WorldWideScience

Sample records for adeno-associated virus vector

  1. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  2. Biosafety of recombinant adeno-associated virus vectors.

    Science.gov (United States)

    Dismuke, David J; Tenenbaum, Liliane; Samulski, R Jude

    2013-12-01

    It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

  3. An adeno-associated virus vector-mediated multiple gene transfer for dopamine synthetic enzymes

    Institute of Scientific and Technical Information of China (English)

    樊东升; 沈扬

    2000-01-01

    Objective: To explore a multiple gene transfer approach with separate adeno-associated virus vectors. Methods: The genes of dopamine synthetic enzymes, tyrosine hydroxylasc (TH), GTP cyclohydrolase I (GCH, an enzyme critical for tetrahydrobioptcrin synthesis), and aromatic L-amino acid decarboxylase (AADC), were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, GCH, and AADC were detected by Western blot analysis. L-dopa and dopamine levels in the ceils were assayed by HPLC. Results: TH, GCH, and AADC proteins were effectively cocxpressed in the transduced cells with three separate AAV vectors, AAV-TH, AAV-GCH, and AAV-AADC. Furthermore, the coexpression of these three proteins resulted in an effectively spontaneous dopainc production in the cotransduced cells. Conclusion: The triple transduction of TH, GCH, and AADC genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.

  4. A novel and highly efficient production system for recombinant adeno-associated virus vector

    Institute of Scientific and Technical Information of China (English)

    WU; Zhijian(伍志坚); WU; Xiaobing(吴小兵); CAO; Hui(曹晖); DONG; Xiaoyan(董小岩); WANG; Hong(王宏); HOU; Yunde(侯云德)

    2002-01-01

    Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  5. [Innate immune mechanisms against recombinant adeno-associated virus vectors--a review].

    Science.gov (United States)

    Diao, Yong; Xu, Ruian

    2012-05-04

    Recombinant adeno-associated virus (rAAV) is one of the most commonly used vectors for gene therapy. Despite the promising safety profile demonstrated in preclinical trials, the clinic efficacy of using rAAV was hampered by undesired response from the immune system. It is important to understand the mechanisms that lead to the induction of immune response against rAAV. Although a crucial role for innate immunity is shaping adaptive immune responses, the innate immune to rAAV was ignored in the past. Till now, at least three human cell types (dendritic cells, macrophages and endothelial cells) were discovered to be involved in sensing rAAV infection. The engagement of TLR9 by rAAV vector genomes triggers the activation of NF-kappaB signaling cascades, leading to the induction of pro-inflammatory cytokine genes. The viral capsid components are detected by TLR2, and this leads to the production of type I interferon mediated by interferon regulatory factors (IRFs) pathway. Self-complementary rAAV vectors induced higher TLR9 dependent innate immune response than single stranded rAAV. This review highlights the recent findings regarding the innate immune responses to rAAV vectors, the signaling pathways involved, and the impacts of innate immunity on the adaptive immune response to rAAV and its transgene expression.

  6. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    2001-01-01

    Full Text Available The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE from the erythropoietin gene (Epo, which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1. Under anoxic conditions, nine copies of HIRE (9XHRE yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

  7. A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

    A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

  8. Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.

    Science.gov (United States)

    Strobel, Benjamin; Klauser, Benedikt; Hartig, Jörg S; Lamla, Thorsten; Gantner, Florian; Kreuz, Sebastian

    2015-10-01

    Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

  9. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography.

    Science.gov (United States)

    Lock, Martin; Alvira, Mauricio R; Wilson, James M

    2012-02-01

    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.

  10. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    LENUS (Irish Health Repository)

    Luo, Xiaoyan

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

  11. Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

    Institute of Scientific and Technical Information of China (English)

    Yonglun Luo; Emil Kofod-Olsen; Rikke Christensen; Charlotte Brandt S(φ)rensen; Lars Bolund

    2012-01-01

    Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer (SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis,hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

  12. Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.

    Science.gov (United States)

    Merentie, M; Lottonen-Raikaslehto, L; Parviainen, V; Huusko, J; Pikkarainen, S; Mendel, M; Laham-Karam, N; Kärjä, V; Rissanen, R; Hedman, M; Ylä-Herttuala, S

    2016-03-01

    Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

  13. Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

    Institute of Scientific and Technical Information of China (English)

    Sen Lu; Yue Yu; Yun Gao; Guo-Qiang Li; Xue-Hao Wang

    2008-01-01

    BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS:The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita-tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS:The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively). CONCLUSIONS:pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological ifndings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

  14. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    OpenAIRE

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; FERKOL, THOMAS; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mut...

  15. A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard.

    Science.gov (United States)

    Wagner, Anke; Röhrs, Viola; Kedzierski, Radoslaw; Fechner, Henry; Kurreck, Jens

    2013-12-01

    Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAVStd). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAVStd. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.

  16. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  17. Adeno-associated virus for cystic fibrosis gene therapy

    Directory of Open Access Journals (Sweden)

    S.V. Martini

    2011-11-01

    Full Text Available Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR. The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR to the affected organ (lung. Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

  18. Engineering adeno-associated viruses for clinical gene therapy.

    Science.gov (United States)

    Kotterman, Melissa A; Schaffer, David V

    2014-07-01

    Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

  19. Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

    Science.gov (United States)

    Flotte, Terence R; Conlon, Thomas J; Poirier, Amy; Campbell-Thompson, Martha; Byrne, Barry J

    2007-03-01

    To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

  20. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    Science.gov (United States)

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  1. Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

    Institute of Scientific and Technical Information of China (English)

    Chen Ling; Irene Zolotukhin; Arun Srivastava; Chang-quan Ling; Yuan Wang; Ying-lu Feng; Ya-ni Zhang; Jun Li; Xin-rui Hu; Li-na Wang; Mao-feng Zhong; Xiao-feng Zhai

    2015-01-01

    Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are signiifcant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as wel as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential inlfuence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 > AAV3 = AAVLK03 > AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤ 35, 36–50, and ≥ 51 years old). People in the Qi-deifciency constitution had signiifcantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

  2. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  3. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  4. Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors

    Institute of Scientific and Technical Information of China (English)

    Xiao-dong JU; Si-quan LOU; Wei-guo WANG; Jian-qiang PENG; Hua TIAN

    2004-01-01

    AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10 % FBS or 5 % FBS and dexamethasone 1 μmol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC.After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

  5. Intracranial Injection of Adeno-associated Viral Vectors

    OpenAIRE

    Lowery, Rebecca L.; Ania K Majewska

    2010-01-01

    Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially us...

  6. Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo▿

    OpenAIRE

    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R. Jude

    2009-01-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) i...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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/ (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.

  8. Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy.

    Science.gov (United States)

    Jayandharan, Giridhara R; Aslanidi, George; Martino, Ashley T; Jahn, Stephan C; Perrin, George Q; Herzog, Roland W; Srivastava, Arun

    2011-03-01

    Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

  9. Comparison of murine leukemia virus, human immunodeficiency virus, and adeno-associated virus vectors for gene transfer in multiple myeloma: lentiviral vectors demonstrate a striking capacity to transduce low-proliferating primary tumor cells.

    Science.gov (United States)

    De Vos, John; Bagnis, Claude; Bonnafoux, Lydie; Requirand, Guilhem; Jourdan, Michel; Imbert, Marie-Christine; Jourdan, Eric; Rossi, Jean-François; Mannoni, Patrice; Klein, Bernard

    2003-12-10

    Genetic modification of primary tumor cells by gene transfer is of major interest to study the role of specific genes in the biology of a given malignancy and to modify tumor cells for therapeutic use. Multiple myeloma (MM) is a low-proliferating cancer, with often less than 1% of the cells in the S phase of the cell cycle. As primary myeloma cells are notoriously difficult to transduce, we conducted a comparison of various viral vectors, known to integrate the transgene of interest into the target genome, for their ability to stably promote the expression of an enhanced green fluorescent protein (EGFP) transgene. We compared three murine leukemia virus-based vectors, differing only in their viral envelope, a human immunodeficiency virus (HIV)-based vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and an adeno-associated virus type 2 vector. Transduction characteristics of these vectors were evaluated in human myeloma cell lines and in primary myeloma cells. Unequivocally, we observed that the VSV-G/HIV vector was the most efficient vector for transducing the cell lines and the only one able to transduce primary myeloma cells reproducibly. The mean percentage of transduced primary myeloma cells was 43.6% (range, 16.3-77.6%), with one round of infection at a low multiplicity of infection, including MM cell samples with less than 1% of cells in the S phase. A quantitative polymerase chain reaction assay demonstrated that this more efficient EGFP expression was associated with a higher GFP copy number in the targeted cell. We propose that lentiviral vectors should be used for transduction of nonproliferating primary tumor cells such as myeloma cells.

  10. Cytotoxic-T-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R Jude

    2009-07-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/alpha1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8(+) CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.

  11. Suppressing tumor growth of nasopharyngeal carcinoma by hTERTC27 polypeptide delivered through adeno-associated virus plus adenovirus vector cocktail

    Institute of Scientific and Technical Information of China (English)

    Xiong Liu; Xiang-Ping Li; Ying Peng; Samuel S.Ng; Hong Yao; Zi-Feng Wang; Xiao-Mei Wang; Hsiang-Fu Kung; Marie C.M.Lin

    2012-01-01

    Nasopharyngeal carcinoma(NPC) is a metastatic carcinoma that is highly prevalent in Southeast Asia.Our laboratory has previously demonstrated that the C-terminal 27-kDa polypeptide of human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human glioblastoma and melanoma cells.In this study,we investigated the antitumor effect of hTERTC27 in human C666-1 NPC cells xenografted in a nude mouse model.A cocktail of vectors comprising recombinant adeno-associated virus (rAAV) and recombinant adenovirus (rAdv) that each carry hTERTC27 (rAAV-hTERTC27 and rAdv-hTERTC27; the cocktail was abbreviated to rAAV/rAdv-hTERTC27) was more effective than either rAAV-hTERTC27 or rAdv-hTERTC27 alone in inhibiting the growth of C666-1 NPC xenografts.Furthermore,we established three tumors on each mouse and injected rAAV/rAdv-hTERTC27 into one tumor per mouse.Although hTERTC27 expression could only be detected in the injected tumors,reduced tumor growth was observed in the injected tumor as well as the uninjected tumors,demonstrating that the vector cocktail could provoke an antitumor effect on distant,metastasized tumors.Further studies showed the observed antitumor effects included inducing necrosis and apoptosis and reducing microvessel density.Together,our data suggest that the rAAV/rAdv-hTERTC27 cocktail can potently inhibit NPC tumor growth in both local and metastasized tumors and should be further developed as a novel gene therapy strategy for NPC.

  12. Expression of human nerve growth factor β gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector

    Institute of Scientific and Technical Information of China (English)

    高凯; 吴勇杰; 吴小兵; 饶春明; 王军志

    2004-01-01

    Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

  13. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    Science.gov (United States)

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag

  14. Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

    Science.gov (United States)

    Fleming, Jane; Spinoulas, Afroditi; Zheng, Maolin; Cunningham, Sharon C; Ginn, Samantha L; McQuilty, Robert C; Rowe, Peter B; Alexander, Ian E

    2005-08-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

  15. Adeno-Associated Virus Gene Therapy for Liver Disease

    Science.gov (United States)

    Kattenhorn, Lisa M.; Tipper, Christopher H.; Stoica, Lorelei; Geraghty, Deborah S.; Wright, Teresa L.; Clark, K. Reed; Wadsworth, Samuel C.

    2016-01-01

    The field of adeno-associated virus (AAV) gene therapy has progressed rapidly over the past decade, with the advent of novel capsid serotype and organ-specific promoters, and an increasing understanding of the immune response to AAV administration. In particular, liver-directed therapy has made remarkable strides, with a number of clinical trials currently planned and ongoing in hemophilia A and B, as well as other liver disorders. This review focuses on liver-directed AAV gene therapy, including historic context, current challenges, and future developments. PMID:27897038

  16. NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee.

    Science.gov (United States)

    O'Reilly, Marina; Shipp, Allan; Rosenthal, Eugene; Jambou, Robert; Shih, Tom; Montgomery, Maureen; Gargiulo, Linda; Patterson, Amy; Corrigan-Curay, Jacqueline

    2012-01-01

    In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.

  17. The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

    Institute of Scientific and Technical Information of China (English)

    WANG An-ping; SUN Huai-chang; WANG Jian-ye; WANG Yong-juan; YUAN Wei-feng

    2007-01-01

    To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

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

    Science.gov (United States)

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

    2016-07-01

    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 nervous system.

  19. [Advances in the application of gene therapy for Parkinson's disease with adeno-associated virus].

    Science.gov (United States)

    Chen, Yang; Lü, Ying-Hui; Li, Zhao-Fa

    2014-05-01

    Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.

  20. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  1. Manufacturing of recombinant adeno-associated viral vectors for clinical trials.

    Science.gov (United States)

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings.

  2. Adeno-associated viral vector transduction of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stender, Stefan; Murphy, Mary; O'Brien, Tim

    2007-01-01

    Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector...... in human MSCs and to assess whether AAV transduction affects MSC multipotentiality. The results indicated that human MSCs could indeed be transiently transduced in vitro by the AAV2 vector with efficiencies of up to 65%. The percentage of GFP-positive cells peaked at 4 days post-transduction and declined...... rapidly towards 0% after day 8. The level of transgene expression in the GFP-positive population increased 4-fold over a 10,000 fold viral dose increase. This dose-response contrasted with the 200-fold increase observed in similarly transduced 293-cells, indicating a relatively restricted transgene...

  3. Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus

    OpenAIRE

    Adam, Virginie S.; Crosariol, Marco; Kumar, Sachin; Ge, Moyar Q.; Czack, Sarah E.; Roy, Soumitra; Haczku, Angela; Tretiakova, Anna; Wilson, James M.; Limberis, Maria P.

    2014-01-01

    Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunod...

  4. A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Lkhagvasuren Damdindorj

    Full Text Available The properties of constitutive promoters within adeno-associated viral (AAV vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV, simian virus 40, and herpes simplex virus thymidine kinase, were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

  5. Capsid modification of adeno-associated virus and tumor targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU ZengHu; ZHOU XiuMei; SHI WenFang; QIAN QiJun

    2008-01-01

    Targeting is critical for successful tumor gene therapy. The adeno-associated virus (AAV) has aroused wide concern due to its excellent advantages over other viral vectors in gene therapy. AAV has a broad infection spectrum, which also results in poor specificity towards tissues or cells and low transduction efficiency. Therefore, it is imperative to improve target and transduction efficiency in AAV-mediated gene therapy. Up to now, researchers have developed many strategies to modify AAV capsids for improving targeting or retargeting only desired cells. These strategies include not only traditional chemical modification, phage display technology, modification of AAV capsid genome, chimeric vectors and so on, but also many novel strategies involved in marker rescue strategy, direct evolution of capsid proteins, direct display random peptides on AAV capsid, AAVP (AAV-Phage), and etc. This review will summarize the advances of researches on the capsid modification of AAV to target malignant cells.

