munger, Karl

2008-01-01

It is estimated that viral infections contribute to 15–20% of all human cancers. As obligatory intracellular parasites, viruses encode proteins that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. These cellular processes are governed by complex and redundant regulatory networks and are surveyed by sentinel mechanisms that ensure that aberrant cells are removed from the proliferative pool. Given that the genome size of a virus is highly restricted to ensure packaging within an infectious structure, viruses must target cellular regulatory nodes with limited redundancy and need to inactivate surveillance mechanisms that would normally recognize and extinguish such abnormal cells. In many cases, key proteins in these same regulatory networks are subject to mutation in non-virally associated diseases and cancers. Oncogenic viruses have thus served as important experimental models to identify and molecularly investigate such cellular networks. These include the discovery of oncogenes and tumor suppressors, identification of regulatory networks that are critical for maintenance of genomic integrity, and processes that govern immune surveillance. PMID:18201576

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

PubMed

Griot-Wenk, M E; Cherpillod, P; Koch, A; Zurbriggen, R; Bruckner, L; Wittek, R; Zurbriggen, A

2001-06-01

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Zika virus infection, associated microcephaly, and low yellow fever vaccination coverage in Brazil: is there any causal link?

PubMed

De Góes Cavalcanti, Luciano Pamplona; Tauil, Pedro Luiz; Alencar, Carlos Henrique; Oliveira, Wanderson; Teixeira, Mauro Martins; Heukelbach, Jorg

2016-06-30

Since the end of 2014, Zika virus (ZIKV) infection has been rapidly spreading in Brazil. To analyze the possible association of yellow fever vaccine with a protective effect against ZIKV-related microcephaly, the following spatial analyses were performed, using Brazilian municipalities as units: i) yellow fever vaccination coverage in Brazilian municipalities in individuals aged 15-49; ii) reported cases of microcephaly by municipality; and iii) confirmed cases of microcephaly related to ZIKV, by municipality. SaTScan software was used to identify clusters of municipalities for high risk of microcephaly. There were seven significant high risk clusters of confirmed microcephaly cases, with four of them located in the Northeast where yellow fever vaccination rates were the lowest. The clusters harbored only 2.9% of the total population of Brazil, but 15.2% of confirmed cases of microcephaly. We hypothesize that pregnant women in regions with high yellow fever vaccination coverage may pose their offspring to lower risk for development of microcephaly. There is an urgent need for systematic studies to confirm the possible link between low yellow fever vaccination coverage, Zika virus infection and microcephaly.

Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus.

PubMed

Pereira, Thiago Nunes; Rocha, Marcele Neves; Sucupira, Pedro Henrique Ferreira; Carvalho, Fabiano Duarte; Moreira, Luciano Andrade

2018-05-02

Wolbachia, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

Advances in Virus-Directed Therapeutics against Epstein-Barr Virus-Associated Malignancies

PubMed Central

Ghosh, Sajal K.; Perrine, Susan P.; Faller, Douglas V.

2012-01-01

Epstein-Barr virus (EBV) is the causal agent in the etiology of Burkitt's lymphoma and nasopharyngeal carcinoma and is also associated with multiple human malignancies, including Hodgkin's and non-Hodgkin's lymphoma, and posttransplantation lymphoproliferative disease, as well as sporadic cancers of other tissues. A causal relationship of EBV to these latter malignancies remains controversial, although the episomic EBV genome in most of these cancers is clonal, suggesting infection very early in the development of the tumor and a possible role for EBV in the genesis of these diseases. Furthermore, the prognosis of these tumors is invariably poor when EBV is present, compared to their EBV-negative counterparts. The physical presence of EBV in these tumors represents a potential “tumor-specific” target for therapeutic approaches. While treatment options for other types of herpesvirus infections have evolved and improved over the last two decades, however, therapies directed at EBV have lagged. A major constraint to pharmacological intervention is the shift from lytic infection to a latent pattern of gene expression, which persists in those tumors associated with the virus. In this paper we provide a brief account of new virus-targeted therapeutic approaches against EBV-associated malignancies. PMID:22500168

77 FR 50916 - Safety Zone; Boston Harbor's Rock Removal Project, Boston Inner Harbor, Boston, MA

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-23

... DEPARTMENT OF HOMELAND SECURITY Coast Guard 33 CFR Part 165 [Docket No. USCG-2012-0767] RIN 1625-AA00 Safety Zone; Boston Harbor's Rock Removal Project, Boston Inner Harbor, Boston, MA AGENCY: Coast.... 165.T01-0767 Safety Zone; Boston Harbor's Rock Removal Project, Boston Inner Harbor, Boston, MA. (a...

AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

PubMed

Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

2016-01-01

Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

The evolution of heart gene delivery vectors

PubMed Central

Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

2012-01-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

Neuroimaging Findings of Zika Virus-Associated Neurologic Complications in Adults.

PubMed

Hygino da Cruz, L C; Nascimento, O J M; Lopes, F P P L; da Silva, I R F

2018-05-17

When the first suspected cases of neurologic disorders associated with the Zika virus were noticed in Brazil in late 2015, several studies had been conducted to understand the pathophysiology of the disease and its associated complications. In addition to its well-established association with microcephaly in neonates, the Zika virus infection has also been suggested to trigger other severe neurologic complications in adults, such as Guillain-Barré syndrome, radiculomyelitis, and meningoencephalitis. Hence, the Zika virus should be deemed a global threat that can cause devastating neurologic complications among individuals in all age ranges. The aim of this review was to further describe neuroimaging findings of Zika virus infection and associated neurologic complications found in adults. © 2018 by American Journal of Neuroradiology.

Origins of the rabies viruses associated with an outbreak in Newfoundland during 2002-2003.

PubMed

Nadin-Davis, Susan; Muldoon, Frances; Whitney, Hugh; Wandeler, Alexander I

2008-01-01

After being free of rabies of terrestrial mammals since 1988, an outbreak of rabies occurred on the Island of Newfoundland in December 2002 and continued into the middle of 2003. Twenty-one cases, all due to the arctic fox strain of rabies virus, were reported. To explore the immediate origins of this outbreak, viruses from the Newfoundland epizootic were genetically compared to two other rabies viruses recovered in mid-2002 from Cartwright, a mainland coastal community near the Island. While all Island isolates from the 2002-03 outbreak were genetically very similar, consistent with a single introduction from the mainland, they were phylogenetically quite distant from the two samples from Cartwright. A broader-based study examined the relationships between the Island viruses and arctic fox strain viruses originating from across Canada over a period of 14 yr. This analysis indicated that the Newfoundland outbreak viruses were most similar to a variant identified in Labrador in 2004 but also widely distributed in northern Quebec both before and after the Newfoundland incursion. The eastern coastline of mainland Labrador has harbored a particularly large number of variants during the study period, some of which have not been detected elsewhere. A small number of Greenland isolates included in this study were dispersed within the clades of the Canadian samples rather than forming a discrete cluster, an observation that may underline the relative ease of movement of the rabies arctic lineage between these two countries as a result of animal movements over pack ice.

33 CFR 125.15 - Access to waterfront facilities, and port and harbor areas, including vessels and harbor craft...

Code of Federal Regulations, 2010 CFR

2010-07-01

..., and port and harbor areas, including vessels and harbor craft therein. 125.15 Section 125.15....15 Access to waterfront facilities, and port and harbor areas, including vessels and harbor craft....09 to those waterfront facilities, and port and harbor areas, including vessels and harbor craft...

Analytical and computational modeling of early penetration of non-enveloped icosahedral viruses into cells.

PubMed

Katzengold, Rona; Zaharov, Evgeniya; Gefen, Amit

2016-07-27

As obligate intracellular parasites, all viruses penetrate target cells to initiate replication and infection. This study introduces two approaches for evaluating the contact loads applied to a cell during early penetration of non-enveloped icosahedral viruses. The first approach is analytical modeling which is based on Hertz's theory for the contact of two elastic bodies; here we model the virus capsid as a triangle and the cell as an order-of-magnitude larger sphere. The second approach is finite element modeling, where we simulate three types of viruses: adeno-, papilloma- and polio- viruses, each interacting with a cell section. We find that the peak contact pressures and forces generated at the initial virus-cell contact depend on the virus geometry - that is both size and shape. With respect to shape, we show that the icosahedral virus shape induces greater peak pressures compared to a spherical virus shape. With respect to size, it is shown that the larger the virus is the greater are the contact loads in the attacked cell. Utilization of our modeling can be substantially useful not only for basic science studies, but also in other, more applied fields, such as in the field of gene therapy, or in `phage' virus studies.

Susceptibility of Tissue Cultures of Canine Origin to Viruses

DTIC Science & Technology

1965-08-01

importance. It was realized that dogs may harbor other than the common- ly recognized rabies, distemper and infectious canine hepatitis viruses. Proper... CANINE ORIGIN TO VIRUSES S......... . i Albuquerque, New Mexico by FRANK F. PINDAK AND WILLIAM E. CLAPPER August 1965 DISTRIBUTON STATEMENTA Approved...TID-4500 (43rd Ed.) SUSCEPTIBILITY OF TISSUE CULTURES OF CANINE ORIGIN TO VIRUSES by Frank F. Pindak and William E. Clapper Submitted as a Technical

Serologic Evidence for Influenza C and D Virus among Ruminants and Camelids, Africa, 1991-2015.

PubMed

Salem, Elias; Cook, Elizabeth A J; Lbacha, Hicham Ait; Oliva, Justine; Awoume, Félix; Aplogan, Gilbert L; Hymann, Emmanuel Couacy; Muloi, Dishon; Deem, Sharon L; Alali, Said; Zouagui, Zaid; Fèvre, Eric M; Meyer, Gilles; Ducatez, Mariette F

2017-09-01

Influenza D virus has been identified in America, Europe, and Asia. We detected influenza D virus antibodies in cattle and small ruminants from North (Morocco) and West (Togo and Benin) Africa. Dromedary camels in Kenya harbored influenza C or D virus antibodies, indicating a potential new host for these viruses.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex

PubMed Central

Brass, Volker; Berke, Jan Martin; Montserret, Roland; Blum, Hubert E.; Penin, François; Moradpour, Darius

2008-01-01

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane α-helix that may be involved in intramembrane protein–protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix α0, formed by NS3 residues 12–23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design. PMID:18799730

Papillary urothelial carcinoma with squamous differentiation in association with human papilloma virus: case report and literature review.

PubMed

Guma, Sergei; Maglantay, Remegio; Lau, Ryan; Wieczorek, Rosemary; Melamed, Jonathan; Deng, Fang-Ming; Zhou, Ming; Makarov, Danil; Lee, Peng; Pincus, Matthew R; Pei, Zhi-Heng

2016-01-01

The human papilloma virus (HPV) is a carcinogen known for its strong association with cervical cancers and cervical lesions. It is also known to be associated with a variety of squamous cell carcinomas in other areas, such as the penis, vulva, anus and head and neck. However, the association with urothelial carcinoma remains controversial. Here, we report a case of urothelial carcinoma with squamous differentiation associated with HPV-6/HPV-11. This is a case of a 70 year old man who presented with nocturia and pressure during urination. During the TURP procedure for what was clinically thought to be benign prostate hyperplasia with pathologic diagnosis as prostate carcinoma, a 2 cm papillary mass was found in the distal penile urethra. The papillary mass was found to be a high grade urothelial carcinoma positive for GATA 3 expression, with focal areas of squamous differentiation. The areas with squamous differentiation demonstrated koilocytic differentiation, which were positive for strong p16 expression. The tumor was found to harbor low risk HPV 6/11 by in situ hybridization. This study case demonstrates HPV infection with a low risk subtype (HPV 6/11) associated with an urothelial carcinoma with squamous differentiation and condylomatous features.

Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts.

PubMed

Katz, B Z; Niederman, J C; Olson, B A; Miller, G

1988-02-01

DNA restriction fragment length polymorphisms of Epstein-Barr virus (EBV) DNA were used as a molecular epidemiological tool to study multiple isolates of virus from the same and different individuals. We studied 35 EBV isolates: 19 from seven immunocompromised children and 16 from seven college students with mononucleosis. Analysis of the fragment length polymorphisms in this collection of isolates permitted several conclusions. Sites of polymorphism were most often encountered in regions with repetitive DNA. Epidemiologically unrelated patients harbored viruses that could be readily distinguished; by contrast, two infants and their mothers harbored similar viruses. Isolates from different sites in the same patient were similar. Variations between different clinical isolates of EBV mimic those found between different laboratory strains of the virus. Fragment length polymorphisms thus provide a useful marker for studying transmission and pathogenesis of EBV infections.

The full-length microRNA cluster in the intron of large latency transcript is associated with the virulence of pseudorabies virus.

PubMed

Wang, Xin; Zhang, Mei-Mei; Yan, Kai; Tang, Qi; Wu, Yi-Quan; He, Wen-Bo; Chen, Huan-Chun; Liu, Zheng-Fei

2018-07-01

Pseudorabies virus (PRV), the etiological pathogen of Aujeszky's disease, belongs to the Alphaherpesvirus subfamily. Large latency transcript (LLT), the most abundant PRV transcript, harbors a ~ 4.6 kb microRNA (miRNA) cluster-encoding intron. To investigate the function of the LLT miRNA cluster during the life cycle of PRV, we generated a miRNA cluster mutation virus (PRV-∆miR cluster) and revertant virus. Analysis of the growth kinetics of PRV-ΔmiR cluster-infected cells revealed significantly smaller plaques and lower titers than the wild-type and revertant viruses. The mutation virus exhibited increased IE180 and decreased EP0 expression. The clinical symptoms observed in mice infected with PRV-ΔmiR cluster revealed that the miRNA cluster is involved in the pathogenesis of PRV. Physical parameters, virus shedding assays, and the SN 50 titers revealed that the miRNA cluster enhances PRV virulence in pigs. Collectively, our findings suggest that the full-length miRNA cluster is involved in PRV replication and virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

PubMed

Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2016-07-15

The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Predicting dredging-associated effects to coral reefs in Apra Harbor, Guam - Part 1: Sediment exposure modeling.