  6. Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications.

    Science.gov (United States)

    Gaj, Thomas; Epstein, Benjamin E; Schaffer, David V

    2016-03-01

    In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

  7. Comparative efficacy and safety of multiple routes of direct CNS administration of adeno-associated virus gene transfer vector serotype rh.10 expressing the human arylsulfatase A cDNA to nonhuman primates.

    Science.gov (United States)

    Rosenberg, Jonathan B; Sondhi, Dolan; Rubin, David G; Monette, Sébastien; Chen, Alvin; Cram, Sara; De, Bishnu P; Kaminsky, Stephen M; Sevin, Caroline; Aubourg, Patrick; Crystal, Ronald G

    2014-09-01

    Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS

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

    Science.gov (United States)

    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.

  9. 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.

    Science.gov (United States)

    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.

  10. Adeno-associated virus serotype rh.10 displays strong muscle tropism following intraperitoneal delivery.

    Science.gov (United States)

    Ai, Jianzhong; Li, Jia; Gessler, Dominic J; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

    2017-01-09

    Recombinant adeno-associated virus (rAAV) is an attractive tool for basic science and translational medicine including gene therapy, due to the versatility in its cell and organ transduction. Previous work indicates that rAAV transduction patterns are highly dependent on route of administration. Based on this relationship, we hypothesized that intraperitoneal (IP) administration of rAAV produces unique patterns of tissue tropism. To test this hypothesis, we investigated the transduction efficiency of 12 rAAV serotypes carrying an enhanced green fluorescent protein (EGFP) reporter gene in a panel of 12 organs after IP injection. Our data suggest that IP administration emphasizes transduction patterns that are different from previously reported intravascular delivery methods. Using this approach, rAAV efficiently transduces the liver, pancreas, skeletal muscle, heart and diaphragm without causing significant histopathological changes. Of note, rAAVrh.10 showed excellent muscle transduction following IP administration, highlighting its potential as a new muscle-targeting vector.

  11. Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures.

    Science.gov (United States)

    Kallendrusch, Sonja; Schopow, Nikolas; Stadler, Sonja C; Büning, Hildegard; Hacker, Ulrich T

    2016-10-01

    Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.

  12. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

    Directory of Open Access Journals (Sweden)

    Yang Lin

    2013-02-01

    Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

  13. ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  14. Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

    Institute of Scientific and Technical Information of China (English)

    Ke SONG; Nianjing RAO; Meiling CHEN; Yingguang CAO

    2008-01-01

    To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se- quence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombi- nant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated,the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×1011 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

  15. 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.

    Science.gov (United States)

    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; Crystal, Ronald G

    2014-03-01

    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 Cln3(Δex7/8) 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 Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) 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, Cln3(Δex7/8) 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 Cln3(Δex7/8) 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

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

    Science.gov (United States)

    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.

  17. Notch1 augments intracellular trafficking of adeno-associated virus type 2.

    Science.gov (United States)

    Ren, Changchun; White, April F; Ponnazhagan, Selvarangan

    2007-02-01

    We report here the significance of the Notch1 receptor in intracellular trafficking of recombinant adeno-associated virus type 2 (rAAV2). RNA profiling of human prostate cancer cell lines with various degrees of AAV transduction indicated a correlation of the amount of Notch1 with rAAV transgene expression. A definitive role of Notch1 in enhancing AAV transduction was confirmed by developing clonal derivatives of DU145 cells overexpressing either full-length or intracellular Notch1. To discern stages of AAV2 transduction influenced by Notch1, competitive binding with soluble heparin and Notch1 antibody, intracellular trafficking using Cy3-labeled rAAV2, and blocking assays for proteasome and dynamin pathways were performed. Results indicated that in the absence or low-level expression of Notch1, only binding of virus was found on the cell surface and internalization was impaired. However, increased Notch1 expression in these cells allowed efficient perinuclear accumulation of labeled capsids. Nuclear transport of the vector was evident by transgene expression and real-time PCR analyses. Dynamin levels were not found to be different among these cell lines, but blocking dynamin function abrogated AAV2 transduction in DU145 clones overexpressing full-length Notch1 but not in clones overexpressing intracellular Notch1. These studies provide evidence for the role of activated Notch1 in intracellular trafficking of AAV2, which may have implications in the optimal use of AAV2 in human gene therapy.

  18. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons

    Directory of Open Access Journals (Sweden)

    Arpiar eSaunders

    2012-07-01

    Full Text Available Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs whose transgene expression is activated by Cre (Cre-On. Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (Cre-Off and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery.

  19. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  20. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates.

    Science.gov (United States)

    Samaranch, Lluis; Salegio, Ernesto A; San Sebastian, Waldy; Kells, Adrian P; Foust, Kevin D; Bringas, John R; Lamarre, Clementine; Forsayeth, John; Kaspar, Brian K; Bankiewicz, Krystof S

    2012-04-01

    Widespread distribution of gene products at clinically relevant levels throughout the CNS has been challenging. Adeno-associated virus type 9 (AAV9) vector has been reported as a good candidate for intravascular gene delivery, but low levels of preexisting antibody titers against AAV in the blood abrogate cellular transduction within the CNS. In the present study we compared the effectiveness of vascular delivery and cerebrospinal fluid (CSF) delivery of AAV9 in transducing CNS tissue in nonhuman primates. Both delivery routes generated similar distribution patterns, although we observed a more robust level of transduction after CSF delivery. Consistent with previous reports administering AAV9, we found greater astrocytic than neuronal tropism via both routes, although we did find a greater magnitude of CNS transduction after CSF delivery compared with intravascular delivery. Last, we have demonstrated that delivery of AAV9 into the CSF does not shield against AAV antibodies. This has obvious implications when developing and/or implementing any clinical trial studies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    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 of 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 of 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.

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

    Science.gov (United States)

    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.

  4. Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery.

    Science.gov (United States)

    Zhang, Chuanling; Yao, Tianzhuo; Zheng, Yongxiang; Li, Zhongjun; Zhang, Qiang; Zhang, Lihe; Zhou, Demin

    2016-02-01

    Virus-based nanoparticles have shown promise as vehicles for delivering therapeutic genes. However, the rational design of viral vectors that enable selective tropism towards particular types of cells and tissues remains challenging. Here, we explored structural-functional relationships of the adeno-associated virus 2 (AAV2) vector by expanding its genetic code during production. As a proof-of-principle, an azide moiety was strategically displayed on the vector capsid as a bioorthogonal chemical reporter. Upon bioorthogonal conjugation of AAV2 with fluorophores and cyclic arginyl-glycyl-aspartic acid ligands at certain modifiable sites, we characterized in vitro and in vivo AAV2 movement and enhanced tropism selectivity towards integrin-expressing tumor cells. Targeting AAV2 vectors resulted in selective killing of U87 glioblastoma cells and derived xenografts via the herpes simplex virus suicide gene thymidine kinase, with the potency of ganciclovir being increased by 25-fold. Our results demonstrated successful rational modification of AAV2 as a targeting delivery vehicle, establishing a facile platform for precision engineering of virus-based nanoparticles in basic research and therapeutic applications.

  5. Establishment of a recombinant adeno-associated virus expressing hVEGF165

    Institute of Scientific and Technical Information of China (English)

    Xianghui Huang; Zhibin Shi; Xiaoqian Dang; Chen Zhang; Pengbo Yu; Kunzheng Wang

    2008-01-01

    BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion

  6. Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery.

    Science.gov (United States)

    Jang, J-H; Koerber, J T; Gujraty, K; Bethi, S R; Kane, R S; Schaffer, D V

    2010-11-01

    Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

  7. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

    2010-01-01

    Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic....... Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

  8. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

    Directory of Open Access Journals (Sweden)

    Daniel J. Schuster

    2014-08-01

    Full Text Available We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5. Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

  9. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  10. Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.

    LENUS (Irish Health Repository)

    Flotte, Terence R

    2011-10-01

    Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes\\/kg (n=3 subjects\\/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg\\/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

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

    Science.gov (United States)

    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…

  12. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  13. Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

    Science.gov (United States)

    Dane, A P; Cunningham, S C; Kok, C Y; Logan, G J; Alexander, I E

    2015-11-01

    Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

  14. Adeno-Associated Viral Vectors Serotype 8 for Cell-Specific Delivery of Therapeutic Genes in the Central Nervous System

    Science.gov (United States)

    Pignataro, Diego; Sucunza, Diego; Vanrell, Lucia; Lopez-Franco, Esperanza; Dopeso-Reyes, Iria G.; Vales, Africa; Hommel, Mirja; Rico, Alberto J.; Lanciego, Jose L.; Gonzalez-Aseguinolaza, Gloria

    2017-01-01

    Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters. PMID:28239341

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

    Science.gov (United States)

    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.

  16. Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis

    Institute of Scientific and Technical Information of China (English)

    Sander van der Marel; Anna Majowicz; Karin Kwikkers; Richard van Logtenstein; Anje A te Velde; Anne S De Groot; Sybren L Meijer

    2012-01-01

    AIM:To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.METHODS:The trinitrobenzene sulfonate (TNBS) model of induced colitis was used in Balb/c mice.Subsequently after intravenous adeno-associated virusmediated regulatory T-cell epitopes (Tregitope) delivery,acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS (0.75 mg in 20% ethanol) 8 d later.Control groups included mice not treated with TNBS (healthy control group) and mice treated by TNBS only (diseased group).At the time of sacrifice colon weight,the disease activity index and histology damage score were determined.Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3 (Foxp3).Thymus,mesenteric lymph nodes,liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.RESULTS:The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice.In contrast,the mice treated with TNBS alone (no Tregitope) developed colitis,and lost 4% of their initial body weight at the time of sacrifice (P < 0.01).The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group (P < 0.01 and P < 0.001,respectively).Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells.For both TNBS treated groups CD4+ T cell infiltrates were

  17. Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

    OpenAIRE

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; Wang, HongLi; Xiao, Weidong

    2008-01-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FV...

  18. Adeno-associated virus activates an innate immune response in normal human cells but not in osteosarcoma cells.

    Science.gov (United States)

    Laredj, Leila N; Beard, Peter

    2011-12-01

    Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

  19. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

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

    Science.gov (United States)

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

    1996-03-01

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

  1. Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles.

    Science.gov (United States)

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

    2014-11-28

    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.

  2. 介导p73去阻抑肽表达的重组腺伴病毒的构建和鉴定%Construction and identification of recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53 (N37)

    Institute of Scientific and Technical Information of China (English)

    白艳霞; 闫利英; 马清涌; 杨广笑; 王全颖

    2012-01-01

    目的 构建编码融合基因NT4-p53(N37)-HA2-TAT的重组腺伴病毒表达载体,为恶性肿瘤基因治疗的实验研究奠定基础.方法 采用互补引物二次PCR法以及T载体克隆法获得p53(N37)基因克隆,酶切后将其连同穿膜肽HA2-TAT片段一起连入pUC19/NT4质粒,再将融合基因NT4-p53(N37)-HA2-TAT亚克隆至腺伴病毒的穿梭质粒pSSHG-CMV中,构建重组质粒pSSHG-CMV/NT4-p53(N37)-HA2-TAT并进行酶切鉴定;应用磷酸钙沉淀法,pSSHCCMV/NT4-p53(N37)-HA2-TAT、辅助质粒pAAV-Ad,腺病毒全基因组质粒pFG140三种质粒共转染HEK293细胞,包装出重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT并用斑点杂交法测定重组病毒的滴度;MTT比色法、流式细胞仪观察重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT对HepG2细胞的抑制作用.结果 克隆出p53(N37)基因,经酶切及测序证实结果正确;得到高滴度的(2×1013pfu/L)重组腺伴病毒表达载体并对HepG2细胞有明显的抑制作用,且这一作用是通过诱导肿瘤细胞凋亡实现的.结论 通过分子克隆体外重组技术成功制备了rAAV/NT4-p53(N37)-HA2-TAT复制缺陷型重组腺伴病毒,为下一步开展在p53突变或缺失肿瘤中针对p73的靶向性肿瘤基因治疗研究奠定了基础.%Objective To construct a recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) so as to lay a foundation for further research on gene therapy of malignant tumors. Methods The p53(N37) gene was obtained by self-complementary primer PCR and T-vector cloning techniques; then the p53(N37) gene and HA2-TAT segment were cloned into pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N37)-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated pSSHG-CMV, and recombinant plasmid pSSHG-CMV/NT4-p53 (N37)-HA2-TAT was constructed and identified by enzyme cutting analysis. The rAAV/NT4-p53 (N37 )-HA2-TAT was produced by using calcium

  3. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  4. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    Science.gov (United States)

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

  5. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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 but 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.

  7. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

    Science.gov (United States)

    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    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 pH 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.

  9. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    Science.gov (United States)

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    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 pH 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.

  10. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Takeuchi

    Full Text Available 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.

  11. Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene

    Institute of Scientific and Technical Information of China (English)

    陈德杰; 谭最; 谢友利; 刘芳

    2004-01-01

    Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165 (VEGF165) gene in adeno-associated virus (AAV) gene delivery system.Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind Ⅲ, and the downstream of angiopoietin 1contained restriction enzyme site BamH Ⅰ. The upstream of VEGF165 contained restriction enzyme site Bgl Ⅱ, and the downstream of VEGF165 contained restriction enzyme site BamH Ⅰ . Using the multiple cloning sites (MCS) in plasmid pZero ++ such as BamH Ⅰ , Bgl Ⅱ, Hind Ⅲ, Not Ⅰ , XhoⅠ,Xba Ⅰ , Sal Ⅰ , BspH Ⅰ , Ksp Ⅰ and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.

  12. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

    Directory of Open Access Journals (Sweden)

    Jennifer L Daily

    Full Text Available Angelman syndrome (AS, a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286 and Thr(305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  13. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    Science.gov (United States)

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  14. Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

    DEFF Research Database (Denmark)

    Woldbye, David Paul Drucker; Ängehagen, Mikael; Gøtzsche, Casper René;

    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 predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

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

    Science.gov (United States)

    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.

  16. Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

    Science.gov (United States)

    Arruda, Valder R; Stedman, Hansell H; Haurigot, Virginia; Buchlis, George; Baila, Stefano; Favaro, Patricia; Chen, Yifeng; Franck, Helen G; Zhou, Shangzhen; Wright, J Fraser; Couto, Linda B; Jiang, Haiyan; Pierce, Glenn F; Bellinger, Dwight A; Mingozzi, Federico; Nichols, Timothy C; High, Katherine A

    2010-06-10

    Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

  17. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  18. The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.

    Science.gov (United States)

    Hutson, Thomas Haynes; Kathe, Claudia; Menezes, Sean Christopher; Rooney, Marie-Claire; Bueler, Hansruedi; Moon, Lawrence David Falcon

    2014-01-01

    Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

  19. Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

    OpenAIRE

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A.

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefol...

  20. Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.

    Science.gov (United States)

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; Wang, Hongli; Xiao, Weidong

    2008-06-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

  1. Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

    Directory of Open Access Journals (Sweden)

    Stefan Michelfelder

    Full Text Available Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV, we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.

  2. Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer

    Directory of Open Access Journals (Sweden)

    Choi Jin-Ok

    2010-04-01

    Full Text Available Abstract Background Enzyme replacement therapy (ERT with α-galactosidase A (α-Gal A is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding α-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The α-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of α-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the α-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. α-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher α-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more α-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The α-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated α-Gal A gene

  3. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.

    Science.gov (United States)

    Allay, James A; Sleep, Susan; Long, Scott; Tillman, David M; Clark, Rob; Carney, Gael; Fagone, Paolo; McIntosh, Jenny H; Nienhuis, Arthur W; Davidoff, Andrew M; Nathwani, Amit C; Gray, John T

    2011-05-01

    To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2 × 10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

  4. Attenuation of Dengue Virus Infection by Adeno-Associated Virus-Mediated siRNA Delivery

    Science.gov (United States)

    2004-08-09

    safe and efficacious in Phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B and are regarded as a potential alternative to...retroviral and adenoviral vectors for gene therapy in humans. The AAV vectors have a number of advantages over other vectors. They are not pathogenic and...did not induce significant acute inflammatory responses and, therefore, is useful in gene therapy for DEN infection in humans. In conclusion, we

  5. Construction and identification of SHH-N gene adeno-associated virus vector and its impact on genes related to proliferation in neural stem cells%SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响

    Institute of Scientific and Technical Information of China (English)

    刘东升; 崔岩; 申仑; 杜延平; 李桂林; 王任直; 王岩; 张波

    2011-01-01

    Objective To construct and identify SHH-N gene adeno-associated virus vector and to detect its effect on genes controlling to proliferation in neural stem cells(NSCs). Methods The neural stem cells in the subventricular zone of postnatal rat brain were used to collect SHH-N. pSNAV2. 0-CMV-SHH-N-IRES-EGFP was established by enzyme cutting and ligation , and then transfected packaging cell line 293T. Real time PCR was performed after SHH-N transfection of neural stem cells by rAAV-SHH-N-EGFP vector for 48 hours, SHH-N gene expression of infected cells after 2 week examined by fluorescence microscopy. Results ( 1 ) SHH-N gene was coincident with NCBI report. (2) pSNAV2. 0-CMV-SHH-N-IRES-EGFP expression vector and rAAV-SHH-N-EGFP vector was successful established and packaged. (3)Real time PCR was performed after SHH-N infection for 48 hour, induction of Nmyc and Glil in rAAV-SHH-N-EGFP-treated group was enhanced compared to control group. Conclusion rAAV-SHH-N-EGFP vector has been successfully established and it can stably express in neural stem cells. Forced expression of SHH-N in NSCs resulted in enhanced induction of Nmyc and Glil.%目的 构建rAAV-SHH-N-EGFP载体并检测其对神经干细胞增殖相关基因影响.方法 分离并培养大鼠脑室下区神经干细胞,提取RNA、反转录、PCR得到SHH-N的cDNA,克隆入pSNAV2.0-CMV-IRES-EGFP,包装得到腺相关病毒rAAV-SHH-N-EGFP,感染神经干细胞48 h后,采用实时定量PCR检测SHH信号通路下游相关基因的mRNA水平变化,并观察rAAV-SHH-N-EGFP载体在神经干细胞内的表达情况.结果 (1)克隆得到SHH-N基因,测序结果与NCBI报道序列一致.(2)成功构建pSNAV2.0-CMV-SHH-N-EGFP表达载体并鉴定,包装得到rAAV-SHH-N-EGFP.(3)实时定量PCR分析rAAV-SHH-N-EGFP感染神经干细胞48 h后,rAAV-SHH-N-EGFP处理组较感染rAAV-EGFP的对照组,Glil和Nmyc的mRNA水平上调.(4)rAAV-SHH-N-EGFP可有效感染神经干细胞,在感染14 d后稳定表达SHH-N.结论

  6. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.

    Science.gov (United States)

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

  7. Recombinant adeno-associated virus-mediated delivery of antisense angiotensin Ⅱ receptor 1 gene attenuates hypertension development

    Institute of Scientific and Technical Information of China (English)

    Xu-guang LI; Jiang-tao YAN; Xi-zheng XU; Jia-ning WANG; Li-ming CHENG; Tao WANG; Ping ZUO; Dao-wen WANG

    2007-01-01

    Aim:The renin-angiotensin system plays a crucial role in the development and establishment of hypertension,and the pharmacological blockade of the system results in a reduction in blood pressure. In the present study,we investigated whether the effects of a novel,double-stranded,recombinant adeno-associated virus vector (rAAV)-mediated antisense angiotensin Ⅱ receptor l (AT1R) gene efficiently prevents the development of hypertension induced by a high-salt diet in adult,male Sprague-Dawley (SD) rats. Methods:A rAAV was prepared with a cassette containing a cytomegalovirus promoter and partial cDNA (660 base pairs) for the AT1R inserted in the antisense direction (rAAV-AT1AS). A single tail vein injection of the rAAV-AT1-AS or rAAV-GFP (green fluorescent protein,a reporter gene) was performed in adult,male SD rats. Two weeks after injection,the animals were fed a diet containing 8% NaCI,and the systolic blood pressure was measured weekly using the tail-cuff method for 12 weeks. Results:The high-salt diet induced a significant rise in systolic blood pressure in the rAAV-GFP-treated animals;however,the rAAV-AT:AS treatment attenuated the rise in blood pressure (142.7±4.5 mmHg vs 117±3.8 mmHg,P<0.01),and the hypotensive effect was maintained until the experiments ended at 12 weeks. In the rAAV-GFP-treated animals AT1 was overexpressed in various tissues,especially in the aorta and kidney at mRNA levels;in contrast,rAAV-AT:AS treatment markedly attenuated AT1 expression. Furthermore,rAAV-AT:AS treatment prevented target organ damages from hypertension,including cardiac dysfunction and renal injury compared to the rAAV-GFP group. Conclusion:These results suggest that rAAVmediated anti-AT1 delivery attenuates the development of hypertension and protects against renal injury and cardiac remodeling.

  8. Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Miao Chen; Guang-Ji Wang; Yong Diao; Rui-An Xu; Hai-Tang Xie; Xin-Yan Li; Jian-Guo Sun

    2005-01-01

    AIM: To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosisin vitro and in vivo.METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-L, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected.RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ,, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group,the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0,458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L,respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13.CONCLUSION: All these results indicated that rAAV-INF-γhas potential effects for gene therapy of hepatic fibrosis,which could inhibit the progression of hepatic fibrosis.

  9. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Directory of Open Access Journals (Sweden)

    Lina Li

    Full Text Available Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA. CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9 Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  10. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Science.gov (United States)

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

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

    Science.gov (United States)

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

    2017-02-01

    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 × 10(14) 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 × 10(15) VG in crude cell harvests and an average of 3.5 × 10(14) total VG of purified rAAV9 material, resulting in greater than 1 × 10(5) VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

  12. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  13. Adeno-associated viral vector serotype 5 poorly transduces liver in rat models.

    Directory of Open Access Journals (Sweden)

    Paula S Montenegro-Miranda

    Full Text Available Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.

  14. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    2008-01-01

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman prim

  15. 人血管内皮生长因子与绿色荧光蛋白标记双基因共表达AAV载体的构建及鉴定%Construction and identification of recombinant adeno- associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    黄向辉; 时志斌; 王坤正; 党晓谦; 杨佩; 余鹏博

    2008-01-01

    取重组病毒基因组成功扩增出外源目的基因片段hVEGF165,证实重组病毒rAAV-VEGF165-GFP包装成功.结论:成功构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒rAAV-VEGF165-GFP.收获的病毒具有较高滴度和感染活性.%BACKGROUND: Vascular endothelial growth factor (VEGF) can specifically promote the division and proliferation of endothelial cells and the revascularization, finally induce angiopoiesis. Recently, VEGF-based gene therapy has been gradually used in clinical trials, but some limits on usually used vectors deserve further studies, including the low transfection efficiency of plasmid vector, the immunogenicity of adnovirus vector to host cells and the potential risk of infection. OBJECTIVE: To construct the non-pathogenic recombinant adeno-associated vires (AAV) co-expressing human vascular endethelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label and measure the virus titer and assess its biological activity.DESIGN, TIME AND SETTING: The open experiment was performed at the Vitrus Laboratory of Shanxi Provincial Center for Disease Control and Prevention from March to September 2007.MATERIALS: AAV-293 virus packaging cell line, AAV HT-1080 cells were purchased from Swatagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. AAV Helper Free System (pAAV-IRES-GFP vector containing GFP label) was purchased from Stratagene, USA. Plasmid pUC18-hVEGF165 was constructed previously by Dr.Shi from Department of Orthopaedics of Second Affiliated Hospital of Xi'an Jiaotong University.METHODS: The hVEGF165 gone from plasmid pUC18-hVEGF165 was amplified and inserted into plasmid pAAV-IRES-hrGFP. Then recombinant plasmid pAAV-hVEGF165-IRES-hrGFP, pAAV-RC and pAAV-Helper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of A

  16. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

    Full Text Available Abstract Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV and lentiviral (LV vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein and LV-Cre-EGFP (enhanced GFP were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  17. Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

    Science.gov (United States)

    Wang, X S; Yoder, M C; Zhou, S Z; Srivastava, A

    1995-01-01

    The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies. Images Fig. 2 Fig. 3 Fig. 4 PMID:8618912

  18. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Directory of Open Access Journals (Sweden)

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  19. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    Science.gov (United States)

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.

  20. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    Science.gov (United States)

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

  1. Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound.

    Science.gov (United States)

    Thévenot, Emmanuel; Jordão, Jessica F; O'Reilly, Meaghan A; Markham, Kelly; Weng, Ying-Qi; Foust, Kevin D; Kaspar, Brian K; Hynynen, Kullervo; Aubert, Isabelle

    2012-11-01

    Noninvasive drug delivery to the brain remains a major challenge for the treatment of neurological disorders. Transcranial focused ultrasound combined with lipid-coated gas microspheres injected into the bloodstream has been shown to increase the permeability of the blood-brain barrier locally and transiently. Coupled with magnetic resonance imaging, ultrasound can be guided to allow therapeutics administered in the blood to reach brain regions of interest. Using this approach, we perform gene transfer from the blood to specific regions of the mouse brain. Focused ultrasound was targeted to the right hemisphere, at multiple foci, or restricted to one focal point of the hippocampus or the striatum. Doses from 5 × 10(8) to 1.25 × 10(10) vector genomes per gram (VG/g) of self-complementary adeno-associated virus serotype 9 carrying the green fluorescent protein were injected into the tail vein. A dose of 2.5 × 10(9) VG/g was optimal to express the transgene, 12 days later, in neurons, astrocytes, and oligodendrocytes in brain regions targeted with ultrasound, while minimizing the infection of peripheral organs. In the hippocampus and striatum, predominantly neurons and astrocytes were infected, respectively. Transcranial focused ultrasound applications could fulfill a long-term goal of gene therapy: delivering vectors to diseased brain areas directly from the circulation, in a noninvasive manner.

  2. Prevalence of adeno-associated virus and human papillomavirus DNA in Iranian women with and without cervical cancer.

    Science.gov (United States)

    Shafiei-Jandaghi, Nazanin Zahra; Yavarian, Jila; Faghihloo, Ebrahim; Ghavami, Nastaran; Yousefi Ghalejoogh, Zohreh; Kiani, Seyed Jalal; Shatizadeh Malekshahi, Somayeh; Shahsiah, Reza; Jahanzad, Eisa; Hosseini, Mostafa; Mokhtari Azad, Talat

    2017-02-24

    There is plenty of substantial evidence to support anti-tumor activity of viruses. Adeno-associated virus (AAV) may interact with human papillomavirus (HPV) to modify the risk of cervical neoplasia. The seroprevalence of AAV among women with cervical cancer has been reported to be lower than healthy ones. In spite of this finding, detection of AAV DNA in cervical biopsies does not entirely support the inverse association between AAV seropositivity and cervical cancer. This association is still controversial and requires more thorough evaluation in different countries. The aim of this case-control study was to find the prevalence of AAV and HPV DNA sequences in Iranian women with and without cervical cancer to assess the probable association of AAV infection and cervical cancer. In this study, paraffin-embedded tissue samples of 61 cervical cancer cases and 50 healthy controls (HCs) were investigated for AAV and HPV DNA by semi-nested and nested PCRs respectively. AAV DNA was detected in 7 cases (14%) of HCs and 9 specimens (14.8%) of case group. According to the branching in the phylogenetic tree, AAV2 was the only type detected in this study. Moreover, HPV DNA was detected in 8 cases (16%) of HCs and 44 specimens (72.13%) of case group. In conclusion, a low proportion of cervical biopsies from Iranian women contained AAV-2 genome. No significant difference in correlation between HPV and cervical cancer in presence or absence of AAV genome in cervix was found.

  3. Delivery of human EV71 receptors by adeno-associated virus increases EV71 infection-induced local inflammation in adult mice.

    Science.gov (United States)

    Hsiao, Hung-Bo; Chou, Ai-Hsiang; Lin, Su-I; Lien, Shu-Pei; Liu, Chia-Chyi; Chong, Pele; Chen, Chih-Yeh; Tao, Mi-Hua; Liu, Shih-Jen

    2014-01-01

    Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

  4. Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis.