PubMed

Gailani, Joseph Z; Lackey, Tahirih C; King, David B; Bryant, Duncan; Kim, Sung-Chan; Shafer, Deborah J

2016-03-01

Model studies were conducted to investigate the potential coral reef sediment exposure from dredging associated with proposed development of a deepwater wharf in Apra Harbor, Guam. The Particle Tracking Model (PTM) was applied to quantify the exposure of coral reefs to material suspended by the dredging operations at two alternative sites. Key PTM features include the flexible capability of continuous multiple releases of sediment parcels, control of parcel/substrate interaction, and the ability to efficiently track vast numbers of parcels. This flexibility has facilitated simulating the combined effects of sediment released from clamshell dredging and chiseling within Apra Harbor. Because the rate of material released into the water column by some of the processes is not well understood or known a priori, the modeling approach was to bracket parameters within reasonable ranges to produce a suite of potential results from multiple model runs. Sensitivity analysis to model parameters is used to select the appropriate parameter values for bracketing. Data analysis results include mapping the time series and the maximum values of sedimentation, suspended sediment concentration, and deposition rate. Data were used to quantify various exposure processes that affect coral species in Apra Harbor. The goal of this research is to develop a robust methodology for quantifying and bracketing exposure mechanisms to coral (or other receptors) from dredging operations. These exposure values were utilized in an ecological assessment to predict effects (coral reef impacts) from various dredging scenarios. Copyright © 2015. Published by Elsevier Ltd.

Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes

PubMed Central

Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

2016-01-01

The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

INFECTIOUS DISEASE AND TOXICOLOGICAL MONITORING OF STRANDED PACIFIC HARBOR SEALS (PHOCA VITULINA RICHARDSI) IN COOK INLET AS SURROGATES FOR MONITORING ENDANGERED BELUGAS (DELPHINAPTERUS LEUCAS).

PubMed

Bauer, Kendra L; Goertz, Caroline E C; Belovarac, Jane A; Walton, Robert W; Dunn, J Lawrence; Tuomi, Pamela

2016-09-01

  Pacific harbor seals ( Phoca vitulina richardsi) and belugas ( Delphinapterus leucas ) eat many of the same prey species, occupy the same geographic area, and demonstrate site fidelity in Cook Inlet, Alaska. Although most direct research involving the critically endangered belugas is currently prohibited, studying harbor seals may provide important information about this beluga population. In recent years, harbor seal populations in Alaska have declined for unknown reasons. As part of its stranding program, the Alaska SeaLife Center (ASLC) managed 59 cases of live and dead stranded harbor seals from Cook Inlet between 1997 and 2011. Animals were screened for a variety of diseases and contaminants of concern. Animals were negative by serology to the following diseases: avian influenza, canine distemper virus, dolphin morbillivirus, porpoise morbillivirus, Leptospira canicola, L. grippotyphosa, L. pomona, Neospora caninum , Sarcocystis neurona , and Toxoplasma gondii . Positive titers were found against Brucella spp., phocine distemper virus, seal herpesvirus-1, L. bratislava, L. hardjo, and L. icterohemorrhagiae. All titers were stable or declining except in one animal with an increasing titer for seal herpesvirus-1. Fecal pathogen screenings identified normal flora as well as stable or declining low levels of potentially pathogenic and opportunistic bacteria, though most were of little concern for seal health. In most animals, toxicology screening showed that the majority of tested contaminants were below detectable limits. The level of evidence of exposure to pathogens of concern was low in harbor seals. Although the infectious disease burden and contaminant levels in belugas in Cook Inlet cannot be definitively determined without direct testing, pathogen and contaminant exposure is expected to be similar to that found in harbor seals in this region, as the harbor seals and belugas share the habitat and food resources.

Novel B19-Like Parvovirus in the Brain of a Harbor Seal

PubMed Central

Bodewes, Rogier; Rubio García, Ana; Wiersma, Lidewij C. M.; Getu, Sarah; Beukers, Martijn; Schapendonk, Claudia M. E.; van Run, Peter R. W. A.; van de Bildt, Marco W. G.; Poen, Marjolein J.; Osinga, Nynke; Sánchez Contreras, Guillermo J.; Kuiken, Thijs; Smits, Saskia L.; Osterhaus, Albert D. M. E.

2013-01-01

Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection. PMID:24223918

Novel B19-like parvovirus in the brain of a harbor seal.

PubMed

Bodewes, Rogier; Rubio García, Ana; Wiersma, Lidewij C M; Getu, Sarah; Beukers, Martijn; Schapendonk, Claudia M E; van Run, Peter R W A; van de Bildt, Marco W G; Poen, Marjolein J; Osinga, Nynke; Sánchez Contreras, Guillermo J; Kuiken, Thijs; Smits, Saskia L; Osterhaus, Albert D M E

2013-01-01

Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.

BK virus-associated hemorrhagic cystitis after pediatric stem cell transplantation

PubMed Central

Han, Seung Beom; Kang, Jin Han

2014-01-01

Hemorrhagic cystitis is a common stem cell transplantation-related complication. The incidence of early-onset hemorrhagic cystitis, which is related to the pretransplant conditioning regimen, has decreased with the concomitant use of mesna and hyperhydration. However, late-onset hemorrhagic cystitis, which is usually caused by the BK virus, continues to develop. Although the BK virus is the most common pathogenic microorganism of poststem cell transplantation late-onset hemorrhagic cystitis, pediatricians outside the hemato-oncology and nephrology specialties tend to be unfamiliar with hemorrhagic cystitis and the BK virus. Moreover, no standard guidelines for the early diagnosis and treatment of BK virus-associated hemorrhagic cystitis after stem cell transplantation have been established. Here, we briefly introduce poststem cell transplantation BK virus-associated hemorrhagic cystitis. PMID:25653684

The evolution of heart gene delivery vectors.

PubMed

Wasala, Nalinda B; Shin, Jin-Hong; Duan, Dongsheng

2011-10-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. Copyright © 2011 John Wiley & Sons, Ltd.

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

PubMed Central

Drouin, Lauren M.; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni

2016-01-01

ABSTRACT The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCE The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

PubMed

Drouin, Lauren M; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni; Baker, Timothy S; Agbandje-McKenna, Mavis

2016-10-01

The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid

T-cell reconstitution during murine acquired immunodeficiency syndrome (MAIDS) produces neuroinflammation and mortality in animals harboring opportunistic viral brain infection.

PubMed

Mutnal, Manohar B; Schachtele, Scott J; Hu, Shuxian; Lokensgard, James R

2013-07-31

Highly active antiretroviral therapy (HAART) restores inflammatory immune responses in AIDS patients which may unmask previous subclinical infections or paradoxically exacerbate symptoms of opportunistic infections. In resource-poor settings, 25% of patients receiving HAART may develop CNS-related immune reconstitution inflammatory syndrome (IRIS). Here we describe a reliable mouse model to study underlying immunopathological mechanisms of CNS-IRIS. Utilizing our HSV brain infection model and mice with MAIDS, we investigated the effect of immune reconstitution on MAIDS mice harboring opportunistic viral brain infection. Using multi-color flow cytometry, we quantitatively measured the cellular infiltrate and microglial activation. Infection with the LP-BM5 retroviral mixture was found to confer susceptibility to herpes simplex virus (HSV)-1 brain infection to normally-resistant C57BL/6 mice. Increased susceptibility to brain infection was due to severe immunodeficiency at 8 wks p.i. and a marked increase in programmed death-1 (PD-1) expression on CD4+ and CD8+ T-cells. Both T-cell loss and opportunistic brain infection were associated with high level PD-1 expression because PD-1-knockout mice infected with LP-BM5 did not exhibit lymphopenia and retained resistance to HSV-1. In addition, HSV-infection of MAIDS mice stimulated peripheral immune cell infiltration into the brain and its ensuing microglial activation. Interestingly, while opportunistic herpes virus brain infection of C57BL/6 MAIDS mice was not itself lethal, when T-cell immunity was reconstituted through adoptive transfer of virus-specific CD3+ T-cells, it resulted in significant mortality among recipients. This immune reconstitution-induced mortality was associated with exacerbated neuroinflammation, as determined by MHC class II expression on resident microglia and elevated levels of Th1 cytokines in the brain. Taken together, these results indicate development of an immune reconstitution disease

Restoring neuronal progranulin reverses deficits in a mouse model of frontotemporal dementia

PubMed Central

Arrant, Andrew E.; Filiano, Anthony J.; Unger, Daniel E.; Young, Allen H.

2017-01-01

Loss-of-function mutations in progranulin (GRN), a secreted glycoprotein expressed by neurons and microglia, are a common autosomal dominant cause of frontotemporal dementia, a neurodegenerative disease commonly characterized by disrupted social and emotional behaviour. GRN mutations are thought to cause frontotemporal dementia through progranulin haploinsufficiency, therefore, boosting progranulin expression from the intact allele is a rational treatment strategy. However, this approach has not been tested in an animal model of frontotemporal dementia and it is unclear if boosting progranulin could correct pre-existing deficits. Here, we show that adeno-associated virus-driven expression of progranulin in the medial prefrontal cortex reverses social dominance deficits in Grn+/– mice, an animal model of frontotemporal dementia due to GRN mutations. Adeno-associated virus-progranulin also corrected lysosomal abnormalities in Grn+/– mice. The adeno-associated virus-progranulin vector only transduced neurons, suggesting that restoring neuronal progranulin is sufficient to correct deficits in Grn+/– mice. To further test the role of neuronal progranulin in the development of frontotemporal dementia-related deficits, we generated two neuronal progranulin-deficient mouse lines using CaMKII-Cre and Nestin-Cre. Measuring progranulin levels in these lines indicated that most brain progranulin is derived from neurons. Both neuronal progranulin-deficient lines developed social dominance deficits similar to those in global Grn+/– mice, showing that neuronal progranulin deficiency is sufficient to disrupt social behaviour. These data support the concept of progranulin-boosting therapies for frontotemporal dementia and highlight an important role for neuron-derived progranulin in maintaining normal social function. PMID:28379303

Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis

PubMed Central

Fishbein, Ilia; Guerrero, David T.; Alferiev, Ivan S.; Foster, Jonathan B.; Minutolo, Nicholas G.; Chorny, Michael; Mas Monteys, Alejandro; Driesbaugh, Kathryn H.; Nagaswami, Chandrasekaran; Levy, Robert J.

2017-01-01

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty. PMID:28832561

Protective Immunity and Safety of a Genetically Modified Influenza Virus Vaccine

PubMed Central

Garcia, Cristiana Couto; Filho, Bruno Galvão; Gonçalves, Ana Paula de Faria; Lima, Braulio Henrique Freire; Lopes, Gabriel Augusto Oliveira; Rachid, Milene Alvarenga; Peixoto, Andiara Cristina Cardoso; de Oliveira, Danilo Bretas; Ataíde, Marco Antônio; Zirke, Carla Aparecida; Cotrim, Tatiane Marques; Costa, Érica Azevedo; Almeida, Gabriel Magno de Freitas; Russo, Remo Castro; Gazzinelli, Ricardo Tostes; Machado, Alexandre de Magalhães Vieira

2014-01-01

Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors. To this aim, one of the most promising approaches consists of generating recombinant viruses harboring partially truncated neuraminidase (NA) segments. To date, all studies have pointed to safety and usefulness of this viral platform. However, some aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to immunocompromised hosts remained to be elucidated. In the present study, we generated a recombinant influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and the safety of this recombinant virus in wild type or knock-out (KO) mice with impaired innate (Myd88 -/-) or acquired (RAG -/-) immune responses. Infection using truncated neuraminidase influenza virus was harmless regarding lung and systemic inflammatory response in wild type mice and was highly attenuated in KO mice. We also demonstrated that vNA-Δ infection does not induce unbalanced cytokine production that strongly contributes to lung damage in infected mice. In addition, the recombinant influenza virus was able to trigger both local and systemic virus-specific humoral and CD8+ T cellular immune responses which protected immunized mice against the challenge with a lethal dose of homologous A/PR8/34 influenza virus. Taken together, our findings suggest and reinforce the safety of using NA deleted influenza viruses as antigen delivery vectors against human or veterinary pathogens. PMID:24927156

Syngeneic AAV pseudo-particles potentiate gene transduction of AAV vectors

USDA-ARS?s Scientific Manuscript database

Gene delivery vectors based on adeno-associated virus (AAV) have emerged as safe and efficient therapeutic platform for numerous diseases. Excessive empty particles were generated as impurities during AAV vector production, but their effects on clinical outcome of AAV gene therapy are unclear. Here,...

Study of the Metatranscriptome of Eight Social and Solitary Wild Bee Species Reveals Novel Viruses and Bee Parasites

PubMed Central

Schoonvaere, Karel; Smagghe, Guy; Francis, Frédéric; de Graaf, Dirk C.

2018-01-01

Bees are associated with a remarkable diversity of microorganisms, including unicellular parasites, bacteria, fungi, and viruses. The application of next-generation sequencing approaches enables the identification of this rich species composition as well as the discovery of previously unknown associations. Using high-throughput polyadenylated ribonucleic acid (RNA) sequencing, we investigated the metatranscriptome of eight wild bee species (Andrena cineraria, Andrena fulva, Andrena haemorrhoa, Bombus terrestris, Bombus cryptarum, Bombus pascuorum, Osmia bicornis, and Osmia cornuta) sampled from four different localities in Belgium. Across the RNA sequencing libraries, 88–99% of the taxonomically informative reads were of the host transcriptome. Four viruses with homology to insect pathogens were found including two RNA viruses (belonging to the families Iflaviridae and Tymoviridae that harbor already viruses of honey bees), a double stranded DNA virus (family Nudiviridae) and a single stranded DNA virus (family Parvoviridae). In addition, we found genomic sequences of 11 unclassified arthropod viruses (related to negeviruses, sobemoviruses, totiviruses, rhabdoviruses, and mononegaviruses), seven plant pathogenic viruses, and one fungal virus. Interestingly, nege-like viruses appear to be widespread, host-specific, and capable of attaining high copy numbers inside bees. Next to viruses, three novel parasite associations were discovered in wild bees, including Crithidia pragensis and a tubulinosematid and a neogregarine parasite. Yeasts of the genus Metschnikowia were identified in solitary bees. This study gives a glimpse of the microorganisms and viruses associated with social and solitary wild bees and demonstrates that their diversity exceeds by far the subset of species first discovered in honey bees. PMID:29491849

Study of the Metatranscriptome of Eight Social and Solitary Wild Bee Species Reveals Novel Viruses and Bee Parasites.

PubMed

Schoonvaere, Karel; Smagghe, Guy; Francis, Frédéric; de Graaf, Dirk C

2018-01-01

Bees are associated with a remarkable diversity of microorganisms, including unicellular parasites, bacteria, fungi, and viruses. The application of next-generation sequencing approaches enables the identification of this rich species composition as well as the discovery of previously unknown associations. Using high-throughput polyadenylated ribonucleic acid (RNA) sequencing, we investigated the metatranscriptome of eight wild bee species ( Andrena cineraria, Andrena fulva, Andrena haemorrhoa, Bombus terrestris, Bombus cryptarum, Bombus pascuorum, Osmia bicornis , and Osmia cornuta ) sampled from four different localities in Belgium. Across the RNA sequencing libraries, 88-99% of the taxonomically informative reads were of the host transcriptome. Four viruses with homology to insect pathogens were found including two RNA viruses (belonging to the families Iflaviridae and Tymoviridae that harbor already viruses of honey bees), a double stranded DNA virus (family Nudiviridae ) and a single stranded DNA virus (family Parvoviridae ). In addition, we found genomic sequences of 11 unclassified arthropod viruses (related to negeviruses, sobemoviruses, totiviruses, rhabdoviruses, and mononegaviruses), seven plant pathogenic viruses, and one fungal virus. Interestingly, nege-like viruses appear to be widespread, host-specific, and capable of attaining high copy numbers inside bees. Next to viruses, three novel parasite associations were discovered in wild bees, including Crithidia pragensis and a tubulinosematid and a neogregarine parasite. Yeasts of the genus Metschnikowia were identified in solitary bees. This study gives a glimpse of the microorganisms and viruses associated with social and solitary wild bees and demonstrates that their diversity exceeds by far the subset of species first discovered in honey bees.

The Role of Epigenetic Regulation in Epstein-Barr Virus-Associated Gastric Cancer

PubMed Central

Nishikawa, Jun; Iizasa, Hisashi; Nakamura, Munetaka; Saito, Mari; Sasaki, Sho; Shimokuri, Kanami; Yanagihara, Masashi; Sakai, Kouhei; Suehiro, Yutaka; Yamasaki, Takahiro; Sakaida, Isao

2017-01-01

The Epstein–Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma (EBVaGC), all tumor cells harbor the clonal EBV genome. The expression of latent EBV genes is strictly regulated through the methylation of EBV DNA. The methylation of viral DNA regulates the type of EBV latency, and methylation of the tumor suppressor genes is a key abnormality in EBVaGC. The methylation frequencies of several tumor suppressor genes and cell adhesion molecules are significantly higher in EBVaGC than in control cases. EBV-derived microRNAs repress translation from viral and host mRNAs. EBV regulates the expression of non-coding RNA in gastric carcinoma. With regard to the clinical application of demethylating agents against EBVaGC, we investigated the effects of decitabine against the EBVaGC cell lines. Decitabine inhibited the cell growth of EBVaGC cells. The promoter regions of p73 and Runt-related transcription factor 3(RUNX3) were demethylated, and their expression was upregulated by the treatment. We review the role of epigenetic regulation in the development and maintenance of EBVaGC and discuss the therapeutic application of DNA demethylating agents for EBVaGC. PMID:28757548

The Role of Epigenetic Regulation in Epstein-Barr Virus-Associated Gastric Cancer.

PubMed

Nishikawa, Jun; Iizasa, Hisashi; Yoshiyama, Hironori; Nakamura, Munetaka; Saito, Mari; Sasaki, Sho; Shimokuri, Kanami; Yanagihara, Masashi; Sakai, Kouhei; Suehiro, Yutaka; Yamasaki, Takahiro; Sakaida, Isao

2017-07-25

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma (EBVaGC), all tumor cells harbor the clonal EBV genome. The expression of latent EBV genes is strictly regulated through the methylation of EBV DNA. The methylation of viral DNA regulates the type of EBV latency, and methylation of the tumor suppressor genes is a key abnormality in EBVaGC. The methylation frequencies of several tumor suppressor genes and cell adhesion molecules are significantly higher in EBVaGC than in control cases. EBV-derived microRNAs repress translation from viral and host mRNAs. EBV regulates the expression of non-coding RNA in gastric carcinoma. With regard to the clinical application of demethylating agents against EBVaGC, we investigated the effects of decitabine against the EBVaGC cell lines. Decitabine inhibited the cell growth of EBVaGC cells. The promoter regions of p73 and Runt-related transcription factor 3(RUNX3) were demethylated, and their expression was upregulated by the treatment. We review the role of epigenetic regulation in the development and maintenance of EBVaGC and discuss the therapeutic application of DNA demethylating agents for EBVaGC.

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies.

PubMed

Yashchenko, Varvara V; Gavrilova, Olga V; Rautian, Maria S; Jakobsen, Kjetill S

2012-05-01

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed. Copyright © 2011 Elsevier GmbH. All rights reserved.

Possible Association Between Zika Virus Infection and Microcephaly - Brazil, 2015.

PubMed

Schuler-Faccini, Lavinia; Ribeiro, Erlane M; Feitosa, Ian M L; Horovitz, Dafne D G; Cavalcanti, Denise P; Pessoa, André; Doriqui, Maria Juliana R; Neri, Joao Ivanildo; Neto, Joao Monteiro de Pina; Wanderley, Hector Y C; Cernach, Mirlene; El-Husny, Antonette S; Pone, Marcos V S; Serao, Cassio L C; Sanseverino, Maria Teresa V

2016-01-29

In early 2015, an outbreak of Zika virus, a flavivirus transmitted by Aedes mosquitoes, was identified in northeast Brazil, an area where dengue virus was also circulating. By September, reports of an increase in the number of infants born with microcephaly in Zika virus-affected areas began to emerge, and Zika virus RNA was identified in the amniotic fluid of two women whose fetuses had been found to have microcephaly by prenatal ultrasound. The Brazil Ministry of Health (MoH) established a task force to investigate the possible association of microcephaly with Zika virus infection during pregnancy and a registry for incident microcephaly cases (head circumference ≥2 standard deviations [SD] below the mean for sex and gestational age at birth) and pregnancy outcomes among women suspected to have had Zika virus infection during pregnancy. Among a cohort of 35 infants with microcephaly born during August-October 2015 in eight of Brazil's 26 states and reported to the registry, the mothers of all 35 had lived in or visited Zika virus-affected areas during pregnancy, 25 (71%) infants had severe microcephaly (head circumference >3 SD below the mean for sex and gestational age), 17 (49%) had at least one neurologic abnormality, and among 27 infants who had neuroimaging studies, all had abnormalities. Tests for other congenital infections were negative. All infants had a lumbar puncture as part of the evaluation and cerebrospinal fluid (CSF) samples were sent to a reference laboratory in Brazil for Zika virus testing; results are not yet available. Further studies are needed to confirm the association of microcephaly with Zika virus infection during pregnancy and to understand any other adverse pregnancy outcomes associated with Zika virus infection. Pregnant women in Zika virus-affected areas should protect themselves from mosquito bites by using air conditioning, screens, or nets when indoors, wearing long sleeves and pants, using permethrin-treated clothing and gear

Characterization of membrane association of Rinderpest virus matrix protein

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

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge.

PubMed

O'Donnell, Vivian; Holinka, Lauren G; Krug, Peter W; Gladue, Douglas P; Carlson, Jolene; Sanford, Brenton; Alfano, Marialexia; Kramer, Edward; Lu, Zhiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R; Borca, Manuel V

2015-08-01

African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 10(6) 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 10(4) HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (10(2) to 10(3) HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 10(2) HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 10(3) conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge

PubMed Central

O'Donnell, Vivian; Holinka, Lauren G.; Krug, Peter W.; Gladue, Douglas P.; Carlson, Jolene; Sanford, Brenton; Alfano, Marialexia; Kramer, Edward; Lu, Zhiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R.

2015-01-01

ABSTRACT African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 106 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 104 HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (102 to 103 HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 102 HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 103 conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce

Detection of viruses in used ventilation filters from two large public buildings.

PubMed

Goyal, Sagar M; Anantharaman, Senthilvelan; Ramakrishnan, M A; Sajja, Suchitra; Kim, Seung Won; Stanley, Nicholas J; Farnsworth, James E; Kuehn, Thomas H; Raynor, Peter C

2011-09-01

Viral and bacterial pathogens may be present in the air after being released from infected individuals and animals. Filters are installed in the heating, ventilation, and air-conditioning (HVAC) systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. These filters process enormous volumes of air. This study was undertaken to determine the utility of sampling used ventilation filters to assess the types and concentrations of virus aerosols present in buildings. The HVAC filters from 2 large public buildings in Minneapolis and Seattle were sampled to determine the presence of human respiratory viruses and viruses with bioterrorism potential. Four air-handling units were selected from each building, and a total of 64 prefilters and final filters were tested for the presence of influenza A, influenza B, respiratory syncytial, corona, parainfluenza 1-3, adeno, orthopox, entero, Ebola, Marburg, Lassa fever, Machupo, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis viruses. Representative pieces of each filter were cut and eluted with a buffer solution. Attempts were made to detect viruses by inoculation of these eluates in cell cultures (Vero, MDCK, and RK-13) and specific pathogen-free embryonated chicken eggs. Two passages of eluates in cell cultures or these eggs did not reveal the presence of any live virus. The eluates were also examined by polymerase chain reaction or reverse-transcription polymerase chain reaction to detect the presence of viral DNA or RNA, respectively. Nine of the 64 filters tested were positive for influenza A virus, 2 filters were positive for influenza B virus, and 1 filter was positive for parainfluenza virus 1. These findings indicate that existing building HVAC filters may be used as a method of detection for airborne viruses. As integrated long-term bioaerosol sampling devices, they may yield valuable information on the epidemiology and aerobiology of

77 FR 38482 - Safety Zone; Oswego Independence Celebration Fireworks, Oswego Harbor, Oswego, NY

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-28

...-AA00 Safety Zone; Oswego Independence Celebration Fireworks, Oswego Harbor, Oswego, NY AGENCY: Coast... Oswego Harbor during the Oswego Independence Celebration Fireworks display. This temporary safety zone is necessary to protect spectators and vessels from the hazards associated with a fireworks display. DATES...

Influence of Herpes Simplex Virus 1 Latency-Associated Transcripts on the Establishment and Maintenance of Latency in the ROSA26R Reporter Mouse Model

PubMed Central

Nicoll, M. P.; Proença, J. T.; Connor, V.

2012-01-01

Herpes simplex virus 1 (HSV-1) can establish life-long latent infection in sensory neurons, from which periodic reactivation can occur. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs). While not essential for any phase of latency, to date the LATs have been shown to increase the efficiency of both establishment and reactivation of latency in small-animal models. We sought to investigate the role of LAT expression in the frequency of latency establishment within the ROSA26R reporter mouse model utilizing Cre recombinase-encoding recombinant viruses harboring deletions of the core LAT promoter (LAP) region. HSV-1 LAT expression was observed to influence the number of latently infected neurons in trigeminal but not dorsal root ganglia. Furthermore, the relative frequencies of latency establishment of LAT-positive and LAT-negative viruses are influenced by the inoculum dose following infection of the mouse whisker pads. Finally, analysis of the infected cell population at two latent time points revealed a relative loss of latently infected cells in the absence of LAT expression. We conclude that the HSV-1 LATs facilitate the long-term stability of the latent cell population within the infected host and that interpretation of LAT establishment phenotypes is influenced by infection methodology. PMID:22696655

Changes associated with Ebola virus adaptation to novel species.