    Science.gov (United States)

    Watanabe, M; Kashiwakura, Y; Kusumi, N; Tamayose, K; Nasu, Y; Nagai, A; Shimada, T; Daida, H; Kumon, H

    2005-07-01

    Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN- and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.

  5. Effect and Mechanism of Mitomycin C Combined with Recombinant Adeno-Associated Virus Type II against Glioma

    Directory of Open Access Journals (Sweden)

    Hong Ma

    2013-12-01

    Full Text Available The effect of chemotherapy drug Mitomycin C (MMC in combination with recombinant adeno-associated virus II (rAAV2 in cancer therapy was investigated, and the mechanism of MMC affecting rAAV2’s bioactivity was also studied. The combination effect was evaluated by the level of GFP and TNF expression in a human glioma cell line, and the mechanism of MMC effects on rAAV mediated gene expression was investigated by AAV transduction related signal molecules. C57 and BALB/c nude mice were injected with rAAV-EGFP or rAAV-TNF alone, or mixed with MMC, to evaluate the effect of MMC on AAV-mediated gene expression and tumor suppression. MMC was shown to improve the infection activity of rAAV2 both in vitro and in vivo. Enhancement was found to be independent of initial rAAV2 receptor binding stage or subsequent second-strand synthesis of target DNA, but was related to cell cycle retardation followed by blocked genome degradation. In vivo injection of MMC combined with rAAV2 into the tumors of the animals resulted in significant suppression of tumor growth. It was thus demonstrated for the first time that MMC could enhance the expression level of the target gene mediated by rAAV2. The combination of rAAV2 and MMC may be a promising strategy in cancer therapy.

  6. Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

    Institute of Scientific and Technical Information of China (English)

    徐卫国; 陈安民; 张衣北; 易成腊

    2004-01-01

    Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μA) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively,w ith the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

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

    Science.gov (United States)

    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.

  8. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yi Wang; Qing-San Zhu; Yi-Wei Wang; Ruo-Feng Yin

    2015-01-01

    Background:Thymosin beta-4 (TB-4) is considered key roles in tissue development,maintenance and pathological processes.The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation.Methods:TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells.Cell of same group were cultured without gene modification as controlled group.Proliferation capacity and cell apoptosis were observed during 6 passages of the cells.Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage.Results:NP cells with TB-4 transfection has normal TB-4 expression and exocytosis.NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation.TB-4 recombinant AAV-transfected human NP cells also show slower cell aging,lower cell apoptosis and higher cell proliferation than control group.Conclusions:TB-4 can prevent NP cell apoptosis,slow NP cell aging and promote NP cell proliferation.AAV transfection technique was able to highly and stably express TB-4 in human NP cells,which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases.

  9. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Amin Hajitou

    2013-10-01

    Full Text Available 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.

  10. An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

    Directory of Open Access Journals (Sweden)

    Abdelwahed Chtarto

    Full Text Available Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS. This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

  11. Osteogenic differentiation of recombinant adeno-associated virus 2-transduced murine mesenchymal stem cells and development of an immunocompetent mouse model for ex vivo osteoporosis gene therapy.

    Science.gov (United States)

    Kumar, Sanjay; Mahendra, Gandham; Nagy, Tim R; Ponnazhagan, Selvarangan

    2004-12-01

    Gene therapy for osteopenic conditions including osteoporosis is a potential alternative to pharmacotherapy for cost effectiveness, long-term viability, and the ability to enhance bone mass by anabolic approaches. Increased understanding of mesenchymal stem cell (MSC) lineage differentiation during osteogenesis, and of the molecular pathways involved in bone cell production, provides an opportunity for the advancement of gene therapy approaches for osteopenic conditions. The potential of MSCs in osteoblast differentiation and the relative ease of MSC isolation and culturing offer a promising resource for the development of ex vivo gene therapy for bone defects. In an effort to develop ex vivo gene therapy for osteoporosis, we used gene-modified MSCs in a preclinical mouse model to determine the efficiency of transduction of murine MSCs by recombinant adeno-associated virus 2 (AAV) vectors carrying reporter genes and determined their osteogenic potential after recombinant AAV-mediated expression of bone morphogenic protein 2, known to induce osteoblast differentiation. Although surgical ovariectomy is believed to induce progressive bone loss in mouse models, similar to an osteoporosis-like phenotype in humans, several factors, including hormonal alteration and dietary habits, significantly affect both the onset and progression of the disease. Thus, in the present study, we determined the influence of these factors and developed an immunocompetent mouse model of osteoporosis with degenerative bone loss as in the human pathology.

  12. Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo

    Directory of Open Access Journals (Sweden)

    Mette Christensen

    2010-01-01

    Full Text Available Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV. rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.

  13. Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

    Science.gov (United States)

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

  14. Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

    Directory of Open Access Journals (Sweden)

    Janssen William GM

    2006-01-01

    Full Text Available Abstract Background Intrathecal (IT gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV, one of the most promising vector types for clinical use. Results Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. Conclusion sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2

  15. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

    Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

  16. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    陆华中; 陈立; 王红卫; 伍志坚; 吴小兵; 王学峰; 王鸿利; 卢大儒; 邱信芳; 薛京伦

    2001-01-01

    A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

  17. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  18. Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.

    Science.gov (United States)

    Foust, Kevin D; Poirier, Amy; Pacak, Christina A; Mandel, Ronald J; Flotte, Terence R

    2008-01-01

    Targeting lower motor neurons (LMNs) for gene delivery could be useful for disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. LMNs reside in the ventral gray matter of the spinal cord and send axonal projections to innervate skeletal muscle. Studies have used intramuscular injections of adeno-associated virus type 2 (AAV2) to deliver viral vectors to LMNs via retrograde transport. However, treating large areas of the spinal cord in a human would require numerous intramuscular injections, thereby increasing viral titer and risk of immune response. New AAV serotypes, such as AAV8, have a dispersed transduction pattern after intravenous or intraperitoneal injection in neonatal mice, and may transduce LMNs by retrograde transport or through entry into the nervous system. To test LMN transduction after systemic injection, we administered recombinant AAV8 (rAAV8) carrying the green fluorescent protein (GFP) gene by intravenous or intraperitoneal injection to neonatal mice on postnatal day 1. Tissues were harvested 5 and 14 days postinjection and analyzed by real-time polymerase chain reaction and GFP immunohistochemistry to assess the presence of AAV genomes and GFP expression, respectively. Spinal cords were positive for AAV genomes at both time points. GFP immunohistochemistry revealed infrequent labeling of LMNs across all time points and injection routes. Somewhat surprisingly, there was extensive labeling of fibers in the dorsal horns and columns, indicating dorsal root ganglion transduction across all time points and injection routes. Our data suggest that systemic injection of rAAV8 is not an effective delivery route to target lower motor neurons, but could be useful for targeting sensory pathways in chronic pain.

  19. A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

    Directory of Open Access Journals (Sweden)

    Fengqin Zhu

    Full Text Available More than 170 million individuals worldwide are infected with hepatitis C virus (HCV, and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

  20. A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts

    Science.gov (United States)

    McClements, Michelle E.; Charbel Issa, Peter; Blouin, Véronique; MacLaren, Robert E.

    2017-01-01

    Objective Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. Methods Oversized (>8kb) transgene constructs containing ABCA4 coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes. In vitro transductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. Results Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion Despite previous success shown with the fAAV approach, the lack of repeatability and identification of stable hybrid transcripts capable of protein production suggests there is more refinement required before considering this approach in a clinical setting. PMID:28239514

  1. Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats.

    Science.gov (United States)

    Andsberg, Gunnar; Kokaia, Zaal; Klein, Ronald L; Muzyczka, Nicholas; Lindvall, Olle; Mandel, Ronald J

    2002-03-01

    Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were continuously delivered to the striatum at biologically active levels via recombinant adeno-associated viral (rAAV) gene transfer 4-5 weeks prior to 30 min of middle cerebral artery occlusion (MCAO). The magnitude of the deficits in a battery of behavioral tests designed to assess striatal function was highly correlated to the extent of ischemic damage determined by unbiased stereological estimations of striatal neuron numbers. The delivery of neurotrophins lead to mild functional improvements in the ischemia-induced motor impairments assessed 3-5 weeks after the insult, in agreement with a small but significant increase of the survival of dorsolateral striatal neurons. Detailed phenotypic analysis demonstrated that the parvalbumin-containing interneurons were spared to a greater extent by the neurotrophin treatment as compared to the projection neurons, which agreed with the specificity for interneuron transduction by the rAAV vector. These data show the advantage of the never previously performed combination of precise quantification of the ischemia-induced neuropathology along with detailed behavioural analysis for assessing neuroprotection after stroke. We observe that intrastriatal delivery of NGF and BDNF using a viral vector system can mitigate, albeit only moderately, neuronal death following stroke, which leads to detectable functional sparing.

  2. 腺相关病毒介导的狨猴 P53基因沉默%Adeno-associated virus mediated p53 gene silence in marmosets

    Institute of Scientific and Technical Information of China (English)

    石亮; 张晨; 向志光; 邓仪晨; 苏静芬; 刘云波

    2016-01-01

    目的:在细胞和整体动物水平,利用RNA干扰技术下调绒猴p53基因表达。方法对狨猴p53基因做生物信息学分析,针对靶序列设计shRNA干扰序列,构建在腺相关病毒载体上,转染非洲绿猴肾细胞( cos-7),在细胞水平用荧光定量PCR检测p53mRNA抑制效果,以Western blot 方法检测p53蛋白水平表达变化;优选shRNA干扰序列,包装含shRNA干扰序列的8型腺相关病毒,静脉注射感染狨猴;手术取少量肝脏组织,用Western blot和免疫组化方法检测p53蛋白水平的变化。结果细胞水平研究发现2个有效RNA干扰靶点,mRNA干扰效率分别为(82.7±8.1)%和(80.7±7.5)%(P<0.05);蛋白表达下调(77.3±11.5)%和(73.7±10.7)%(P<0.05);2只绒猴感染病毒后,经活体荧光成像分析可见病毒在肝脏、睾丸、颈部等位置分布,狨猴肝脏P53蛋白经Western blot、免疫组化分析未见明显变化。结论本研究在细胞水平实现绒猴P53基因表达下调,但整体动物水平狨猴肝脏P53蛋白表达未发现明显变化;今后需在感染方式等方面做进一步优化。%Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique.Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis.The plasmids were transfected into African green monkey kidney cos-7 cells.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt.The adeno-associated virus-8 was injected through the hind leg vein.The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry.Results We screened two RNA interference effective arget sequences.The expression of p53 mRNA was suppressed ( 82.7 ±8.1 )% and ( 80.7 ± 7.5)%, respectively (P<0

  3. A Regulatory Element Near the 3′ End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts

    Science.gov (United States)

    Hammer, Eva; Gonsior, Melanie; Stutika, Catrin; Heilbronn, Regine

    2016-01-01

    ABSTRACT Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3′ end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RIS-Ad). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5′ half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCE Insertion of sequences from the 3′ part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so

  4. Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol

    Science.gov (United States)

    Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

    2016-01-01

    Background Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. Objective The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. Methods We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. Results The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype

  5. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Hélène Hall

    Full Text Available Intraneuronal inclusions containing alpha-synuclein (a-syn constitute one of the pathological hallmarks of Parkinson's disease (PD and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  6. Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.

    Science.gov (United States)

    Aoyama, Yutaka; Kobayashi, Koichi; Morishita, Yoshihiro; Maeda, Kengo; Murohara, Toyoaki

    2015-07-01

    The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackievirus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ 2 weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11 treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines.

  7. Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jiang-tao YAN; Tao WANG; Dao-wen WANG

    2009-01-01

    Aim: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pres-sure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-rnediated HK gene therapy protects against organ damage by inhibiting cell apoptosis. Methods: rAAV encoding HK(rAAV-HK) or LacZ(rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells. Results: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-in-duced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x_L, and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apopto-sis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT. Conclusion: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

  8. CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFp3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ1

    Institute of Scientific and Technical Information of China (English)

    Jia-ming Sai; You-gu Hu; De-chun Wang

    2007-01-01

    Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1.Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site Sal Ⅰ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plas-mid AAV-TGFβ3 was transfected into H293 cells with Lipofectamine(tm) 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ, in the earlier and later dedifferentiated NP cells.Results For the earlier dedifferentiated NP cells, AAV-TGFβ slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ, rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ, inhibited its synthesis.Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

  9. Comparison of efficacy of the disease-specific LOX1- and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice.

    Directory of Open Access Journals (Sweden)

    Hongqing Zhu

    Full Text Available The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of "disease-specific promoters" has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2 using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.