PubMed

Pappalardo, Morena; Reddin, Ian G; Cantoni, Diego; Rossman, Jeremy S; Michaelis, Martin; Wass, Mark N

2017-07-01

Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24 and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin α5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses. m.n.wass@kent.ac.uk , m.michaelis@kent.ac.uk or j.s.rossman@kent.ac.uk. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

PubMed Central

Zhang, Wenli; Solanki, Manish; Müther, Nadine; Ebel, Melanie; Wang, Jichang; Sun, Chuanbo; Izsvak, Zsuzsanna; Ehrhardt, Anja

2013-01-01

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells. PMID:24116154

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

PubMed

Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

2015-12-01

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Harbor, to the northwest point. (b) Effective times and dates. This safety and security zone will be in... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone...

The versatile nature of coral-associated viruses.

PubMed

Bettarel, Yvan; Bouvier, Thierry; Nguyen, Hanh Kim; Thu, Pham The

2015-10-01

A recent hypothesis considers that many coral pathologies are the result of a sudden structural alteration of the epibiotic bacterial communities in response to environmental disturbances. However, the ecological mechanisms that lead to shifts in their composition are still unclear. In the ocean, viruses represent a major bactericidal agent but little is known on their occurrence within the coral holobiont. Recent reports have revealed that viruses are abundant and diversified within the coral mucus and therefore could be decisive for coral health. However, their mode of action is still unknown, and there is now an urgent need to shed light on the nature of the relationships they might have with the other prokaryotic and eukaryotic members of the holobiont. In this opinion letter, we are putting forward the hypothesis that coral-associated viruses (mostly bacterial and algal viruses), depending on the environmental conditions might either reinforce coral stability or conversely fasten their decline. We propose that these processes are presumably based on an environmentally driven shift in infection strategies allowing viruses to regulate, circumstantially, both coral symbionts (bacteria or Symbiodinium) and surrounding pathogens. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Adeno-Associated Viral Vector (Serotype 2)-Nerve Growth Factor for Patients With Alzheimer Disease: A Randomized Clinical Trial.

PubMed

Rafii, Michael S; Tuszynski, Mark H; Thomas, Ronald G; Barba, David; Brewer, James B; Rissman, Robert A; Siffert, Joao; Aisen, Paul S

2018-03-26

Nerve growth factor (NGF) is an endogenous neurotrophic factor that prevents the death and augments the functional state of cholinergic neurons of the basal forebrain, a cell population that undergoes extensive degeneration in Alzheimer disease (AD). To determine whether stereotactically guided intracerebral injections of adeno-associated viral vector (serotype 2)-nerve growth factor (AAV2-NGF) are well tolerated and exhibit preliminary evidence of impact on cognitive decline in mild to moderate AD-associated dementia. In a multicenter phase 2 trial, 49 participants with mild to moderate AD were randomly assigned in a 1:1 ratio to receive stereotactically guided intracerebral injections of AAV2-NGF or sham surgery. Participants were enrolled between November 2009 and December 2012. Analyses began in February 2015. The study was conducted at 10 US academic medical centers. Eligibility required a diagnosis of mild to moderate dementia due to AD and individuals aged 55 to 80 years. A total of 39 participants did not pass screening; the most common reason was Mini-Mental State Examination scores below cutoff. Analyses were intention-to-treat. Stereotactically guided intracerebral injections of AAV2-NGF into the nucleus basalis of Meynert of each hemisphere or sham surgery. Change from baseline on the Alzheimer Disease Assessment Scale-cognitive subscale at month 24. Among 49 participants, 21 (43%) were women, 42 (86%) self-identified as white, and the mean (SD) age was 68 (6.4) years. AAV2-NGF was safe and well-tolerated through 24 months. No significant difference was noted between the treatment group and placebo on the primary outcome measure, the Alzheimer Disease Assessment Scale-cognitive subscale (mean [SD] score, 14.52 [4.66] vs 9.11 [4.65], P = .17). This multicenter randomized clinical trial demonstrated the feasibility of sham-surgery-controlled stereotactic gene delivery studies in patients with AD. AAV2-NGF delivery was well-tolerated but did not

Microbial associates of the vine mealybug Planococcus ficus (Hemiptera: Pseudococcidae) under different rearing conditions.

PubMed

Iasur-Kruh, Lilach; Taha-Salaime, Leena; Robinson, Wyatt E; Sharon, Rakefet; Droby, Samir; Perlman, Steve J; Zchori-Fein, Einat

2015-01-01

Sap-feeding insects harbor diverse microbial endosymbionts that play important roles in host ecology and evolution, including contributing to host pest status. The vine mealybug, Planococcus ficus, is a serious pest of grapevines, vectoring a number of pathogenic grape viruses. Previous studies have shown that virus transmission is abolished when mealybugs are raised in the laboratory on potato. To examine the possible role of microbial symbionts in virus transmission, the archaeal, bacterial, and fungal microbiota of field and laboratory P. ficus were characterized using molecular and classical microbiological methods. Lab and field colonies of P. ficus harbored different microbiota. While both were dominated by the bacterial obligate nutritional symbionts Moranella and Tremblaya, field samples also harbored a third bacterium that was allied with cluster L, a lineage of bacterial symbionts previously identified in aphids. Archaea were not found in any of the samples. Fungal communities in field-collected mealybugs were dominated by Metschnikowia and Cladosporium species, while those from laboratory-reared mealybugs were dominated by Alternaria and Cladosporium species. In conclusion, this study has identified a diverse set of microbes, most of which appear to be facultatively associated with P. ficus, depending on environmental conditions. The role of various members of the mealybug microbiome, as well as how the host plant affects microbial community structure, remains to be determined.

Implicated vectors and spread of grapevine red blotch-associated virus in Oregon vineyards

USDA-ARS?s Scientific Manuscript database

Grapevine viruses have detrimental consequences for wine grape production, as is known for Grapevine leafroll -associated viruses (GLRaVs) and Grapevine red blotch -associated virus (GRBaV). From 2013-2016, vineyards in three wine grape production regions of Oregon were surveyed for the presence of ...

Hepatitis A Virus and Hepatitis E Virus: Emerging and Re-Emerging Enterically Transmitted Hepatitis Viruses.

PubMed

Lemon, Stanley M; Walker, Christopher M

2018-05-07

Over the past two decades, progress in understanding human infections with hepatitis A virus (HAV) and hepatitis E virus (HEV) has been eclipsed by the priority of combating persistent hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. During that time, the global burden of liver disease caused by enteric hepatitis viruses has not abated. Because of vaccines, hepatitis A has become increasingly a disease of adults instead of early childhood in many regions of the world, resulting in an age-related shift toward more severe disease. HEV has remained endemic in many developing countries, and in well-developed, economically advanced countries it is now recognized as a cause of chronic, progressive liver disease in individuals with compromised immunity. The goal of this collection of articles is to review recent progress and to shine a bright light on gaps in our understanding of how these viruses replicate, cause disease, interact with the liver and host immune system, and are transmitted, along with prospects for improved control in human populations. Renewed efforts to study and compare HAV and HEV biology in humans and animal models have high potential to enhance our understanding of host-pathogen balance in the liver, and may contribute ultimately to the control of other infectious diseases of the liver. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Lack of association between ectoparasite intensities and rabies virus neutralizing antibody seroprevalence in wild big brown bats (Eptesicus fuscus), Fort Collins, Colorado

USGS Publications Warehouse

Pearce, R.D.; O'Shea, T.J.; Shankar, V.; Rupprecht, C.E.

2007-01-01

Recently, bat ectoparasites have been demonstrated to harbor pathogens of potential importance to humans. We evaluated antirabies antibody seroprevalence and the presence of ectoparasites in big brown bats (Eptesicus fuscus) sampled in 2002 and 2003 in Colorado to investigate if an association existed between ectoparasite intensity and exposure to rabies virus (RV). We used logistic regression and Akaike's Information Criteria adjusted for sample size (AICc) in a post-hoc analysis to investigate the relative importance of three ectoparasite species, as well as bat colony size, year sampled, age class, colony size, and year interaction on the presence of rabies virus neutralizing antibodies (VNA) in serum of wild E. fuscus. We obtained serum samples and ectoparasite counts from big brown bats simultaneously in 2002 and 2003. Although the presence of ectoparasites (Steatonyssus occidentalis and Spinturnix bakeri) were important in elucidating VNA seroprevalence, their intensities were higher in seronegative bats than in seropositive bats, and the presence of a third ectoparasite (Cimex pilosellus) was inconsequential. Colony size and year sampled were the most important variables in these AICc models. These findings suggest that these ectoparasites do not enhance exposure of big brown bats to RV. ?? 2007 Mary Ann Liebert, Inc.

Restoring neuronal progranulin reverses deficits in a mouse model of frontotemporal dementia.

PubMed

Arrant, Andrew E; Filiano, Anthony J; Unger, Daniel E; Young, Allen H; Roberson, Erik D

2017-05-01

Loss-of-function mutations in progranulin (GRN), a secreted glycoprotein expressed by neurons and microglia, are a common autosomal dominant cause of frontotemporal dementia, a neurodegenerative disease commonly characterized by disrupted social and emotional behaviour. GRN mutations are thought to cause frontotemporal dementia through progranulin haploinsufficiency, therefore, boosting progranulin expression from the intact allele is a rational treatment strategy. However, this approach has not been tested in an animal model of frontotemporal dementia and it is unclear if boosting progranulin could correct pre-existing deficits. Here, we show that adeno-associated virus-driven expression of progranulin in the medial prefrontal cortex reverses social dominance deficits in Grn+/- mice, an animal model of frontotemporal dementia due to GRN mutations. Adeno-associated virus-progranulin also corrected lysosomal abnormalities in Grn+/- mice. The adeno-associated virus-progranulin vector only transduced neurons, suggesting that restoring neuronal progranulin is sufficient to correct deficits in Grn+/- mice. To further test the role of neuronal progranulin in the development of frontotemporal dementia-related deficits, we generated two neuronal progranulin-deficient mouse lines using CaMKII-Cre and Nestin-Cre. Measuring progranulin levels in these lines indicated that most brain progranulin is derived from neurons. Both neuronal progranulin-deficient lines developed social dominance deficits similar to those in global Grn+/- mice, showing that neuronal progranulin deficiency is sufficient to disrupt social behaviour. These data support the concept of progranulin-boosting therapies for frontotemporal dementia and highlight an important role for neuron-derived progranulin in maintaining normal social function. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

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

Brodzik, R.; Bandurska, K.; Deka, D.

2005-12-16

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles weremore » efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.« less

Influenza A(H6N1) Virus in Dogs, Taiwan

PubMed Central

Lin, Hui-Ting; Wang, Ching-Ho; Chueh, Ling-Ling; Su, Bi-Ling

2015-01-01

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species. PMID:26583707

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

PubMed Central

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

2016-01-01

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin. PMID:27028521

Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

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

McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie

2004-12-05

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95%more » of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.« less

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

PubMed Central

Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

77 FR 69866 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-21

... for Diabetes and Obesity Description of Technology: This invention is directed to adeno- associated virus (AAV) vector delivery of exendin-4 (Ex-4) to salivary glands as treatment for diabetes and obesity... and weight profile in two rat models of obesity and type 2 diabetes. Further, AAV-mediated delivery of...

Polyomavirus-Specific Cellular Immunity: From BK-Virus-Specific Cellular Immunity to BK-Virus-Associated Nephropathy?

PubMed Central

Dekeyser, Manon; François, Hélène; Beaudreuil, Séverine; Durrbach, Antoine

2015-01-01

In renal transplantation, BK-virus (BKV)-associated nephropathy has emerged as a major complication, with a prevalence of 1–10% and graft loss in >50% of cases. BKV is a member of the polyomavirus family and rarely induces apparent clinical disease in the general population. However, replication of polyomaviruses, associated with significant organ disease, is observed in patients with acquired immunosuppression. Monitoring of specific immunity combined with viral load could be used to individually assess the risk of viral reactivation and virus control. We review the current knowledge on BKV-specific cellular immunity and, more specifically, in immunocompromised patients. In the future, immune-based therapies could allow us to treat and prevent BKV-associated nephropathy. PMID:26136745

75 FR 78601 - Drawbridge Operation Regulation; Gulf Intracoastal Waterway, New Orleans Harbor, Inner Harbor...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-16

... Operation Regulation; Gulf Intracoastal Waterway, New Orleans Harbor, Inner Harbor Navigation Canal, New Orleans, Orleans Parish, LA AGENCY: Coast Guard, DHS. ACTION: Notice of temporary deviation from... Harvey Lock), at New Orleans, Orleans Parish, Louisiana. This deviation is necessary to adjust the...

Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

PubMed

O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

2018-04-15

JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo.

PubMed

Santangelo, Kelly S; Baker, Sarah A; Nuovo, Gerard; Dyce, Jonathan; Bartlett, Jeffrey S; Bertone, Alicia L

2010-02-01

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. (c) 2009 Orthopaedic Research Society.

Drug-induced hypersensitivity syndrome associated with Epstein-Barr virus infection.