  10. Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

    Institute of Scientific and Technical Information of China (English)

    Sung Yi Hong; Myun Hee Lee; Kyung Sup Kim; Hyun Cheol Jung; Jae Kyung Roh; Woo Jin Hyung; Sung Hoon Noh; Seung Ho Choi

    2004-01-01

    AIM: rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer. However,a sustained and high protein delivery is required to achieve the desired therapeutic effects. We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model.METHODS: rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines. To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin, Western blotting and ELISA were performed. The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays.The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor, etoposide, were evaluated in a mouse liver tumor model.RESULTS: Topoisomerase inhibitors, including camptothecin and etoposide, were found to increase the endostatin expression level in vitro. The over-expressed endostatin,as a result of pretreatment with a topoisomerase inhibitor,was also biologically active. In animal experiments, the combined therapy of topoisomerase inhibitor, etoposide with the rAAV-endostatin vector had the best tumorsuppressive effect and tumor foci were barely observed in livers of the treated mice. Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice. Finally, the mice treated with rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models.CONCLUSION: rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

  11. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Zhan SUN; Yi-Tong MA; Bang-Dang CHEN; Fen LIU

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

  12. Package of recombinant adeno-associated virus encoding cell division cycle 2-siRNA%携带cdc2-siRNA重组腺相关病毒的包装

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 王卓然

    2009-01-01

    背景:细胞分裂周期2(cell division cycle 2,cdc2)在神经变性疾病如阿尔茨海默病的神经元变性过程中扮演了重要角色.以腺相关病毒为载体对cdc2基因进行沉默,可能对神经变性疾病的神经元起保护作用.目的:包装携带cdc2-siRNA的重组腺相关病毒(recombinant adeno-associated virus,rAAV).设计、时间及地点:空白对照实验,于2007-10/2008-08在武汉同济医院神经科实验室完成.材料:Helper Free腺相关病毒系统(pAAV-MCS-EGFP、p-RC、p-Helper)及AAV-293细胞为Stratagene公司产品.方法:应用分子生物学方法构建生成pAAV-MCS-U6-cdc2-slRNA-EGFP表达质粒;用磷酸钙法将该质粒和p-RC、p-Helper质粒共转染AAV-293细胞,包装生成携带cdc2-siRNA的重组腺相关病毒(rAAV-U6-cdc2-siRNA-EGFP).病毒感染AAV-293细胞后行Western Blot检测cdc2-siRNA对细胞内cdc2基因的沉默效果,并用斑点杂交方法测定该病毒的滴度.主要观察指标:pAAV-MCS-U6-cdc2-siRNA-EGFP质粒中插入片段U6-cdc2-siRNA的测序;3质粒pAAV-MCS-U6-cdc2-siRNA-EGFP、p-RC、p-Helper共转染AAV-293细胞; rAAV-U6-cdc2-siRNA-EGFP感染AAV-293细胞后cdc2的表达.结果:①DNA测序证明U6-cdc2-siRNA已成功构建到表达质粒pAAV-MCS-EGFP中.②AAV-293细胞表达绿色荧光蛋白,共转染成功.③包装得到的重组腺相关病毒(rAAV-U6-cdC2-siRNA-EGFP)感染AAV-293细胞后能显著下调AAV-293细胞cdc2基因的表达量.④rAAV-U6-cdc2-siRNA-EGFP的滴度为1×1012v.g/mL.结论:携带cdc2-siRNA重组腺相关病毒的包装获得成功.它具有沉默cdc2基因表达的功能.%BACKGROUND: Cell division cycle 2 (cdc2) plays an important role in the course of neuronal degeneration in neurodegenerative diseases such as Alzheimer's disease. The silencing of cdc2 gene with adeno-associated virus vector might protect the neurons in neurodegenerative diseases.OBJECTIVE: To pack recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. DESIGN, TIME AND

  13. Recombinant adeno-associated virus-mediated global anterograde delivery of glial cell line-derived neurotrophic factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in the rat.

    Science.gov (United States)

    Foust, Kevin D; Flotte, Terence R; Reier, Paul J; Mandel, Ronald J

    2008-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery.

  14. hTRX-PR39重组腺相关病毒载体的构建%Construction of a hTRX-PR39 recombinant adeno-associated vires vector

    Institute of Scientific and Technical Information of China (English)

    阮喜云; 魏敏; 毕建忠; 杨广笑; 王全颍

    2011-01-01

    Objective To construct a vector of recombinant adeno-associated virus (AAV) containing the fusion gene hTRX-PR39, and explore the role of fusion gene hTRX-PR39 as a target gene in the treatment of cerebral ischemic disease. Methods The constructed fusion gene hTRX-PR39 was inserted into the EcoRI-BamHI site of vector plasmid pSSCMV, and the vector of hTRX-PR39 recombinant AAV was constructed. The recombinant AAV stock was packaged. Renal embryo 293 cells were co-transfected with the recombinant AAV vector of plasmid pSSCMV/hTRX-PR39,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140. Recombinant AAV was produced by homologous recombination of the 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization. Results The vector of hTRX-PR39 recombinant AAV was successfully constructed. High titer of recombinant AAV was obtained by homologous recombination in renal embryo 293 cells (3.46 x 1012-3.46 × 1013 PFU/mL).Conclusion The recombinant AAV vector encoding the gene hTRX-PR39 was successfully constructed in this study by molecular cloning and in vitro recombination techniques, laying the foundation for further research into genetic therapy of cerebral ischemic disease.%目的 探讨hTRX-PR39融合基因在脑缺血疾病中的治疗作用.方法 将已经构建好的hTRX-PR39融合基因插入到具有相应酶切位点的pSSCMV病毒载体质粒中,得到重组腺相关病毒载体质粒,酶切电泳鉴定.将已构建的重组腺相关病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad,三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得hTRX-PR39重组腺相关病毒载体,收集病毒,斑点杂交(Dot blot)法测定病毒滴度.结果 成功构建重组腺相关病毒质粒pSSCMV/hTRX-PR39.包装、回收病毒后,Dot b1ot法测定重组病毒滴度为3.46×(1012~1013)PFU/mL.结论 成功构建pSSCMV/hTRX-PR39重组腺相关病毒载体,并包装较高浓度的重组病毒.

  15. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt;

    2010-01-01

    . Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

  16. Cloning of avian adeno-associated virus genome and rescue of the infectious virus%禽腺联病毒全基因组的克隆及感染性病毒的拯救

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2007-01-01

    为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7 kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序.序列分析表明,AAAV YZ-1株的基因组为4 684 bp,两端具有141 bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%.将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建.

  17. Recombinant adeno-associated virus-mediated human kallikrein gene therapy prevents high-salt diet-induced hypertension without effect on basal blood pressure

    Institute of Scientific and Technical Information of China (English)

    Jiang-tao YAN; Tao WANG; Juan LI; Xiao XIAO; Dao-wen WANG

    2008-01-01

    Aim: To investigate the effects of the expression of human kallikrein (HK) on basal level blood pressure and high-salt diet-induced hypertension. Methods: We delivered the recombinant adeno-associated viral (rAAV)-mediated HK (rAAV-HK) gene and rAAV-LacZ (as the control) to normal, adult Sprague-Dawley rats. The animals were administered a normal diet in the first 4 weeks, followed by a high-salt diet. The expression of HK in the rats was assessed by ELISA and RT-PCR. Blood pressure and Na~ and K~ urinary excretion were monitored. Results: Under the normal diet, no obvious changes in blood pressure and Na+ and K+ urinary excretion were observed. When the high-salt diet was administered, sys-tolic blood pressure in the control animals receiving rAAV-LacZ increased from 122.3±1. 13 mmHg to a stable 142.4±1.77 mmHg 8 weeks after the high-salt diet. In contrast, there was no significant increase in the blood pressure in the rAAV-HK-treated group, in which the blood pressure remained at 121.9±1.73 mmHg. In the rAAV-HK-treated group, Na+ and K+ urinary excretion were higher compared to those of the control group. The morphological analysis showed that HK delivery remarkably protected against renal damage induced by a high-salt intake. Conclusion: Our study indicates that rAAV-mediated human tissue kallikrein gene delivery is a potentially safe method for the long-term treatment of hypertension. More importantly, it could be applied in the salt-sensitive population to prevent the occurrence of hypertension.

  18. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    Science.gov (United States)

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  19. In vitro and in vivo induction of bone formation based on adeno-associ-ated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yan KANG; Wei-ming LIAO; Zhen-hua YUAN; Pu-yi SHENG; Long-juan ZHANG; Xiang-wei YUAN; Lei LEI

    2007-01-01

    Aim: To determine whether adeno-associated virus (AAV)-2-mediated, bone mor-phogenetic protein (BMP)-7-expressing human adipose-derived mesenchymal stem cells (ADMS) cells would induce bone formation in vitro and in vivo.Methods:ADMS cells were harvested from patients undergoing selective suction-assisted fipectomy and transduced with AAV carrying the human BMP-7 gene. Non-trans-duced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualita-tively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase (ALP) and Chinalizarin staining. A total of 2.5x 106 cells mixed with type Ⅰ collagen were implanted into the hind limb of severe combined immune-deficient (SCID) mice and subjected to a histological analysis 3 weeks post implantation.Results: Transfection of the ADMS cells achieved an effi-ciency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further con-firmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7-expressing ADMS cells could induce new bone formation in vivo.Conclusion: The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.

  20. 重组腺相关病毒介导遗传性色盲基因治疗的研究进展%Advance in recombinant adeno-associated virus mediated gene therapy for color blindness

    Institute of Scientific and Technical Information of China (English)

    杨红霞; 邱一果

    2013-01-01

    色盲是缺乏或完全没有辨色能力的一类遗传性疾病,长期被认为是不可治愈性疾病.近年来以腺相关病毒(AAV)为载体介导的基因疗法主要用于对由视蛋白缺乏引起的红绿色盲及由视锥细胞环核苷酸门控离子通道A3(CNGA3)A或B(CNGB3)亚单位基因缺失引起的全色盲的治疗,已在动物实验中获得成功.人类色盲患者与一些实验动物存在着相同的基因缺陷,因此相关的动物实验研究结果用AAV介导的基因疗法为色盲患者进行治疗提供了有用的信息.%Color blindness represents a group of vision disorders characterized by lack of ability to distinguish different colors.The inherited color blindness has been regarded as incurable for a long period of time.Recently,adeno-associated virus(AAV) mediated gene therapy has successfully restored cone system vision in animal models with color blindness caused by different gene mutations.These mutations are presented in human color blindness patients.It is predicted that gene therapy will become a novel treatment for these color blindness victims.In addition,a single gene transfer may achieve long-term correction of color deficiency.

  1. 腺相关病毒2-ND4基因转染细胞线粒体的研究%Study on transfection of adeno associated virus 2-ND4 gene into mitochondria

    Institute of Scientific and Technical Information of China (English)

    杨硕; 刘磊; 裴晗; 万幸; 陆朵朵; 胡维琨; 李斌

    2014-01-01

    Background Leber hereditary optic neuropathy (LHON) is mitochondrial DNA (mtDNA) disease and mainly leads to optical nerve degeneration.Its primary mechanism is synthesis disorder of DN4 protein due to variation of mtDNA 11778 locus.So to construct a vector with exogenous normal ND4 and transfect into mitochondria is a key of gene therapy for LHON.Objective This study was to investigate the in vitro transfection of adeno-associated virus (AAV)-ND4 gene into mitochondria.Methods Human renal epithelial cell lines transfected adenovirus E1A (293 cells) were regularly cultured and divided into two groups.Framework plasmids of recombinant AAV-ND4 or simple AAV2 were added to the cell medium respectively.The expression of ND4 in cells were located 12,24,36 and 48 hours after transfected by Y03 dual fluorescent quantum dots staining.The positive response for ND4 showed the green fluorescence.Results Cultured 293 cells grew well with 80% confluence.Abundant green fluorescence particles were seen in cytoplasm in the AAV-ND4 transfected group,but only red fluorescence from mitochondrial protein was seen in the simple AAV transfected group under the fluorescence microscope.Conclusions Exogenous ND4 protein can been successfully transfected into mitochondria using the ND4 gene constructed AAV.This result provides experimental evidence for the further study on gene therapy of LHON.%背景 Leber遗传性视神经病变(LHON)是导致视神经退行性变的线粒体遗传性疾病,主要与线粒体11778位点突变导致ND4蛋白合成异常有关,构建含正常ND4蛋白的载体是基因治疗的关键.由于ND4 DNA存在于线粒体,而转染的外源基因只能进入细胞核,不能作用于突变的线粒体DNA.探讨将ND4基因成功转染到线粒体是LHON的基因治疗的关键. 目的 验证人工合成的ND4基因所构建的重组腺相关病毒(AAV)转染细胞后产生的ND4蛋白能否进入线粒体.方法 常规体外培养转染腺病毒E1A

  2. 重组腺相关病毒2型/人凝血因子IX的质量研究%Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    高凯; 王军志; 饶春明; 吴小兵

    2003-01-01

    目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准.方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒(wtAAV)的残留片段.SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度,TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度.以斑点杂交法测定病毒颗粒数.一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性,并通过ELISA法测定感染BHK-21细胞后hFIX的表达量.结果 PCR法确证病毒的重组核酸结构与构建预期相同;在1×107 VG的rAAV-2/hFIX颗粒中,残留辅助病毒的基因片段数少于1个拷贝;在1×108 VG的rAAV-2/hFIX颗粒中,野生型AAV-2基因片段数少于1个拷贝.病毒颗粒及外壳蛋白纯度均大于98%,病毒颗粒数大于1.0×1015 VG*L-1(virus genome*L-1).IX因子剔除小鼠肌肉注射病毒后21 d,小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%,IX因子的体外表达水平大于20.0 μg*L-1.其他各项检测指标均符合规定.结论建立了rAAV-2/hFIX的质量标准,用于控制产品质量.

  3. Culture of 293 cells for the package of adeno-associated viruses%用于包装腺相关病毒293细胞的培养

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 张苏明; 徐金枝

    2007-01-01

    BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei

  4. Bac-to-Bac杆状病毒昆虫表达系统介导的重组腺相关病毒制备%Production of adeno-associated virus mediated by Bac-to-Bac baculovirus insect expression system

    Institute of Scientific and Technical Information of China (English)

    陈雅宁; 王春荣; 杨宗灿; 姬朝霞; 张延瑞; 韩双印

    2012-01-01

    目的 建立高效的临床级重组腺相关病毒( rAAV)制备方法.方法 将rAAV的反向末端重复序列( ITR)及目的基因表达框(2053 bp)从pAAV-MCS剪切引入杆状病毒载体中构建顺式载体,rAAV血清型2的衣壳和复制蛋白基因在反式载体pFastBacDual中利用真核基因遗漏扫描原理表达,两者分别制备成杆状病毒,共感染昆虫细胞sf9包装rAAV.增强绿色荧光蛋白(EGFP)作为报告基因验证其可行性.结果 顺式和反式杆状病毒载体分别在DH 10BacTM中转座形成杆粒bacmid、昆虫细胞sf9中制备成杆状病毒,病毒滴度可达1×108~3 ×108 v.g./ml.二杆状病毒共感染昆虫细胞sf9,完成rAAV拯救、复制和包装过程,经rAAV-EGFP感染293T细胞测试具有良好的生物学活性.结论 该系统具有高效、安全、简便等特点,为临床级rAAV制备和基因治疗奠定了基础.%Objective To establish an efficient and safe method for production of recombinant adeno-associated viruses (rAAV) of clinical scale.Methods The 2053 bp of rAAV inverted terminal repeat (ITR) and foreign gene expression cassette was cloned into baculovirus vector as cis-acting vector.The capsid and replication proteins of rAAV serum type 2 were expressed in a trans-acting baculovirus vector (pFastBacDual) by leaky scanning mechanism of eukaryotic gene transcription.Both constructs were made into baculovirus which infected insect cells st9 for packaging rAAV.Enhanced green fluorescent protein (EGFP) was used as reporter gene to verify the feasibility of system.Results Transposition of cis-acting and trans-acting vector into bacmid was carried out in DH10BacTM reswctively.Two baculoviruses were prepared in insect cells sf9 and the titers were about 1×108-3×108 v.g./mL.rAAV were rescued,replicated and packaged by co-infection of two baculoviruses into sf9.The biological activity of rAAV-EGFP was proved by infecting 293T cells.Conclusion This system provides an experimental data for making

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

    Science.gov (United States)

    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.