PubMed

Descamps, V; Mahe, E; Houhou, N; Abramowitz, L; Rozenberg, F; Ranger-Rogez, S; Crickx, B

2003-05-01

Association of drug-induced hypersensitivity syndrome with viral infection is debated. Human herpesvirus 6 (HHV-6) reactivation has been the most frequently reported infection associated with this syndrome. However, a case of cytomegalovirus (CMV) infection was recently described associated with anticonvulsant-induced hypersensitivity syndrome. We report a case of severe allopurinol-induced hypersensitivity syndrome with pancreatitis associated with Epstein-Barr virus (EBV) infection. Active EBV infection was demonstrated in two consecutive serum samples by the presence of anti-EBV early antigen (EA) IgM antibodies and an increase in anti-EBV EA IgG antibodies, whereas no anti-EBV nuclear antigen IgG antibodies were detected. EBV DNA was detected by polymerase chain reaction (PCR) in peripheral blood mononuclear cells. Reactivation of HHV-6 was suggested only by the presence of anti-HHV-6 IgM antibodies, but HHV-6 DNA was not detected by PCR in the serum. Other viral investigations showed previous infection (CMV, rubella, measles, parvovirus B19), immunization after vaccination (hepatitis B virus), or absence of previous infection (hepatitis C virus, human immunodeficiency virus). We suggest that EBV infection may participate in some cases, as do the other herpesviruses HHV-6 or CMV, in the development of drug-induced hypersensitivity syndrome.

Occurrence and Partial Characterization of Lettuce big vein associated virus and Mirafiori lettuce big vein virus in Lettuce in Iran.

PubMed

Alemzadeh, E; Izadpanah, K

2012-12-01

Mirafiori lettuce big vein virus (MiLBVV) and lettuce big vein associated virus (LBVaV) were found in association with big vein disease of lettuce in Iran. Analysis of part of the coat protein (CP) gene of Iranian isolates of LBVaV showed 97.1-100 % nucleotide sequence identity with other LBVaV isolates. Iranian isolates of MiLBVV belonged to subgroup A and showed 88.6-98.8 % nucleotide sequence identity with other isolates of this virus when amplified by PCR primer pair MiLV VP. The occurrence of both viruses in lettuce crop was associated with the presence of resting spores and zoosporangia of the fungus Olpidium brassicae in lettuce roots under field and greenhouse conditions. Two months after sowing lettuce seed in soil collected from a lettuce field with big vein affected plants, all seedlings were positive for LBVaV and MiLBVV, indicating soil transmission of both viruses.

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors

PubMed Central

Hui, Kwai Fung; Tam, Kam Pui

2017-01-01

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers. PMID:29160853

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors.

PubMed

Hui, Kwai Fung; Tam, Kam Pui; Chiang, Alan Kwok Shing

2017-11-21

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers.

33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

Code of Federal Regulations, 2012 CFR

2012-07-01

... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone... entrance of the harbor connecting coordinates 41°51′09″ N, 087°36′36″W and 41°51′11″ N, 087°36′22″ W. (b...

33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

Code of Federal Regulations, 2014 CFR

2014-07-01

... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone... entrance of the harbor connecting coordinates 41°51′09″ N, 087°36′36″ W and 41°51′11″ N, 087°36′22″ W. (b...

33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

Code of Federal Regulations, 2013 CFR

2013-07-01

... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone... entrance of the harbor connecting coordinates 41°51′09″ N, 087°36′36″W and 41°51′11″ N, 087°36′22″ W. (b...

Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization.

PubMed

Reisinger, Jürgen; Rumpler, Silvia; Lion, Thomas; Ambros, Peter F

2006-04-01

For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV.

Probable Zika virus-associated Guillain-Barré syndrome: Challenges with clinico-laboratory diagnosis.

PubMed

Miller, Eliza; Becker, Zoe; Shalev, Daniel; Lee, Christopher T; Cioroiu, Comana; Thakur, Kiran

2017-04-15

A 55year old woman in New York City presented in May 2016 with progressive weakness, ataxia, paresthesia, and areflexia, shortly after returning from the Dominican Republic. Lumbar puncture revealed cytoalbuminological dissociation. Due to her recent travel, Zika-associated Guillain Barré syndrome (GBS) was suspected and she underwent evaluation for recent flavivirus exposure. Zika virus RNA was not detected in serum, but Zika virus immunoglobulin M (IgM) was detected in both serum and cerebrospinal fluid. Dengue virus IgM in serum was equivocal and dengue virus IgG was detected in the serum. Plaque-reduction neutralization testing showed elevated titers to both Zika virus and dengue virus, providing evidence of recent infection with a flavivirus. The patient was diagnosed with probable Zika virus-associated GBS based on clinical findings, ancillary testing, and laboratory assays according to current guidance from the Centers for Disease Control and Prevention and the Council of State and Territorial Epidemiologists. Zika virus transmission in the Americas is resulting in increasing numbers of patients presenting with Zika virus-associated neurological syndromes. Clinical and laboratory diagnosis in these cases can be challenging and may be aided by consultation with CDC, and state and local public health agencies. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA Viruses in Hymenopteran Pollinators: Evidence of Inter-Taxa Virus Transmission via Pollen and Potential Impact on Non-Apis Hymenopteran Species

PubMed Central

Rajotte, Edwin G.; Holmes, Edward C.; Ostiguy, Nancy; vanEngelsdorp, Dennis; Lipkin, W. Ian; dePamphilis, Claude W.; Toth, Amy L.; Cox-Foster, Diana L.

2010-01-01

Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non

Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

PubMed

Zhou, Bin; Ma, Jingjiao; Liu, Qinfang; Bawa, Bhupinder; Wang, Wei; Shabman, Reed S; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B; Richt, Juergen A; Wentworth, David E; Ma, Wenjun

2014-10-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

PubMed Central

Bawa, Bhupinder; Wang, Wei; Shabman, Reed S.; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B.; Richt, Juergen A.; Wentworth, David E.; Ma, Wenjun

2014-01-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. PMID:25275541

77 FR 40628 - Draft Safe Harbor Agreement and Application for an Enhancement of Survival Permit for the Tres...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-10

...-FF02ENEH00] Draft Safe Harbor Agreement and Application for an Enhancement of Survival Permit for the Tres... permit application and the associated draft Safe Harbor Agreement (SHA). DATES: Comment Period: To ensure... . SUPPLEMENTARY INFORMATION: Under a Safe Harbor Agreement, participating property owners voluntarily undertake...

Modulation of extracellular matrix in cancer is associated with enhanced tumor cell targeting by bacteriophage vectors.

PubMed

Yata, Teerapong; Lee, Eugene L Q; Suwan, Keittisak; Syed, Nelofer; Asavarut, Paladd; Hajitou, Amin

2015-06-03

Gene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM). We used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student's t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups. We demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the

Immunology of Bats and Their Viruses: Challenges and Opportunities

PubMed Central

Schountz, Tony

2014-01-01

Bats are reservoir hosts of several high-impact viruses that cause significant human diseases, including Nipah virus, Marburg virus and rabies virus. They also harbor many other viruses that are thought to have caused disease in humans after spillover into intermediate hosts, including SARS and MERS coronaviruses. As is usual with reservoir hosts, these viruses apparently cause little or no pathology in bats. Despite the importance of bats as reservoir hosts of zoonotic and potentially zoonotic agents, virtually nothing is known about the host/virus relationships; principally because few colonies of bats are available for experimental infections, a lack of reagents, methods and expertise for studying bat antiviral responses and immunology, and the difficulty of conducting meaningful field work. These challenges can be addressed, in part, with new technologies that are species-independent that can provide insight into the interactions of bats and viruses, which should clarify how the viruses persist in nature, and what risk factors might facilitate transmission to humans and livestock. PMID:25494448

Immunology of bats and their viruses: challenges and opportunities.

PubMed

Schountz, Tony

2014-12-01

Bats are reservoir hosts of several high-impact viruses that cause significant human diseases, including Nipah virus, Marburg virus and rabies virus. They also harbor many other viruses that are thought to have caused disease in humans after spillover into intermediate hosts, including SARS and MERS coronaviruses. As is usual with reservoir hosts, these viruses apparently cause little or no pathology in bats. Despite the importance of bats as reservoir hosts of zoonotic and potentially zoonotic agents, virtually nothing is known about the host/virus relationships; principally because few colonies of bats are available for experimental infections, a lack of reagents, methods and expertise for studying bat antiviral responses and immunology, and the difficulty of conducting meaningful field work. These challenges can be addressed, in part, with new technologies that are species-independent that can provide insight into the interactions of bats and viruses, which should clarify how the viruses persist in nature, and what risk factors might facilitate transmission to humans and livestock.

Pearl Harbor Biological Survey

DTIC Science & Technology

1974-08-30

properties, uses, and driving mechanisms affecting the harbor is given. The methods of obtaining current data, salinity profiles, and temperature... salinities were used for each calibration In order to check the salinity computation mechanism of the Instrument. Temperature calibrations were...Water Temperature Contours for Navy Thermal Discharges 3.2-23 3.2-7. General Layout of Pearl Harbor Showing Mean Monthly Salinity (3L) Variation

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

PubMed

Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption

PubMed Central

Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae

2013-01-01

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications, but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions. PMID:23899192

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors

PubMed Central

Bouamama-Gzara, Badra; Selmi, Ilhem; Chebil, Samir; Melki, Imene; Mliki, Ahmed; Ghorbel, Abdelwahed; Carra, Angela; Carimi, Francesco; Mahfoudhi, Naima

2017-01-01

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. ‘Hencha’ were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1−1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1−1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% ‘Hencha’ somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination. PMID:29238279

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors.

PubMed

Bouamama-Gzara, Badra; Selmi, Ilhem; Chebil, Samir; Melki, Imene; Mliki, Ahmed; Ghorbel, Abdelwahed; Carra, Angela; Carimi, Francesco; Mahfoudhi, Naima

2017-12-01

Prospecting of local grapevine ( Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. 'Hencha' were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1 -1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1 -1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% 'Hencha' somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

Sediment toxicity in Savannah Harbor

USGS Publications Warehouse

Winger, P.V.; Lasier, P.J.

1995-01-01

Savannah Harbor, located near the mouth of the Savannah River, Georgia and South Carolina, is impacted by industrial and municipal effluents. Potential release of contaminants stored in harbor sediments through dredging and shipping operations requires that contaminated areas be identified for proper management of the system and protection of wildlife resources. During 1991, Hyalella azteca were exposed in 10-d static-renewal toxicity tests to pore-water and solid-phase sediment samples collected from 26 sites within Savannah Harbor. Pore-water toxicity was more pronounced than that for solidphase sediment. Toxicity and reduced leaf consumption demonstrated impaired sediment quality at specific sites within Savannah Harbor and Back River. Factors responsible for the decreased sediment quality were ammonia, alkalinity, and metal concentrations (cadmium, chromium, lead, molybdenum, and nickel). Elevated concentrations of metals and toxicities in Back River sediments indicated impacts from adjacent dredge-spoil areas.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

PubMed

Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

2016-07-29

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Epstein-Barr Virus in Gastric Carcinoma

PubMed Central

Nishikawa, Jun; Yoshiyama, Hironori; Iizasa, Hisashi; Kanehiro, Yuichi; Nakamura, Munetaka; Nishimura, Junichi; Saito, Mari; Okamoto, Takeshi; Sakai, Kouhei; Suehiro, Yutaka; Yamasaki, Takahiro; Oga, Atsunori; Yanai, Hideo; Sakaida, Isao

2014-01-01

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma, all tumor cells harbor the clonal EBV genome. Gastric carcinoma associated with EBV has distinct clinicopathological features, occurs predominately in men and in younger-aged individuals, and presents a generally diffuse histological type. Most cases of EBV-associated gastric carcinoma exhibit a histology rich in lymphocyte infiltration. The immunological reactiveness in the host may represent a relatively preferable prognosis in EBV-positive cases. This fact highlights the important role of EBV in the development of EBV-associated gastric carcinoma. We have clearly proved direct infection of human gastric epithelialcells by EBV. The infection was achieved by using a recombinant EBV. Promotion of growth by EBV infection was observed in the cells. Considerable data suggest that EBV may directly contribute to the development of EBV-associated GC. This tumor-promoting effect seems to involve multiple mechanisms, because EBV affects several host proteins and pathways that normally promote apoptosis and regulate cell proliferation. PMID:25386788

Gene Therapy of Human Breast Cancer.

DTIC Science & Technology

1998-10-01

gene product of human papilloma virus . They transduced this modified cell line with B7 and showed that immunization with the B7- transduced cell...adeno-LacZ virus , aliquots of 106 human breast cancer cells, purified using methods described above, will be incubated in suspension with adeno-LacZ...v.- Final Report:«DAMD17-94-J-4385 "Gene Therapy of Human Cancer" Page 1 AD GRANT NUMBER DAMD17-94-J-4385 TITLE: Gene Therapy of Human

Genome-wide analysis of Epstein-Barr virus identifies variants and genes associated with gastric carcinoma and population structure.

PubMed

Yao, Youyuan; Xu, Miao; Liang, Liming; Zhang, Haojiong; Xu, Ruihua; Feng, Qisheng; Feng, Lin; Luo, Bing; Zeng, Yi-Xin

2017-10-01

Epstein-Barr virus is a ubiquitous virus and is associated with several human malignances, including the significant subset of gastric carcinoma, Epstein-Barr virus-associated gastric carcinoma. Some Epstein-Barr virus-associated diseases are uniquely prevalent in populations with different geographic origins. However, the features of the disease and geographically associated Epstein-Barr virus genetic variation as well as the roles that the variation plays in carcinogenesis and evolution remain unclear. Therefore, in this study, we sequenced 95 geographically distinct Epstein-Barr virus isolates from Epstein-Barr virus-associated gastric carcinoma biopsies and saliva of healthy donors to detect variants and genes associated with gastric carcinoma and population structure from a genome-wide spectrum. We demonstrated that Epstein-Barr virus revealed the population structure between North China and South China. In addition, we observed population stratification between Epstein-Barr virus strains from gastric carcinoma and healthy controls, indicating that certain Epstein-Barr virus subtypes are associated with different gastric carcinoma risks. We identified that the BRLF1, BBRF3, and BBLF2/BBLF3 genes had significant associations with gastric carcinoma. LMP1 and BNLF2a genes were strongly geographically associated genes in Epstein-Barr virus. Our study provides insights into the genetic basis of oncogenic Epstein-Barr virus for gastric carcinoma, and the genetic variants associated with gastric carcinoma can serve as biomarkers for oncogenic Epstein-Barr virus.

Crop-associated virus reduces the rooting depth of non-crop perennial native grass more than non-crop-associated virus with known viral suppressor of RNA silencing (VSR).

PubMed

Malmstrom, Carolyn M; Bigelow, Patrick; Trębicki, Piotr; Busch, Anna K; Friel, Colleen; Cole, Ellen; Abdel-Azim, Heba; Phillippo, Colin; Alexander, Helen M

2017-09-15

As agricultural acreage expanded and came to dominate landscapes across the world, viruses gained opportunities to move between crop and wild native plants. In the Midwestern USA, virus exchange currently occurs between widespread annual Poaceae crops and remnant native perennial prairie grasses now under consideration as bioenergy feedstocks. In this region, the common aphid species Rhopalosiphum padi L. (the bird cherry-oat aphid) transmits several virus species in the family Luteoviridae, including Barley yellow dwarf virus (BYDV-PAV, genus Luteovirus) and Cereal yellow dwarf virus (CYDV-RPV and -RPS, genus Polerovirus). The yellow dwarf virus (YDV) species in these two genera share genetic similarities in their 3'-ends, but diverge in the 5'-regions. Most notably, CYDVs encode a P0 viral suppressor of RNA silencing (VSR) absent in BYDV-PAV. Because BYDV-PAV has been reported more frequently in annual cereals and CYDVs in perennial non-crop grasses, we examine the hypothesis that the viruses' genetic differences reflect different affinities for crop and non-crop hosts. Specifically, we ask (i) whether CYDVs might persist within and affect a native non-crop grass more strongly than BYDV-PAV, on the grounds that the polerovirus VSR could better moderate the defenses of a well-defended perennial, and (ii) whether the opposite pattern of effects might occur in a less defended annual crop. Because previous work found that the VSR of CYDV-RPS possessed greater silencing suppressor efficiency than that of CYDV-RPV, we further explored (iii) whether a novel grass-associated CYDV-RPS isolate would influence a native non-crop grass more strongly than a comparable CYDV-RPV isolate. In growth chamber studies, we found support for this hypothesis: only grass-associated CYDV-RPS stunted the shoots and crowns of Panicum virgatum L. (switchgrass), a perennial native North American prairie grass, whereas crop-associated BYDV-PAV (and coinfection with BYDV-PAV and CYDV-RPS) most

77 FR 63732 - Safety Zone; Bridge Demolition Project; Indiana Harbor Canal, East Chicago, IN

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-17

...-AA00 Safety Zone; Bridge Demolition Project; Indiana Harbor Canal, East Chicago, IN AGENCY: Coast Guard... from a portion of the Indiana Harbor Canal due to the Demolition Project on the Cline Avenue Bridge... vessels from the hazards associated with the demolition project on the Cline Avenue bridge, which are...

77 FR 72957 - Safety Zone; Bridge Demolition Project; Indiana Harbor Canal, East Chicago, IN

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-07

...-AA00 Safety Zone; Bridge Demolition Project; Indiana Harbor Canal, East Chicago, IN AGENCY: Coast Guard... from a portion of the Indiana Harbor Canal due to the demolition Project on the Cline Avenue Bridge... associated with the demolition project on the Cline Avenue bridge, which are discussed further below. Under 5...

Charge detection mass spectrometry: Instrumentation & applications to viruses

NASA Astrophysics Data System (ADS)

Pierson, Elizabeth E.

virus capsids. Finally, CDMS has been used to characterize the purity of adeno-associated viral vectors for potential gene therapy applications.

33 CFR 80.1122 - Channel Islands Harbor, CA.

Code of Federal Regulations, 2012 CFR

2012-07-01

... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Channel Islands Harbor, CA. 80...

33 CFR 80.1122 - Channel Islands Harbor, CA.

Code of Federal Regulations, 2010 CFR

2010-07-01

... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Channel Islands Harbor, CA. 80...

33 CFR 80.1122 - Channel Islands Harbor, CA.

Code of Federal Regulations, 2014 CFR

2014-07-01

... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Channel Islands Harbor, CA. 80...

33 CFR 80.1122 - Channel Islands Harbor, CA.

Code of Federal Regulations, 2013 CFR

2013-07-01

... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Channel Islands Harbor, CA. 80...

33 CFR 80.1122 - Channel Islands Harbor, CA.

Code of Federal Regulations, 2011 CFR

2011-07-01

... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Channel Islands Harbor, CA. 80...

No association between Epstein-Barr Virus and Mouse Mammary Tumor Virus with Breast Cancer in Mexican Women

NASA Astrophysics Data System (ADS)

Morales-Sánchez, Abigail; Molina-Muñoz, Tzindilú; Martínez-López, Juan L. E.; Hernández-Sancén, Paulina; Mantilla, Alejandra; Leal, Yelda A.; Torres, Javier; Fuentes-Pananá, Ezequiel M.

2013-10-01

Breast cancer is the most frequent malignancy affecting women worldwide. It has been suggested that infection by Epstein Barr Virus (EBV), Mouse Mammary Tumor Virus or a similar virus, MMTV-like virus (MMTV-LV), play a role in the etiology of the disease. However, studies looking at the presence of these viruses in breast cancer have produced conflicting results, and this possible association remains controversial. Here, we used polymerase chain reaction assay to screen specific sequences of EBV and MMTV-LV in 86 tumor and 65 adjacent tissues from Mexican women with breast cancer. Neither tumor samples nor adjacent tissue were positive for either virus in a first round PCR and only 4 tumor samples were EBV positive by a more sensitive nested PCR. Considering the study's statistical power, these results do not support the involvement of EBV and MMTV-LV in the etiology of breast cancer.

The Boston Harbor Project, and large decreases in loadings of eutrophication-related materials to Boston Harbor.

PubMed

Taylor, David I

2010-04-01

Boston Harbor, a bay-estuary in the north-east USA, has recently been the site of one of the largest wastewater infrastructure projects conducted in the USA, the Boston Harbor Project (BHP). The BHP, which was conducted from 1991 to 2000, ended over a century of direct wastewater treatment facility discharges to the harbor. The BHP caused the loadings of total nitrogen (TN), total phosphorus (TP), total suspended solids (TSS) and particulate organic carbon (POC) to the harbor, to decrease by between 80% and 90%. Approximately one-third of the decreases in TSS and POC loadings occurred between 1991 and 1992; the remaining two-thirds, between 1995 and 2000. For TN and TP, the bulk of the decreases occurred between 1997 or 1998, and 2000. (c) 2009 Elsevier Ltd. All rights reserved.

Metal concentrations in surface sediments of Boston Harbor: Changes with time

USGS Publications Warehouse

Bothner, Michael H.; Buchholtz ten Brink, Marilyn R.; Manheim, F.T.

1998-01-01

The concentrations of metals in surface sediments of Boston Harbor have decreased during the period 1977–1993. This conclusion is supported by analysis of: (1) surface sediments collected at monitoring stations in the outer harbor between 1977 and 1993; (2) metal concentration profiles in sediment cores from depositional areas of the harbor; and (3) historical data from a contaminated-sediment database, which includes information on metal and organic contaminants and sediment texture. The background and matrix-corrected concentrations of lead (Pb) measured in the surficial layer (0–2 cm) of cores decreased by an average of 46%±12% among four locations in the outer harbor during the 16 y period. Chromium (Cr), copper (Cu), mercury (Hg), silver (Ag), and zinc (Zn) exhibited similar trends. Results from our sediment sampling are supported by historical data that were compiled from diverse sources into a regional sediment database. This sediment database contains approximately 3000 samples; of these, about 460 samples were collected and analyzed for Cu, Hg, or Zn and many other sediment parameters in Boston Harbor surface sediments between 1971–1993. The database indicates that the concentrations of these three metals also decreased with time in Boston’s Inner Harbor. The decreases in metal concentrations that are observed in more recent years parallel a general decrease in the flux of metals to the harbor, implemented by: (1) ending the sewage sludge discharge to the Harbor in December, 1991; (2) greater source reduction (e.g. recovery of silver from photographic processing) and closing or moving of industries; (3) improvements in wastewater handling and sewage treatment; and (4) diminishing use of lead in gasoline beginning about 1973. Despite the general decrease in metal concentrations in Boston Harbor surface sediments, the concentrations of Ag and Hg measured at some outer harbor stations in 1993 were still at, or above, the level associated with

Methods of treating Parkinson's disease using viral vectors

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

Bankiewicz, Krystof; Cunningham, Janet

Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson's Disease.

33 CFR 110.38 - Edgartown Harbor, Mass.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Edgartown Harbor, Mass. 110.38 Section 110.38 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.38 Edgartown Harbor, Mass. An area in the inner harbor...

33 CFR 110.38 - Edgartown Harbor, Mass.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Edgartown Harbor, Mass. 110.38 Section 110.38 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.38 Edgartown Harbor, Mass. An area in the inner harbor...

33 CFR 110.38 - Edgartown Harbor, Mass.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Edgartown Harbor, Mass. 110.38 Section 110.38 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.38 Edgartown Harbor, Mass. An area in the inner harbor...

33 CFR 110.38 - Edgartown Harbor, Mass.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Edgartown Harbor, Mass. 110.38 Section 110.38 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.38 Edgartown Harbor, Mass. An area in the inner harbor...

33 CFR 110.38 - Edgartown Harbor, Mass.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Edgartown Harbor, Mass. 110.38 Section 110.38 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.38 Edgartown Harbor, Mass. An area in the inner harbor...

THE SWINE LUNGWORM AS A RESERVOIR AND INTERMEDIATE HOST FOR SWINE INFLUENZA VIRUS

PubMed Central

Shope, Richard E.

1941-01-01

Multiple intramuscular injections of H. influenzae suis were found to precipitate swine influenza virus infections in a group of apparently normal swine. The most likely explanation of the phenomenon seemed to be that the animals, though healthy and susceptible, harbored the virus in some unknown manner. The factors possibly determining the phenomenon were explored experimentally but without success. PMID:19871114

Filaggrin gene polymorphism associated with Epstein-Barr virus-associated tumors in China.

PubMed

Yang, Yang; Liu, Wen; Zhao, Zhenzhen; Zhang, Yan; Xiao, Hua; Luo, Bing

2017-08-01

Mutations of filaggrin gene (FLG) have been identified as the cause of ichthyosis vulgaris, while recently FLG mutations were found to be associated with gastric cancer. This study aimed to investigate the association of filaggrin polymorphism with Epstein-Barr virus-associated tumors in China. A total of 200 patients with three types of tumors and 117 normal control samples were genotyped at three common FLG mutation loci (rs3126085, K4671X, R501X) by using Sequenom MassARRAY technique. The χ 2 test was used to evaluate the relationship between the mutation and the three kinds of tumors. A two-sided P value of <0.05 was considered statistically significant. The results showed that two single-nucleotide polymorphism (SNP) loci (rs3126085, K4671X) were significantly associated with nasopharyngeal carcinoma in genetic model. In addition, the two SNPs K4671X and rs3126085 were related to Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC), respectively. Furthermore, allele distributions in EBVaGC and EBVnGC were verified to be different in both SNP loci.

Neuroprotective effects of voluntary running on cognitive dysfunction in an α-synuclein rat model of Parkinson's disease.

PubMed

Crowley, Erin K; Nolan, Yvonne M; Sullivan, Aideen M

2018-05-01

Parkinson's disease (PD) is no longer primarily classified as a motor disorder due to increasing recognition of the impact on patients of several nonmotor PD symptoms, including cognitive dysfunction. These nonmotor symptoms are highly prevalent and greatly affect the quality of life of patients with PD, and so, therapeutic interventions to alleviate these symptoms are urgently needed. The aim of this study was to investigate the potential neuroprotective effects of voluntary running on cognitive dysfunction in an adeno-associated virus-α-synuclein rat model of PD. Bilateral intranigral administration of adeno-associated virus-α-synuclein was found to induce motor dysfunction and a significant loss of nigral dopaminergic neurons, neither of which were rescued by voluntary running. Overexpression of α-synuclein also resulted in significant impairment on hippocampal neurogenesis-dependent pattern separation, a cognitive task; this was rescued by voluntary running. This was substantiated by an effect of running on neurogenesis levels in the dorsal dentate gyrus, suggesting that the functional effects of running on pattern separation were mediated via increased neurogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

33 CFR 110.130 - Bar Harbor, Maine.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the following...

Zika Virus-associated Ocular and Neurologic Disorders: The Emergence of New Evidence.