  6. Potent anti-melanoma effect by combination of mytomycin C with recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO%丝裂霉素C与表达Smac/DIABLO的肿瘤靶向腺相关病毒联用对抗恶性黑色素瘤的效果

    Institute of Scientific and Technical Information of China (English)

    王坚; 林茂; 吴平; 何惠娟; 刘新垣

    2012-01-01

    To investigate whether recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO can improve sensitivity of melanoma to mitomycin C both in vitro and in vivo. Methods Tumor-targeting expressed Smac/DIABLO or green fluorescence protein (EGFP) in recombinant adeno-associated virus vectors (rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP) was constructed. The culture cells were transfected with rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP. The expression of EGFP in culture cells was observed with fluorescence microscope. Smac/DIABLO expression was detected by RT-PCR method. Proliferation of tumor cells was measured by MTT method. Apoptosis of tumor cells at different drug concentrations was examined by flow cytometry. Synergistic anti-tumor activity of mitomycin C combined with rAAV-hTERT/ Smac/DIABLO was measured by MTT in vitro and animal experiment in vivo. Data was evaluated by SPSS statisticssoftware analysis. Results Green fluorescence could be observed in tumor cells but not in normal cells 48 h after rAAV-hTERT/EGFP transfection. Almost all tumor cells displayed bright yellow-green fluorescence after 96 h. The expression of Smac/DIABLO in rAAV-hTERT/Smac/DIABLO transfected tumor cells showed Smac/DIABLO mRNA band 24 h after transfection and stabilized 48 h after transfection. Tumor cell inhibition rate was increased obviously higher in group of combination of mitomycin C with rAAV-hTERT/Smac/DIABLO than mitomycin C alone (P<0.01). Flow cytometry results indicated that mitomycin C combined with rAAV-hTERT/Smac/DIABLO group had the highest apoptosis-induced effect in groups of negative, mitomycin C, and rAAV-hTERT/Smac/ DIABLO (P<0.01). Animal experiment result indicated that tumor growth was inhibited and survival rate was improved significantly in mitomycin C combined with rAAV-hTERT/Smac/DIABLO group compared to rAAV-hTERT/Smac/DIABLO or mitomycin C aione. Conclusion Tumor-targeting rAAV-hTERT/Smac/DIABLO can improve sensitivity of tumor cells

  7. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Prasadan, Krishna; Shiota, Chiyo; Peirish, Lauren; Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Wiersch, John; El-Gohary, Yousef; Husain, Sohail Z; Gittes, George K

    2014-12-01

    Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

  8. Methods of treating Parkinson's disease using viral vectors

    Science.gov (United States)

    Bankiewicz, Krystof; Cunningham, Janet

    2016-11-15

    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.

  9. The Contrast Tranfecting Efficiency of a Double Stranded Recombinant Adeno -associated Virus Expressing Exendin -4 Compared with Single Stranded AAV%重组双链及单链腺相关病毒介导Exendin -4表达效率的比较

    Institute of Scientific and Technical Information of China (English)

    王俊红; 问姣; 董鹏; 张静; 焦杨; 郭永红

    2015-01-01

    To construct a recombinant double -strands adeno -associated virus(dsAAV)expressing Exendin -4,compared trans-fecting efficiency in cells with a single stranded AAV(ssAAV).The recombinant dsAAV vector pSSHG/Exendin -4 was reconstructed which Exendin -4 by gene engineering method.The recombinant virus was packaged by human embryo kidney 293 cells and the virus titer was measured .The expression GFPs of dsAAV and ssAAV in NIH3T3 cell were detected by fluorescence microscope.Exendin -4 level in the superment of NIH3T3 transferred by dsAAV /pSSHG/Exendin -4or ssAAV /pSSHG/Exendin -4 were determined by ELISA.The expression Exendin -4 of dsAAV and ssAAV in NIH3T3 cell were detected by immunohistochemical.Recombinant dsAAV vector was successfμl constructed and confirmed by restriction enzymes and DNA sequencing.The more higher titer of recombi-nant virus was by homologous recombinationin dsAAV (2.5 ×10 11 pfu)than by ssAAV(2.5 ×109 pfu).There was higher fluorescence intensity in transfected NIH3T3 cells by dsAAV.The Exendin -4 level was higer in supernatant of dsAAV than in ssAAV(181.1 ± 8.75 pmol/mlvs133.81 ±8.09 pmol/ml).The Exendin -4 expression was positive both in dsAAV and ssAAV.The Exendin -4 ex-pression system in dsAAV has relatively high transfecting ability.It should play a significant and fundamental role for further researches about diabetes gene therapy.%比较双链腺相关病毒(Double -stranded adeno -associated virus,dsAAV)及单链 AAV(Single -stranded adeno -associated virus,ssAAV)介导 Exendin -4分泌表达效率。应用基因工程方法构建 dsAAV /pSSHG/Exendin -4,与重组 ssAAV 比较转染 NIH3T3细胞效率及转染后细胞上清 Exendin -4浓度,免疫组化检测 Exendin -4表达。经限制性内切酶及测序证实载体构建成功,测定重组 dsAAV pSSHG/Exendin -4滴度为2.5×1011 pfu,较重组 ssAA 滴度高(2.5×109 pfu);dsAAV /pSSHG/GFP 转染 NIH3T3细

  10. 禽腺联病毒Rep78和VP3蛋白的原核表达及抗血清制备%Prokaryotic expression of the Rep78 and VP3 proteins of avian adeno-associated virus and preparation of specific antisera

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2008-01-01

    分别将禽腺联病毒(Avian adeno-associated virus,AAAV)的Rep78基因和VP3基因克隆入pET-47b原核表达载体并转化BL21(DE3)大肠杆菌,在1PTG的诱导下2种目的蛋白均成功得到了表达.SDS-PAGE显示,Rep78蛋白的相对分子质量约为85 000,而VP3蛋白相对分子质量约为60 000.Western-blot分析显示,表达产物均能与抗AAAV的阳性血清反应.将目的蛋白切胶免疫BALB/c小鼠分别制备了针对2种蛋白的多克隆血清.间接免疫荧光试验显示制备的抗血清能够与AAAV抗原特异反应.不与鸡胚致死孤儿病毒(CELO)抗原反应.结果表明,制备的抗Rep78和VP3蛋白的血清可以用于检测重组AAAV载体制备过程中Rep和Cap基因的表达水平.

  11. Enhanced transduction of mouse salivary glands with AAV5-based vectors

    NARCIS (Netherlands)

    H. Katano; M.R. Kok; A.P. Cotrim; S. Yamano; M. Schmidt; S. Afione; B.J. Baum; J.A. Chiorini

    2006-01-01

    We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4

  12. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Science.gov (United States)

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  13. Methods of treating Parkinson's disease using viral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Bankiewicz, Krystof; Cunningham, Janet

    2016-11-15

    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.

  14. Effects of adeno-associated virus-mediated klotho gene delivery on the expression of runx2 and MMP-13 gene in the bone of the ovariectomy rats%腺相关病毒介导的klotho基因表达对去势大鼠骨Runx2及MMP-13表达的影响

    Institute of Scientific and Technical Information of China (English)

    王艳娇; 马厚勋; 李宝善; 吴平

    2012-01-01

    目的 探讨腺相关病毒介导的klotho(KL)基因表达对去势骨质疏松大鼠的调控作用.方法 SD雌性大鼠随机分为假手术组(S组)和手术组,外科去势术后12周再随机分为模型组(O组)、17β-雌二醇组(E组)、KL基因组(KO组)和空质粒组(GO组),实验12周后处死.取股骨、胫骨测骨密度;冰冻切片及免疫组化法观察肾KL荧光及KL蛋白表达;RT-PCR和免疫组化法检测骨Runx2、MMP-13 mRNA及蛋白表达;HE染色观察骨组织形态学变化.结果 KO组和E组骨密度高于O组和GO组(P<0.05);KO组大鼠肾有小鼠KL基因特异性表达;与O组相比,KO组Runx2 mRNA表达明显上调,MMP-13 mRNA表达显著下调(P<0.05);免疫组化分析KO组Runx2吸光度值为411±96,显著高于O组的353±50(P<0.05);KO组MMP-13吸光度值为397±84,显著低于O组的656±89(P<0.05).KO组、E组和S组大鼠骨小梁排列紧密,连接成网,形态结构较完整,明显优于O组和GO组.结论 KL基因表达上调可减缓去势大鼠骨质疏松症的发展及骨组织微结构的破坏,提示KL基因可能在骨质疏松症的发展中扮演重要角色.%Objective To research the effect of the recombinant adeno-associated virus vector containing klotho gene delivery on the regulating of osteoporosis in ovariectomized rats. Methods Female SD rats were randomly divided into sham operation group (S group) and model group. Model was successfully constructed with ovariectomy after 12 weeks,they were randomly divided into model group (0 group), 17(β-estradiol (E group), klotho gene group (K0 group), empty vector group (GO group), all were sacrificed after 12 weeks. Bone mineral density (BMD) of the femurs and tibia were measured. The fluorescent expression of renal klotho was observed by Cryo-sectioning technique. The Runx2 and MMP-13 mRNA expression of bone tissue were detected by reverse transcription-polymerase chain reaction(RT-PCR). Expression of klotho protein in kidney and Runx

  15. Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

    OpenAIRE

    Lewis, Travis B.; Glasgow, Joel N.; Glandon, Anya M.; Curiel, David T.; Standaert, David G.

    2010-01-01

    BACKGROUND: Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector ove...

  16. 重组8型腺相关病毒介导双荧光素酶基因在小鼠体内的表达%Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

    Institute of Scientific and Technical Information of China (English)

    王刚; 尉迟捷; 董小岩; 田文洪; 吴小兵

    2012-01-01

    目的 利用共表达的分泌型荧光素酶Gluc(gaussia princeps luciferase)和非分泌型荧光素酶Fluc(firefly luciferase)研究重组8型腺相关病毒(recombinant adeno-associated virus type 8,rAAV8)介导的转基因在小鼠体内的表达特点.方法 制备携带双荧光素酶基因的重组8型腺相关病毒rAAV8-Gluc/Fluc,体外感染HEK293细胞并检测上清和胞内Gluc和Fluc活性;将不同剂量的rAAV8-Gluc/Fluc尾静脉注射或肌内注射至BALB/c小鼠,通过尾静脉采血检测Gluc活性,通过活体成像和裂解组织检测Fluc活性.结果 成功制备了rAAV8-Gluc/Fluc,可以有效感染HEK293细胞,同时分泌表达Gluc和胞内表达Fluc;尾静脉注射或肌内注射rAAV8-Gluc/Fluc至小鼠后,外周血Gluc活性均在注射后10 ~20 d达到高峰并稳定持续120 d以上,Gluc活性随注射剂量增加而增高;静脉注射rAAV8-Gluc/Fluc时Fluc主要在肝脏表达,在骨骼肌和心肌有少量表达,而肌内注射时Fluc既在肌内注射局部表达同时也在肝脏中表达.结论 本研究成功制备了携带双荧光素酶基因rAAV8-Gluc/Fluc,研究了其介导的转基因在小鼠体内的表达特点,为rAAV8的临床前应用打下基础.%Objective Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).Methods rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro.BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im).Then Gluc activities in blood were measured,the whole-body images for Fluc activities were performed and Fluc activities of tissue lysate were also detected.Results rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells.The Gluc was mainly detected in the culture media while the Fluc was mainly

  17. 腺伴随病毒介导 ADNF-9基因转染对豚鼠卡那霉素致聋的治疗作用%Treatment of adeno-associated virus mediated ADNF-9 gene transfection on guinea pig cochlea deafened by kanamycin

    Institute of Scientific and Technical Information of China (English)

    景阳; 郑国玺; 祝康; 刘晖; 张文

    2015-01-01

    Objective:To observe the effect of rAAV-NT4-ADNF-9 in treating ototoxicity associated with aminoglycoside antibiotics.Methods:The guinea pigs were used,and its auvicle reflex and auditory brain-stem response(ABR)were normal.The ototoxic animal model of guinea pigs were made by the method of hypodemic injection of Kanamycin(400 mg/kg).In animals under general anesthesia,the guinea pigs were cut off the skin of ringt ear sulcus posterior and opened acoustic capsule,then injected the rAAV-NT4-AD-NF-9 10 μL from fossula rotunda.After 14 days,the right acoustic capsule were dislodged.The cochlear were stained with AgNO3 .The cochlear hair cells were observed by contrast phase microscope.Results:Taking the group for rAAV-NT4-ADNF-9 as example,the threshold ABR decreased remarkably after treat-ment(P <0.05).Through the phase contrast microscope the cochlear basilar membrane was complete and the outer hair cells had dot deletion.The thresholds of ABR had no notable differences for the other three groups before and after treatment.Taking comparison between the artificial endolymph control group and empty vector group,through the phase contrast microscope after treatment,hair cells showed a patchy loss in the former group and there was no such phenomenon in the latter group.The average number of hair cells observed in the group of ADNF was significantly higher than that of the artificial endolymph control group and empty vector group(P <0.05).Conclusion:rAAV-NT4-ADNF-9 is able to be transfected into cochle-ar,and to express secretory NT4-ADNF-9 peptide,which preventing hair cells from impairment.%目的:观察包装重组的腺伴随病毒(adeno-associated virus,AAV)rAAV-NT4-ADNF-9对卡拉霉素致聋豚鼠的治疗作用。方法:选用耳廓反射和听性脑干反应(auditory brainstem response,ABR)正常的白色红目豚鼠,在大腿内侧肌肉注射卡那霉素400 mg/(kg·d),连续注射6 d,制备耳聋模型。在全身麻醉的条件

  18. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    Science.gov (United States)

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

    2016-05-01

    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 intraosseous 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 body

  19. A Targeted Mulifunctional Platform for Imaging and Treatment of Breast Cancer and Its Metastases Based on Adenoviral Vectors and Magnetic Nanoparticles

    Science.gov (United States)

    2008-02-01

    enhanced safety profile. Gene Therapy 12, 579-587 Dong Y, Wen P, Manome Y, Parr M, Hirshowitz A, Chen L, Hirschowitz EA, Crystal R, Weichselbaum R...904 Halbert CL, Rutledge EA, Allen JM, Russell DW, Miller AD (2000) Repeat transduction in the mouse lung by using adeno-associated virus vectors...Haralambieva I, Iankov I, Hasegawa K, Harvey M, Russell SJ, Peng KW (2007) Engineering oncolytic measles virus to circumvent the intracellular innate

  20. Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.