PubMed

Şahiner, Fatih; Siğ, Ali Korhan; Savaşçi, Ümit; Tekin, Kemal; Akay, Fahrettin

2017-12-01

It has been approximately 70 years since the discovery of the Zika virus (ZIKV). It had been established that the virus causes mild infections and is confined to Africa and Asia; however, major changes in the clinical and epidemiologic patterns of ZIKV infection have occurred in recent years. The virus has attracted intense interest because of the possible association of several autoimmune and neurodevelopmental disorders. We present a summary of the articles that attempt to explain the ZIKV unknowns and strengthen the association with some disorders that are thought to be related to ZIKV, by describing the discovery milestones from the initial identification of the virus to the present day. New evidence strengthens the association between ZIKV infections and Guillain-Barré syndrome (GBS), microcephaly and various neurodevelopmental and ophthalmologic disorders as a result of numerous new clinical and experimental studies. The World Health Organization declared the end of the "Public Health Emergency of International Concern" in December 2016, but ZIKV and associated consequences remain a significant enduring public health challenge.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

Preclinical Evaluation of An Anti-HCV miRNA Cluster for Treatment of HCV Infection

PubMed Central

Yang, Xiao; Marcucci, Katherine; Anguela, Xavier; Couto, Linda B.

2013-01-01

We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17–92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection. PMID:23295950

The phylogeography of Myotis bat-associated rabies viruses across Canada.

PubMed

Nadin-Davis, Susan; Alnabelseya, Noor; Knowles, M Kimberly

2017-05-01

As rabies in carnivores is increasingly controlled throughout much of the Americas, bats are emerging as a significant source of rabies virus infection of humans and domestic animals. Knowledge of the bat species that maintain rabies is a crucial first step in reducing this public health problem. In North America, several bat species are known to be rabies virus reservoirs but the role of bats of the Myotis genus has been unclear due to the scarcity of laboratory confirmed cases and the challenges encountered in species identification of poorly preserved diagnostic submissions by morphological traits alone. This study has employed a collection of rabid bat specimens collected across Canada over a 25 year period to clearly define the role of particular Myotis species as rabies virus reservoirs. The virus was characterised by partial genome sequencing and host genetic barcoding, used to confirm species assignment of specimens, proved crucial to the identification of certain bat species as disease reservoirs. Several variants were associated with Myotis species limited in their Canadian range to the westernmost province of British Columbia while others were harboured by Myotis species that circulate across much of eastern and central Canada. All of these Myotis-associated viral variants, except for one, clustered as a monophyletic MYCAN clade, which has emerged from a lineage more broadly distributed across North America; in contrast one distinct variant, associated with the long-legged bat in Canada, represents a relatively recent host jump from a big brown bat reservoir. Together with evidence from South America, these findings demonstrate that rabies virus has emerged in the Myotis genus independently on multiple occasions and highlights the potential for emergence of new viral-host associations within this genus.

The phylogeography of Myotis bat-associated rabies viruses across Canada

PubMed Central

Alnabelseya, Noor; Knowles, M. Kimberly

2017-01-01

As rabies in carnivores is increasingly controlled throughout much of the Americas, bats are emerging as a significant source of rabies virus infection of humans and domestic animals. Knowledge of the bat species that maintain rabies is a crucial first step in reducing this public health problem. In North America, several bat species are known to be rabies virus reservoirs but the role of bats of the Myotis genus has been unclear due to the scarcity of laboratory confirmed cases and the challenges encountered in species identification of poorly preserved diagnostic submissions by morphological traits alone. This study has employed a collection of rabid bat specimens collected across Canada over a 25 year period to clearly define the role of particular Myotis species as rabies virus reservoirs. The virus was characterised by partial genome sequencing and host genetic barcoding, used to confirm species assignment of specimens, proved crucial to the identification of certain bat species as disease reservoirs. Several variants were associated with Myotis species limited in their Canadian range to the westernmost province of British Columbia while others were harboured by Myotis species that circulate across much of eastern and central Canada. All of these Myotis-associated viral variants, except for one, clustered as a monophyletic MYCAN clade, which has emerged from a lineage more broadly distributed across North America; in contrast one distinct variant, associated with the long-legged bat in Canada, represents a relatively recent host jump from a big brown bat reservoir. Together with evidence from South America, these findings demonstrate that rabies virus has emerged in the Myotis genus independently on multiple occasions and highlights the potential for emergence of new viral-host associations within this genus. PMID:28542160

33 CFR 110.9 - Wells Harbor, Maine.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Wells Harbor, Maine. 110.9... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.9 Wells Harbor, Maine. (a) Anchorage “A”. All of the... approximately 5,800 sq. yards, encompassing the central portion of Wells Harbor. (b) Anchorage “B”. All of the...

33 CFR 110.9 - Wells Harbor, Maine.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Wells Harbor, Maine. 110.9... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.9 Wells Harbor, Maine. Link to an amendment published at..., encompassing the central portion of Wells Harbor. (b) Anchorage “B”. All of the waters enclosed by a line...

33 CFR 207.610 - St. Lawrence River, Cape Vincent Harbor, N.Y.; use, administration, and navigation of the harbor...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false St. Lawrence River, Cape Vincent Harbor, N.Y.; use, administration, and navigation of the harbor and U.S. breakwater. 207.610 Section 207... NAVIGATION REGULATIONS § 207.610 St. Lawrence River, Cape Vincent Harbor, N.Y.; use, administration, and...

Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nouri, Shahideh; Salem, Nidà; Falk, Bryce W

2016-07-21

We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus. Copyright © 2016 Nouri et al.

33 CFR 117.272 - Boot Key Harbor.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Boot Key Harbor. 117.272 Section 117.272 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between...

Defense.gov Special Report: Pearl Harbor Anniversary

Science.gov Websites

Department of Defense Submit Search 71th Anniversary of the Attack on Pearl Harbor - World War II News Joint Chiefs of Staff, saluted veterans at the National World War II Memorial in Washington, D.C Attack Video Return To Pearl Harbor Return To Pearl Harbor World War II Timeline The attack on Pearl

Geoscience rediscovers Phoenicia's buried harbors

NASA Astrophysics Data System (ADS)

Marriner, Nick; Morhange, Christophe; Doumet-Serhal, Claude; Carbonel, Pierre

2006-01-01

After centuries of archaeological debate, the harbors of Phoenicia's two most important city states, Tyre and Sidon, have been rediscovered, and including new geoarcheological results reveal how, where, and when they evolved after their Bronze Age foundations. The early ports lie beneath their present urban centers, and we have indentified four harbor phases. (1) During the Bronze Age, Tyre and Sidon were characterized by semi-open marine coves that served as protoharbors. (2) Biostratigraphic and lithostratigraphic data indicate the presence of early artificial basins after the first millennium B.C. (3) The harbors reached their apogees during the Greco-Roman and Byzantine periods. (4) Silting up and coastal progradation led to burial of the medieval basins, lost until now.

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

PubMed

Kyostio-Moore, Sirkka; Berthelette, Patricia; Cornell, Cathleen Sookdeo; Nambiar, Bindu; Figueiredo, Monica Dias

2018-05-01

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Association between simian virus 40 and non-Hodgkin lymphoma

NASA Technical Reports Server (NTRS)

Vilchez, Regis A.; Madden, Charles R.; Kozinetz, Claudia A.; Halvorson, Steven J.; White, Zoe S.; Jorgensen, Jeffrey L.; Finch, Chris J.; Butel, Janet S.

2002-01-01

BACKGROUND: Non-Hodgkin lymphoma has increased in frequency over the past 30 years, and is a common cancer in HIV-1-infected patients. Although no definite risk factors have emerged, a viral cause has been postulated. Polyomaviruses are known to infect human beings and to induce tumours in laboratory animals. We aimed to identify which one of the three polyomaviruses able to infect human beings (simian virus 40 [SV40], JC virus, and BK virus) was associated with non-Hodgkin lymphoma. METHODS: We analysed systemic non-Hodgkin lymphoma from 76 HIV-1-infected and 78 HIV-1-uninfected patients, and non-malignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumours; 54 colon and breast carcinoma samples served as cancer controls. We used PCR followed by Southern blot hybridisation and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. FINDINGS: Polyomavirus T antigen sequences, all of which were SV40-specific, were detected in 64 (42%) of 154 non-Hodgkin lymphomas, none of 186 non-malignant lymphoid samples, and none of 54 control cancers. This difference was similar for HIV-1-infected patients and HIV-1-uninfected patients alike. Few tumours were positive for both SV40 and Epstein-Barr virus. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B-cell and follicular-type lymphomas. INTERPRETATION: SV40 is significantly associated with some types of non-Hodgkin lymphoma. These results add lymphomas to the types of human cancers associated with SV40.

Evolutionary Insights from a Genetically Divergent Hantavirus Harbored by the European Common Mole (Talpa europaea)

PubMed Central

Kang, Hae Ji; Bennett, Shannon N.; Sumibcay, Laarni; Arai, Satoru; Hope, Andrew G.; Mocz, Gabor; Song, Jin-Won; Cook, Joseph A.; Yanagihara, Richard

2009-01-01

Background The discovery of genetically distinct hantaviruses in shrews (Order Soricomorpha, Family Soricidae) from widely separated geographic regions challenges the hypothesis that rodents (Order Rodentia, Family Muridae and Cricetidae) are the primordial reservoir hosts of hantaviruses and also predicts that other soricomorphs harbor hantaviruses. Recently, novel hantavirus genomes have been detected in moles of the Family Talpidae, including the Japanese shrew mole (Urotrichus talpoides) and American shrew mole (Neurotrichus gibbsii). We present new insights into the evolutionary history of hantaviruses gained from a highly divergent hantavirus, designated Nova virus (NVAV), identified in the European common mole (Talpa europaea) captured in Hungary. Methodology/Principal Findings Pair-wise alignment and comparison of the full-length S- and L-genomic segments indicated moderately low sequence similarity of 54–65% and 46–63% at the nucleotide and amino acid levels, respectively, between NVAV and representative rodent- and soricid-borne hantaviruses. Despite the high degree of sequence divergence, the predicted secondary structure of the NVAV nucleocapsid protein exhibited the characteristic coiled-coil domains at the amino-terminal end, and the L-segment motifs, typically found in hantaviruses, were well conserved. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that NVAV formed a distinct clade that was evolutionarily distant from all other hantaviruses. Conclusions Newly identified hantaviruses harbored by shrews and moles support long-standing virus-host relationships and suggest that ancestral soricomorphs, rather than rodents, may have been the early or original mammalian hosts. PMID:19582155

Autoimmune Neurological Conditions Associated With Zika Virus Infection.

PubMed

Acosta-Ampudia, Yeny; Monsalve, Diana M; Castillo-Medina, Luis F; Rodríguez, Yhojan; Pacheco, Yovana; Halstead, Susan; Willison, Hugh J; Anaya, Juan-Manuel; Ramírez-Santana, Carolina

2018-01-01

Zika virus (ZIKV) is an emerging flavivirus rapidly spreading throughout the tropical Americas. Aedes mosquitoes is the principal way of transmission of the virus to humans. ZIKV can be spread by transplacental, perinatal, and body fluids. ZIKV infection is often asymptomatic and those with symptoms present minor illness after 3 to 12 days of incubation, characterized by a mild and self-limiting disease with low-grade fever, conjunctivitis, widespread pruritic maculopapular rash, arthralgia and myalgia. ZIKV has been linked to a number of central and peripheral nervous system injuries such as Guillain-Barré syndrome (GBS), transverse myelitis (TM), meningoencephalitis, ophthalmological manifestations, and other neurological complications. Nevertheless, mechanisms of host-pathogen neuro-immune interactions remain incompletely elucidated. This review provides a critical discussion about the possible mechanisms underlying the development of autoimmune neurological conditions associated with Zika virus infection.

Epstein–Barr Virus in Gliomas: Cause, Association, or Artifact?

PubMed Central

Akhtar, Saghir; Vranic, Semir; Cyprian, Farhan Sachal; Al Moustafa, Ala-Eddin

2018-01-01

Gliomas are the most common malignant brain tumors and account for around 60% of all primary central nervous system cancers. Glioblastoma multiforme (GBM) is a grade IV glioma associated with a poor outcome despite recent advances in chemotherapy. The etiology of gliomas is unknown, but neurotropic viruses including the Epstein–Barr virus (EBV) that is transmitted via salivary and genital fluids have been implicated recently. EBV is a member of the gamma herpes simplex family of DNA viruses that is known to cause infectious mononucleosis (glandular fever) and is strongly linked with the oncogenesis of several cancers, including B-cell lymphomas, nasopharyngeal, and gastric carcinomas. The fact that EBV is thought to be the causative agent for primary central nervous system (CNS) lymphomas in immune-deficient patients has led to its investigations in other brain tumors including gliomas. Here, we provide a review of the clinical literature pertaining to EBV in gliomas and discuss the possibilities of this virus being simply associative, causative, or even an experimental artifact. We searched the PubMed/MEDLINE databases using the following key words such as: glioma(s), glioblastoma multiforme, brain tumors/cancers, EBV, and neurotropic viruses. Our literature analysis indicates conflicting results on the presence and role of EBV in gliomas. Further comprehensive studies are needed to fully implicate EBV in gliomagenesis and oncomodulation. Understanding the role of EBV and other oncoviruses in the etiology of gliomas, would likely open up new avenues for the treatment and management of these, often fatal, CNS tumors. PMID:29732319

Comparison of Four Sampling Gears in Detecting Invasive Invertebrates in the Duluth-Superior Harbor, Oral Presentation

EPA Science Inventory

It is important to conduct non-indigenous species assessments in areas at risk, such as ecosystems associated with Great Lakes harbors, both for the detection of new species and to determine the spread of existing ones. In addition to direct impacts on harbor ecosystems, addition...

Canine distemper virus-associated hypocalcemia.

PubMed

Weisbrode, S E; Krakowka, S

1979-01-01

A retrospective study was done to correlate serum calcium concentrations and parathyroid gland ultrastructure to clinical, immunologic, and pathologic changes experimentally induced in gnotobiotic dogs by canine distemper virus (CDV). Dogs infected with CDV had significantly reduced serum calcium concentrations associated with ultrastructural evidence of parathyroid gland inactivity, degeneration, and viral inclusions. Although CDV-infected dogs exhibited neurologic signs, minimal lesions were present in the central nervous system. It is suggested that viral-induced parathyroid dysfunction may contribute to neutrologic disturbance of CDV infection.

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

The genome organisation and taxonomy of Sugarcane striate mosaic associated virus.

PubMed

Thompson, N; Randles, J W

2001-08-01

Sugarcane striate mosaic associated virus (SCSMaV) has slightly flexuous 950 nm x 15 nm filamentous particles and is associated with sugarcane striate mosaic disease in central Queensland, Australia. We report the full sequence of its RNA genome, which comprises 5 open reading frames representing the polymerase, movement function proteins encoded in a triple gene block and coat protein. Phylogenetic analyses based on either the full nucleotide sequence, the polymerase protein, or the coat protein all placed SCSMaV in an intermediate position between the genera Foveavirus and Carlavirus, but outside both genera. In addition, the absence of a sixth open reading frame excludes it from the genus Carlavirus, and the coat protein is approximately half the size of the type member for the genus Foveavirus. Although SCSMaV was most closely allied to Cherry green ring mottle virus by genome analysis, the two viruses are morphologically and biologically dissimilar. SCSMaV may therefore represent a new plant virus taxon.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

PubMed Central

Webster, Claire L.; Waldron, Fergal M.; Robertson, Shaun; Crowson, Daisy; Ferrari, Giada; Quintana, Juan F.; Brouqui, Jean-Michel; Bayne, Elizabeth H.; Longdon, Ben; Buck, Amy H.; Lazzaro, Brian P.; Akorli, Jewelna; Haddrill, Penelope R.; Obbard, Darren J.

2015-01-01

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research. PMID:26172158

Epidemiology and Transmission of Hepatitis A Virus and Hepatitis E Virus Infections in the United States.

PubMed

Hofmeister, Megan G; Foster, Monique A; Teshale, Eyasu H

2018-04-30

There are many similarities in the epidemiology and transmission of hepatitis A virus (HAV) and hepatitis E virus (HEV) genotype (gt)3 infections in the United States. Both viruses are enterically transmitted, although specific routes of transmission are more clearly established for HAV than for HEV: HAV is restricted to humans and primarily spread through the fecal-oral route, while HEV is zoonotic with poorly understood modes of transmission in the United States. New cases of HAV infection have decreased dramatically in the United States since infant vaccination was recommended in 1996. In recent years, however, outbreaks have occurred among an increasingly susceptible adult population. Although HEV is the most common cause of acute viral hepatitis in developing countries, it is rarely diagnosed in the United States. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Bullous pemphigoid associated with chronic hepatitis C virus infection in a hepatitis B virus endemic area: A case report.

PubMed

Jang, Hyunil; Jin, Young-Joo; Yoon, Chang Hwi; Kim, Cheol-Woo; Kim, Lucia

2018-04-01

Bullous pemphigoid is a type of acute or chronic autoimmune disease that involves subepidermal skin lesions with bulla formation. Although viral infections, such as, human herpes virus (HHV), human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus, HHV-6, hepatitis B virus (HBV), and hepatitis C virus (HCV), are known factors of bullous pemphigoid, HCV infection has only been rarely associated factor, especially in HBV endemic area. A 78-year-old man was admitted to our hospital due to erythematous bulla of onset 3 months before presentation affecting his entire body. Pathologic findings, that is, subepidermal bullae containing eosinophils and neutrophils with superficial perivascular lymphocytic and eosinophilic infiltration, were consistent with bullous pemphigoid. Anti-HCV was positive and HCV quantitative real-time polymerase chain reaction (PCR) was 1.25 x 10 IU/mL. HCV genotype was 2a. After a diagnosis of bullous pemphigoid associated with chronic HCV infection was reached, he was treated with oral methylprednisolone for bullous pemphigoid, and his skin lesions improved. Oral direct-acting antiviral agents (sofosbuvir plus ribavirin) were prescribed for chronic hepatitis C, and sustained viral response was achieved. The authors report a rare case of bullous pemphigoid associated with chronic HCV infection in a HBV endemic area and advise that HCV should be considered in the differential diagnosis of factors precipitating bullous pemphigoid, even in HBV endemic areas.

Behavior associated with forced copulation of juvenile Pacific harbor seals (Phoca vitulina richardsi) by southern sea otters (Enhydra lutris nereis)

USGS Publications Warehouse

Harris, Heather S.; Oates, Stori C.; Staedler, Michelle M.; Tinker, M. Tim; Jessup, David A.; Harvey, James T.; Miller, Melissa A.

2010-01-01

Nineteen occurrences of interspecific sexual behavior between male southern sea otters (Enhydra lutris nereis) and juvenile Pacific harbor seals (Phoca vitulina richardsi) were reported in Monterey Bay, California, between 2000 and 2002. At least three different male sea otters were observed harassing, dragging, guarding, and copulating with harbor seals for up to 7 d postmortem. Carcasses of 15 juvenile harbor seals were recovered, and seven were necropsied in detail by a veterinary pathologist. Necropsy findings from two female sea otters that were recovered dead from male sea otters exhibiting similar behavior are also presented to facilitate a comparison of lesions. The most frequent lesions included superficial skin lacerations; hemorrhage around the nose, eyes, flippers, and perineum; and traumatic corneal erosions or ulcers. The harbor seals sustained severe genital trauma, ranging from vaginal perforation to vagino-cervical transection, and colorectal perforations as a result of penile penetration. One harbor seal developed severe pneumoperitoneum subsequent to vaginal perforation, which was also observed in both female sea otters and has been reported as a postcoital lesion in humans. This study represents the first description of lesions resulting from forced copulation of harbor seals by sea otters and is also the first report of pneu-moperitoneum secondary to forced copulation in a nonhuman animal. Possible explanations for this behavior are discussed in the context of sea otter biology and population demographics.

Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms

USDA-ARS?s Scientific Manuscript database

Star jasmine (Jasminum multiflorum) plants growing in Hawaii expressing a diverse array of virus-like foliar symptoms were examined for the presence of a causal agent. Symptomatic tissues collected from three locations on the island of Oahu, Hawaii consistently harbored double-stranded (ds)RNAs app...

78 FR 38577 - Special Local Regulations; Red Bull Flugtag National Harbor Event, Potomac River; National Harbor...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-27

...-AA08 Special Local Regulations; Red Bull Flugtag National Harbor Event, Potomac River; National Harbor... waters of the Potomac River on September 21, 2013. These special local regulations are necessary to... temporarily restrict vessel traffic in a portion of the Potomac River during the event. DATES: This rule is...

Untangling the origin of viruses and their impact on cellular evolution.

PubMed

Nasir, Arshan; Sun, Feng-Jie; Kim, Kyung Mo; Caetano-Anollés, Gustavo

2015-04-01

The origin and evolution of viruses remain mysterious. Here, we focus on the distribution of viral replicons in host organisms, their morphological features, and the evolution of highly conserved protein and nucleic acid structures. The apparent inability of RNA viral replicons to infect contemporary akaryotic species suggests an early origin of RNA viruses and their subsequent loss in akaryotes. A census of virion morphotypes reveals that advanced forms were unique to viruses infecting a specific supergroup, while simpler forms were observed in viruses infecting organisms in all forms of cellular life. Results hint toward an ancient origin of viruses from an ancestral virus harboring either filamentous or spherical virions. Finally, phylogenetic trees built from protein domain and tRNA structures in thousands of genomes suggest that viruses evolved via reductive evolution from ancient cells. The analysis presents a complete account of the evolutionary history of cells and viruses and identifies viruses as crucial agents influencing cellular evolution. © 2015 New York Academy of Sciences.

Viruses in close associations with free-living amoebae.

PubMed

Scheid, Patrick

2015-11-01

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Exosome-associated hepatitis C virus in cell cultures and patient plasma

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

Liu, Ziqing; Zhang, Xiugen; Yu, Qigui

Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV thanmore » exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.« less

Differential Life History Trait Associations of Aphids with Nonpersistent Viruses in Cucurbits.

PubMed

Angelella, G M; Egel, D S; Holland, J D; Nemacheck, J A; Williams, C E; Kaplan, I

2015-06-01

The diversity of vectors and fleeting nature of virus acquisition and transmission renders nonpersistent viruses a challenge to manage. We assessed the importance of noncolonizing versus colonizing vectors with a 2-yr survey of aphids and nonpersistent viruses on commercial pumpkin farms. We quantified aphid alightment using pan traps, while testing leaf samples with multiplex RT-PCR targeting cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and papaya ringspot virus (PRSV). Overall, we identified 53 aphid species (3,899 individuals), from which the melon aphid, Aphis gossypii Glover, a pumpkin-colonizing species, predominated (76 and 37% of samples in 2010 and 2011, respectively). CMV and ZYMV were not detected, but WMV and PRSV were prevalent, both regionally (WMV: 28/29 fields, PRSV: 21/29 fields) and within fields (infection rates = 69 and 55% for WMV in 2010 and 2011; 28 and 25% for PRSV in 2010 and 2011). However, early-season samples showed extremely low infection levels, suggesting cucurbit viruses are not seed-transmitted and implicating aphid activity as a causal factor driving virus spread. Interestingly, neither noncolonizer and colonizer alightment nor total aphid alightment were good predictors of virus presence, but community analyses revealed species-specific relationships. For example, cowpea aphid (Aphis craccivora Koch) and spotted alfalfa aphid (Therioaphis trifolii Monell f. maculata) were associated with PRSV infection, whereas the oleander aphid (Aphis nerii Bover de Fonscolombe) was associated with WMV spread within fields. These outcomes highlight the need for tailored management plans targeting key vectors of nonpersistent viruses in agricultural systems. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

78 FR 18274 - Special Local Regulations; Red Bull Flugtag National Harbor Event, Potomac River; National Harbor...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-26

...-AA08 Special Local Regulations; Red Bull Flugtag National Harbor Event, Potomac River; National Harbor... event,'' to be held on the waters of the Potomac River on September 21, 2013. These special local... representative. This action is intended to temporarily restrict vessel traffic in a portion of the Potomac River...

Imaging Virus-Associated Cancer

PubMed Central

Fu, De-Xue; Foss, Catherine A.; Nimmagadda, Sridhar; Ambinder, Richard F.; Pomper, Martin G.

2012-01-01

Cancer remains an important and growing health problem. Researchers have made great progress in defining genetic and molecular alterations that contribute to cancer formation and progression. Molecular imaging can identify appropriate patients for targeted cancer therapy and may detect early biochemical changes in tumors during therapy, some of which may have important prognostic implications. Progress in this field continues largely due to a union between molecular genetics and advanced imaging technology. This review details uses of molecular-genetic imaging in the context of tumor-associated viruses. Under certain conditions, and particularly during pharmacologic stimulation, gammaherpesviruses will express genes that enable imaging and therapy in vivo. The techniques discussed are readily translatable to the clinic. PMID:18991718

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

PubMed

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

PubMed Central

Meessen-Pinard, Mathieu; Dubé, Mathieu; Day, Robert; Seidah, Nabil G.; Talbot, Pierre J.

2015-01-01

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G 758 to RRSR↓R 758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies. PMID:26545254

33 CFR 162.155 - Sandusky and Huron Harbors, Ohio.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Harbors, Ohio. (a) In Sandusky Harbor, no vessel greater than 40 feet in length may exceed 10 miles per hour. (b) In Huron Harbor, no vessel greater than 40 feet in length may exceed 6 miles per hour, except in the outer harbor where no vessel greater than 40 feet in length may exceed 10 miles per hour. Note...

33 CFR 162.155 - Sandusky and Huron Harbors, Ohio.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Harbors, Ohio. (a) In Sandusky Harbor, no vessel greater than 40 feet in length may exceed 10 miles per hour. (b) In Huron Harbor, no vessel greater than 40 feet in length may exceed 6 miles per hour, except in the outer harbor where no vessel greater than 40 feet in length may exceed 10 miles per hour. Note...

33 CFR 162.155 - Sandusky and Huron Harbors, Ohio.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Harbors, Ohio. (a) In Sandusky Harbor, no vessel greater than 40 feet in length may exceed 10 miles per hour. (b) In Huron Harbor, no vessel greater than 40 feet in length may exceed 6 miles per hour, except in the outer harbor where no vessel greater than 40 feet in length may exceed 10 miles per hour. Note...

33 CFR 162.155 - Sandusky and Huron Harbors, Ohio.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Harbors, Ohio. (a) In Sandusky Harbor, no vessel greater than 40 feet in length may exceed 10 miles per hour. (b) In Huron Harbor, no vessel greater than 40 feet in length may exceed 6 miles per hour, except in the outer harbor where no vessel greater than 40 feet in length may exceed 10 miles per hour. Note...