    Science.gov (United States)

    Monahan, Paul E; Lothrop, Clinton D; Sun, Junjiang; Hirsch, Matthew L; Kafri, Tal; Kantor, Boris; Sarkar, Rita; Tillson, D Michael; Elia, Joseph R; Samulski, R Jude

    2010-11-01

    Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

  1. 重组腺相关病毒转导人树突状细胞体外诱导抗肝癌免疫应答%Generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells with adeno-associated virus expressing α-fetoprotein

    Institute of Scientific and Technical Information of China (English)

    杜文贞; 于天霞

    2011-01-01

    Objective To investigate the generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells (DC)with recombinant adeno-associated virus expressing α-fetoprotein (rAAV-AFP). Methods Peripheral blood mononuclear cells were isolated from healthy volunteers. Adherent peripheral blood mononuclear cells were transduced with AAV-AFP and cultured in the presence of granulocyte macrophage colony stimulating factor and interleukin-4 to generate dendritic cells.MTS assay was used to measure the ability of DC transduced with AAV-AFP ( AAV-AFP + DC) to stimulate the proliferation of T cell. The phenotype and AFP protein expression of DC and the secretion of IFN (interferon)-γ and IL (interleukin)-4 by T cells were detected by flow cytometry. The killing efficacy of cytotoxic T lymphocytes (CTL) activated by AAV-AFP + DC against AFP positive hepatocellular carcinoma cell lines was detected by lactate dehydrogenase (LDH) release assay. Results AAV-AFP + DC expressed HLA Ⅰ (97. 12%), HLAⅡ (97.32%), CD80(38.94%), CD83(60.84%)and CD86(98. 14%). AFP was secreted by 81.2% of AAV-AFP + DC. And it could stimulate effectively the proliferation of T cell.19. 84% of CD4 + T cells and 18.65% of CD8 + T cells activated by AAV-AFP + DC produced IFN-γbut not IL-4 and showed distinct killing activities against AFP positive hepatocellular carcinoma cell lines HepG2 (56. 45% ) and BEL7402 (78. 84% ). Conclusion AAV-AFP + DC can elicit distinct antitumor responses against AFP positive hepatocellular carcinoma cell lines so as to provide a basis for further researches on the clinical application of AAV-AFP + DC in the treatment of hepatocellular carcinoma.%目的 探讨携带甲胎蛋白基因的重组腺相关病毒(rAAV-AFP)转导人树突状细胞(DC)体外诱导抗肝癌免疫应答.方法 分离健康志愿者外周血单核细胞,贴壁细胞转导rAAV-AFP后,在粒细胞巨噬细胞集落刺激因子(GMCSF)和白细胞介素4(IL-4)的联

  2. 重组腺相关病毒神经肽Y基因转染对癫(癎)大鼠海马病理变化的影响%Effect of recombinant adeno-associated virus-mediated human-derived neuropeptide Y gene transfection on pathological change of the hippocampus in epileptic rat

    Institute of Scientific and Technical Information of China (English)

    董长征; 董秀芳; 李文玲; 岳向勇; 郭韬; 梁传栋; 赵文清

    2012-01-01

    目的 观察重组腺相关病毒介导人源性神经肽Y(rAA V-hNPY-EGFP)基因转染对癫(癎)大鼠海马病理变化的影响.方法 28只Wistar大鼠随机分为点燃组(n=20)和正常对照组(n=8).正常对照组不进行特殊处理,点燃组以大鼠海马内多次注射红藻氨酸(KA)建立慢性癫(癎)模型,造模成功16只,其随机分为模型组和神经肽Y(NPY)治疗组,每组各8只大鼠.NPY治疗组大鼠转染rAA V2/I- hNPY-EGFP基因,模型组未转染.转染4周后,每组取6只大鼠海马行苏木精-伊红染色,2只行电镜观察.结果 苏木精-伊红染色显示:正常对照组大鼠海马CA3区神经元形态正常;模型组海马CA3区神经元丢失,胶质细胞增生;NPY治疗组基因转染后神经元丢失减少.模型组神经元数目为(10.67±7.87)个/视野,正常对照组为(81.42±5.63)个/视野,明显多于模型组(P<0.05);而NPY治疗组神经元数目为(65.73±2.81)个/视野,明显多于模型组(P<0.05).电镜显示:正常对照组神经元结构正常;模型组神经元固缩,线粒体肿胀;NPY治疗组神经元线粒体结构完整.结论 rAA V-hNPY-EGFP基因转染可减轻大鼠癫(癎)发作引起的病理改变,发挥抑制癫(癎)的作用.%Objective To investigate the effect of recombinant adeno-associated virus-mediated human-derived neuropeptide Y gene (rAAV-hNPY-EGFP gene) transfection on pathological change of hippocampus in epileptic rat. Methods A total of 28 Wistar rats were randomly divided into kindling group (n=20) and normal control group (n=8). No special treatment was performed on rats in normal control group. The chronic epileptic models were successfully established in 16 rats by repeated injection of kainic acid into the hippocampi of rats in kindling group which were equally subdivided into two groups: model group and neuropeptide Y (NPY) treatment group. The rats were transfected with rAAV2/1-hNPV-EGFP gene in NPY treatment group and no transfection was made in model

  3. 重组腺相关病毒介导的MDA-7基因对人肝癌细胞的选择性凋亡诱导效应%Selective apoptosis-inducing effect of adeno-associated virus mediated gene transfer of MDA-7 on human hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    徐波; 朱光辉; 夏金堂; 翁杰锋; 李书华; 李雯

    2008-01-01

    目的 探讨PEG启动子调控腺相关病人毒介导的黑色素瘤分化相关基因MDA-7对肝癌细胞的选择性凋亡诱导效应.方法 以重组腺相关病人毒rAAV-PEG-MDA-7表达系统感染人肝癌细胞株HepG2细胞和正常人肝细胞株LO2细胞,Westerm Blot检测转染细胞内MDA-7蛋白,MTT法检测细胞增殖抑制率,流式细胞术分析细胞周期、Annexin-Ⅴ、线粒体跨膜电位(△Ψm),RT-PCR检测bcl-2基因mRNA.结果 重组腺相关病毒rAAV-PEG-MDA7可特异性转染HepG2细胞,MDA7蛋白在HepG2细胞中高效表达,并呈时间依赖性;重组腺相关病毒rAAV-PEC-MDA-7可抑制HepG2细胞增殖并诱导其凋亡,细胞周期分析处于G0/G1期细胞百分比明显增多,处于G2/M期的细胞减少,并且可见到较明显的凋亡峰的形成,从24 h开始Annexin-Ⅴ阳性细胞比例增多,△Ψm降低,抗凋亡基因bcl-2 mRNA表达降低.而重组腺相关病毒rAAV-PEG-MDA-7对LO2细胞无类似效应.结论 构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统可选择性抑制肝癌细胞增殖和诱导其凋亡,其诱导凋亡机制受到bcl-2家族经线粒体途径的调节.%Objective To investigate the selective apoptosis-inducing effect of adeno-associated virus mediated gene transfer of MDA-7 regulated by PEG promoter on human hepatocellular carcinoma cells.Methods We transduced human hepatocellular carcinoma cell line HepG2 and normal human hepatocytes LO2 with rAAV-PEG-MDA-7 and assessed the cell growth inhibition,cell cycle and apoptosis.MDA-7 protein was detected by Western blotting,cell growth-inhibiting rate evaluated by MTT and cell cycle, Annexin-Ⅴ labeling and mitochondrial transmembrane potential were determined by flow eytometry.The expression of bcl-2 mRNA was determined bv RT-PCR.Results rAAV-PEG-MDA-7 was effectively transfected into HepG2 cells.MDA-7 protein was highly expressed in HepG2 cells in a time-dependent manner.rAAV-PEG-MDA-7 suppressed HepG2 cell growth.The cells

  4. Insect Vectors of Rice Yellow Mottle Virus

    Directory of Open Access Journals (Sweden)

    Augustin Koudamiloro

    2015-01-01

    Full Text Available Rice yellow mottle virus (RYMV is the major viral constraint to rice production in Africa. RYMV was first identified in 1966 in Kenya and then later in most African countries where rice is grown. Several studies have been conducted so far on its evolution, pathogenicity, resistance genes, and especially its dissemination by insects. Many of these studies showed that, among RYMV vectors, insects especially leaf-feeders found in rice fields are the major source of virus transmission. Many studies have shown that the virus is vectored by several insect species in a process of a first ingestion of leaf material and subsequent transmission in following feedings. About forty insect species were identified as vectors of RYMV since 1970 up to now. They were essentially the beetles, grasshoppers, and the leafhoppers. For this review, we presented the chronology of their identification. Also, the biology, ecology, host range, distribution, and caused damage of these insects were briefly summarized.

  5. Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virus and evaluation of its safety

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Haoming; ZOU Beiyan; WU Zhijian; WU Xiaobing; LU Daru; XUE Jinglun

    2003-01-01

    The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which was regulated by CMV promoter was constructed. Large quantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by an intramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸ expression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸ expression curve. The QC-PCR method is easier and more accurate than traditional dothybridization for determination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing in produced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.

  6. Plant Virus Expression Vector Development: New Perspectives

    Directory of Open Access Journals (Sweden)

    Kathleen Hefferon

    2014-01-01

    Full Text Available Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants.

  7. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  8. Chikungunya virus and its mosquito vectors.

    Science.gov (United States)

    Higgs, Stephen; Vanlandingham, Dana

    2015-04-01

    Chikungunya virus (CHIKV), a mosquito-borne alphavirus of increasing public health significance, has caused large epidemics in Africa and the Indian Ocean basin; now it is spreading throughout the Americas. The primary vectors of CHIKV are Aedes (Ae.) aegypti and, after the introduction of a mutation in the E1 envelope protein gene, the highly anthropophilic and geographically widespread Ae. albopictus mosquito. We review here research efforts to characterize the viral genetic basis of mosquito-vector interactions, the use of RNA interference and other strategies for the control of CHIKV in mosquitoes, and the potentiation of CHIKV infection by mosquito saliva. Over the past decade, CHIKV has emerged on a truly global scale. Since 2013, CHIKV transmission has been reported throughout the Caribbean region, in North America, and in Central and South American countries, including Brazil, Columbia, Costa Rica, El Salvador, French Guiana, Guatemala, Guyana, Nicaragua, Panama, Suriname, and Venezuela. Closing the gaps in our knowledge of driving factors behind the rapid geographic expansion of CHIKV should be considered a research priority. The abundance of multiple primate species in many of these countries, together with species of mosquito that have never been exposed to CHIKV, may provide opportunities for this highly adaptable virus to establish sylvatic cycles that to date have not been seen outside of Africa. The short-term and long-term ecological consequences of such transmission cycles, including the impact on wildlife and people living in these areas, are completely unknown.

  9. Viral vectors for gene transfer: current status of gene therapeutics.

    Science.gov (United States)

    Heilbronn, Regine; Weger, Stefan

    2010-01-01

    Gene therapy for the correction of inherited or acquired disease has gained increasing importance in recent years. Successful treatment of children suffering from severe combined immunodeficiency (SCID) was achieved using retrovirus vectors for gene transfer. Encouraging improvements of vision were reported in a genetic eye disorder (LCA) leading to early childhood blindness. Adeno-associated virus (AAV) vectors were used for gene transfer in these trials. This chapter gives an overview of the design and delivery of viral vectors for the transport of a therapeutic gene into a target cell or tissue. The construction and production of retrovirus, lentivirus, and AAV vectors are covered. The focus is on production methods suitable for biopharmaceutical upscaling and for downstream processing. Quality control measures and biological safety considerations for the use of vectors in clinical trials are discussed.

  10. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    Science.gov (United States)

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

  11. Data-driven identification of potential Zika virus vectors

    Science.gov (United States)

    Evans, Michelle V; Dallas, Tad A; Han, Barbara A; Murdock, Courtney C; Drake, John M

    2017-01-01

    Zika is an emerging virus whose rapid spread is of great public health concern. Knowledge about transmission remains incomplete, especially concerning potential transmission in geographic areas in which it has not yet been introduced. To identify unknown vectors of Zika, we developed a data-driven model linking vector species and the Zika virus via vector-virus trait combinations that confer a propensity toward associations in an ecological network connecting flaviviruses and their mosquito vectors. Our model predicts that thirty-five species may be able to transmit the virus, seven of which are found in the continental United States, including Culex quinquefasciatus and Cx. pipiens. We suggest that empirical studies prioritize these species to confirm predictions of vector competence, enabling the correct identification of populations at risk for transmission within the United States. DOI: http://dx.doi.org/10.7554/eLife.22053.001 PMID:28244371

  12. Insect vector-mediated transmission of plant viruses.

    Science.gov (United States)

    Whitfield, Anna E; Falk, Bryce W; Rotenberg, Dorith

    2015-05-01

    The majority of plant-infecting viruses are transmitted to their host plants by vectors. The interactions between viruses and vector vary in duration and specificity but some common themes in vector transmission have emerged: 1) plant viruses encode structural proteins on the surface of the virion that are essential for transmission, and in some cases additional non-structural helper proteins that act to bridge the virion to the vector binding site; 2) viruses bind to specific sites in or on vectors and are retained there until they are transmitted to their plant hosts; and 3) viral determinants of vector transmission are promising candidates for translational research aimed at disrupting transmission or decreasing vector populations. In this review, we focus on well-characterized insect vector-transmitted viruses in the following genera: Caulimovirus, Crinivirus, Luteovirus, Geminiviridae, Reovirus, Tospovirus, and Tenuivirus. New discoveries regarding these genera have increased our understanding of the basic mechanisms of virus transmission by arthropods, which in turn have enabled the development of innovative strategies for breaking the transmission cycle.

  13. 腺相关病毒介导转化生长因子β1和血管内皮生长因子联合转染促进糖尿病溃疡愈合的生物学效应%Biological effects of co-transfection of transforming growth factor beta 1 and vascular endothelial growth factor mediated by adeno-associated virus on promoting the dermal ulcer healing in diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    赛佳明; 张慧琴

    2006-01-01

    使溃疡组织中毛细血管密度明显增多,愈合组织中Ⅰ型和Ⅲ型胶原构成比中Ⅰ型胶原的比例明显提高,并有效地促进溃疡愈合.%BACKGROUND: The ulcer wound is hard to heal in diabetic patients,and it is believed to be caused by the microcirculatory disorder of wound and decreased contents of endogenous growth factors in patients with diabetes mellitus.OBJECTIVE: To observe the biological effects of adeno-associated virus (AAV) mediated transforming growth factor beta1 (AAV-TGFβ1) and vascular endothelial growth factor (AAV-VEGF) in promoting the dermal ulcer healing of diabetic rabbits.DESIGN: A randomized controlled animal experiment.SETTINGS: Medical College, Qingdao University; Affiliated Hospital of Medical College, Qingdao University.MATERIALS: The experiments were carried out in the gynecological laboratory, Affiliated Hospital of Medical College, Qingdao University from July 2004 to January 2006. Twenty-four healthy adult New Zealand rabbits were randomly divided into co-transfection group (n=12) and control group (n=12).METHODS: ① The dermal ulcer models of diabetic rabbits was established by injecting alloxan (130 mg/kg) via ear vein, and the ulcer wound was made by operation. ② In the co-transfection group, the wound was locally infiltrated, and injected with AAV-TGFβ1 virus and AAV-VEGF virus (the concentration was 9×106 virus granules/mL respectively). The rabbits in the control group were treated with injection of saline.MAIN OUTCOME MEASURES: ① The levels of TGFβ1 and VEGF gene transcription in the healing tissue were detected with polymerase chain reaction (PCR) at 1 month postoperatively. ② The capillary density in the wound margin was counted with microcirculation microscope at 3 weeks postoperatively. ③ The collagen Ⅰ and Ⅲ were isolated and detected with Western blotting by protein gel electrophoresis and semi-dry electrophoretic transfer. ④ The content of collagen in the ulcer healing issue

  14. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    Science.gov (United States)

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  15. Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States

    Science.gov (United States)

    Hart, Charles E.; Roundy, Christopher M.; Azar, Sasha R.; Huang, Jing H.; Yun, Ruimei; Reynolds, Erin; Leal, Grace; Nava, Martin R.; Vela, Jeremy; Stark, Pamela M.; Debboun, Mustapha; Rossi, Shannan; Vasilakis, Nikos

    2017-01-01

    Zika virus has recently spread throughout the Americas. Although Aedes aegypti mosquitoes are considered the primary vector, Culex quinquefasciatus and mosquitoes of other species may also be vectors. We tested Cx. quinquefasciatus and Ae. taeniorhynchus mosquitoes from the US Gulf Coast; both were refractory to infection and incapable of transmission. PMID:28005002

  16. Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States.

    Science.gov (United States)

    Hart, Charles E; Roundy, Christopher M; Azar, Sasha R; Huang, Jing H; Yun, Ruimei; Reynolds, Erin; Leal, Grace; Nava, Martin R; Vela, Jeremy; Stark, Pamela M; Debboun, Mustapha; Rossi, Shannan; Vasilakis, Nikos; Thangamani, Saravanan; Weaver, Scott C

    2017-03-01

    Zika virus has recently spread throughout the Americas. Although Aedes aegypti mosquitoes are considered the primary vector, Culex quinquefasciatus and mosquitoes of other species may also be vectors. We tested Cx. quinquefasciatus and Ae. taeniorhynchus mosquitoes from the US Gulf Coast; both were refractory to infection and incapable of transmission.

  17. Potential role of ticks as vectors of bluetongue virus

    NARCIS (Netherlands)

    Bouwknegt, C.; Rijn, van P.A.; Schipper, J.M.J.; Holzel, D.R.; Boonstra, J.; Nijhof, A.; Rooij, van E.M.A.; Jongejan, F.

    2010-01-01

    When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not

  18. Herpes simplex virus type 1-derived recombinant and amplicon vectors.

    Science.gov (United States)

    Fraefel, Cornel; Marconi, Peggy; Epstein, Alberto L

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.

  19. Large Animal Models for Foamy Virus Vector Gene Therapy

    Directory of Open Access Journals (Sweden)

    Peter A. Horn

    2012-12-01

    Full Text Available Foamy virus (FV vectors have shown great promise for hematopoietic stem cell (HSC gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit.

  20. Addressing immune tolerance issues in inflammatory bowel disease and adeno-associated virus based gene transfer

    NARCIS (Netherlands)

    Majowicz, Anna

    2014-01-01

    This thesis is focusing on cell-mediated induction of immune tolerance and consists of two parts. The studies described in Part I report the development of strategies for possible treatment of Inflammatory Bowel Diseases (IBD). Induction of immune tolerance, in IBD mouse model, with the use of regul

  1. Potential North American vectors of West Nile virus.

    Science.gov (United States)

    Turell, M J; Sardelis, M R; Dohm, D J; O'Guinn, M L

    2001-12-01

    The outbreak of disease in the New York area in 1999 due to West Nile (WN) virus was the first evidence of the occurrence of this virus in the Americas. To determine potential vectors, more than 15 mosquito species (including Culex pipiens, Cx. nigripalpus, Cx. quinquefasciatus, Cx. salinarius, Aedes albopictus, Ae. vexans, Ochlerotatus japonicus, Oc. sollicitans, Oc. taeniorhynchus, and Oc. triseriatus) from the eastern United States were evaluated for their ability to serve as vectors for the virus isolated from birds collected during the 1999 outbreak in New York. Mosquitoes were allowed to feed on one- to four-day old chickens that had been inoculated with WN virus 1-3 days previously. The mosquitoes were incubated for 12-15 days at 26 degrees C and then allowed to refeed on susceptible chickens and assayed to determine transmission and infection rates. Several container-breeding species (e.g., Ae. albopictus, Oc. atropalpus, and Oc. japonicus) were highly efficient laboratory vectors of WN virus. The Culex species were intermediate in their susceptibility. However, if a disseminated infection developed, all species were able to transmit WN virus by bite. Factors such as population density, feeding preference, longevity, and season of activity also need to be considered in determining the role these species could play in the transmission of WN virus.

  2. Vector competence of Anopheles and Culex mosquitoes for Zika virus

    Science.gov (United States)

    Dodson, Brittany L.

    2017-01-01

    Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. Although Aedes mosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, including Culex and Anopheles species, and we lack a thorough understanding of which mosquito species to target for vector control. We exposed Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint, Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes were refractory to Zika virus infection. We conclude that Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus. PMID:28316896

  3. Vector competence of Anopheles and Culex mosquitoes for Zika virus

    Directory of Open Access Journals (Sweden)

    Brittany L. Dodson

    2017-03-01

    Full Text Available Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. Although Aedes mosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, including Culex and Anopheles species, and we lack a thorough understanding of which mosquito species to target for vector control. We exposed Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint, Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes were refractory to Zika virus infection. We conclude that Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus.

  4. Vector competence of Anopheles and Culex mosquitoes for Zika virus.

    Science.gov (United States)

    Dodson, Brittany L; Rasgon, Jason L

    2017-01-01

    Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. Although Aedes mosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, including Culex and Anopheles species, and we lack a thorough understanding of which mosquito species to target for vector control. We exposed Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint, Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes were refractory to Zika virus infection. We conclude that Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus.

  5. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    OpenAIRE

    Sang-Ho Park; Hoseong Choi; Semin Kim; Won Kyong Cho; Kook-Hyung Kim

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the n...

  6. [Kunjin virus replicon--a novel viral vector].

    Science.gov (United States)

    Li, Shihua; Li, Xiaofeng; Qin, E'de; Qin, Chengfeng

    2011-02-01

    Viral replicon is a kind of self-replicating viral RNA sourced from viral genome, which contains viral non-structural genes that are critical for viral genome replication with structural proteins deleted or replaced by foreign genes. Kunjin virus is a member of the Flavivirida family, Flavivirus genus, and Kunjin virus replicon is the first and the clearly defined flavivirus replicon. Kunjun virus replicon has been regarded as an excellent viral vector on account of its high expression, lower cytotoxicity and genetic stability. These unique characteristics of kunjin virus replicons make them suitable for the study of viral genome replication, recombinant proteins production, vaccine development and gene therapy. In this article, recent progress in the development, properties and applications of kunjin virus replicon system was briefly reviewed.

  7. 'ANOTHER VECTOR BORNE CHALLENGE TO COMBAT- ZIKA VIRUS OUTBREAKS'.

    Science.gov (United States)

    Shoaib, Maria; Faraz, Ahmad; Ahmed, Syed Ahsanuddin

    2016-01-01

    Zika virus is a single-stranded RNA virus of the Flaviviridae family. It is known to transmit to humans primarily through the bite of an infected Aedes species mosquito which is also known to carry dengue, chikungunya & yellow fever virus. Transmission is anthroponotic (human-to-vector-to-human) during outbreaks, Perinatally in utero, sexually and via infected blood transfusion. It is mild and self-limiting infection lasting for several days to a week. However, it is suspected as a cause of Guillain Barre Syndrome. There is a teratogenic association of Zika virus causing congenital birth defects like microcephaly and neurologic abnormalities. Treatment is generally supportive and for symptomatic relief. No specific antiviral treatment or vaccine is yet available for Zika virus disease. It highlights importance of preventive public health measures at the community level and avoids travelling to the endemic areas.

  8. Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus.

    Science.gov (United States)

    Guo, Xiao-Xia; Li, Chun-Xiao; Deng, Yong-Qiang; Xing, Dan; Liu, Qin-Mei; Wu, Qun; Sun, Ai-Juan; Dong, Yan-de; Cao, Wu-Chun; Qin, Cheng-Feng; Zhao, Tong-Yan

    2016-09-07

    Zika virus (ZIKV) has become a threat to global health since the outbreak in Brazil in 2015. Although ZIKV is generally considered an Aedes-transmitted pathogen, new evidence has shown that parts of the virus closely resemble Culex-transmitted viruses. Therefore, it is important to evaluate the competence of Culex species for ZIKV to understand their potential as vectors. In this study, female Culex pipiens quinquefasciatus were orally exposed to ZIKV. Mosquito midguts, salivary glands and ovaries were tested for ZIKV to measure infection and dissemination at 2, 4, 6, 8, 12, 16 and 18 days post exposure (pe). In addition, saliva was collected from mosquitoes after infection and infant mice were bitten by infected mosquitoes to measure the transmission ability of Cx. p. quinquefasciatus. The results showed that the peak time of virus appearance in the salivary glands was day 8 pe, with 90% infection rate and an estimated virus titer of 3.92±0.49 lg RNA copies/mL. Eight of the nine infant mice had positive brains after being bitten by infected mosquitoes, which meant that Cx. p. quinquefasciatus could be infected with and transmit ZIKV following oral infection. These laboratory results clearly demonstrate the potential role of Cx. p. quinquefasciatus as a vector of ZIKV in China. Because there are quite different vector management strategies required to control Aedes (Stegomyia) species and Cx. p. quinquefasciatus, an integrated approach may be required should a Zika epidemic occur.

  9. Vector-mediated cancer gene therapy: an overview.

    Science.gov (United States)

    Seth, Prem

    2005-05-01

    In recent years there has been a dramatic increase in developing gene therapy approaches for the treatment of cancer. The two events that have permitted the formulation of concept of cancer gene therapy are the new understanding of the molecular mechanisms underlying oncogenesis, and the development of the DNA-delivery vehicles or vectors. Many approaches to cancer gene therapy have been proposed, and several viral and non-viral vectors have been utilized. The purpose of this review article is to describe the various strategies of cancer gene therapy (transfer of tumor suppressor genes, suicide genes-enzyme/pro-drug approach, inhibition of dominant oncogenes, immunomodulation approaches, expression of molecules that affect angiogenesis, tumor invasion and metastasis, chemosensitization and radiosensitization approaches, and chemoprotection of stem cells). The chapter also reviews the commonly used vectors (retroviral vectors, adenoviral vectors, adeno-associated viral vectors, pox viruses, herpes simplex viruses, HIV- vectors, non-viral vectors and targetable vectors) for cancer gene therapy. Some of the important issues in cancer gene therapy, and the potential future directions are also being discussed.

  10. Preference by a virus vector for infected plants is reversed after virus acquisition.

    Science.gov (United States)

    Rajabaskar, Dheivasigamani; Bosque-Pérez, Nilsa A; Eigenbrode, Sanford D

    2014-06-24

    Pathogens and their vectors can interact either directly or indirectly via their shared hosts, with implications for the persistence and spread of the pathogen in host populations. For example, some plant viruses induce changes in host plants that cause the aphids that carry these viruses to settle preferentially on infected plants. Furthermore, relative preference by the vector for infected plants can change to a preference for noninfected plants after virus acquisition by the vector, as has recently been demonstrated in the wheat-Rhopalosiphum padi-Barley yellow dwarf virus pathosystem. Here we document a similar dynamic in the potato-Myzus persicae (Sulzer)-Potato leaf roll virus (PLRV) pathosystem. Specifically, in a dual choice bioassay, nonviruliferous apterous M. persicae settled preferentially on or near potato plants infected with PLRV relative to noninfected (sham-inoculated) control plants, whereas viruliferous M. persicae (carrying PLRV) preferentially settled on or near sham-inoculated potato plants relative to infected plants. The change in preference after virus acquisition also occurred in response to trapped headspace volatiles, and to synthetic mimics of headspace volatile blends from PLRV-infected and sham-inoculated potato plants. The change in preference we document should promote virus spread by increasing rates of virus acquisition and transmission by the vector.

  11. Can Viruses be Modified to Achieve Sustained Gene Transfer?

    Directory of Open Access Journals (Sweden)

    Hildegund CJ Ertl

    2011-07-01

    Full Text Available It is very easy to replace a faulty gene in an immunocompromised mouse. First, one takes a well-characterized virus, such as an adenovirus or an adeno-associated virus, and incorporates the correct version of the faulty gene together with some regulatory sequences into the genome. Then, one transduces the recombinant genome into helper cells, which will add the viral capsid. At last, one injects the resulting viral vector into the sick mouse, and the mouse is cured. It is not that easy in an immunocompetent mouse, let alone in a human, as over the eons the immune system evolved to eliminate viruses regardless if they penetrate as dangerous pathogens or are injected by a well-meaning gene therapist. Here we offer our perspective on the potential of how viral vectors achieve sustained gene transfer in the face of a hostile immune system.

  12. Vectors of Crimean Congo Hemorrhagic Fever Virus in Iran

    Directory of Open Access Journals (Sweden)

    Zakkyeh Telmadarraiy

    2015-10-01

    Full Text Available Background: Ticks are important vectors and reservoirs of Crimean Congo Hemorrhagic Fever (CCHF virus. Human beings may be infected whenever the normal life cycle of the infected ticks on non- human vertebrate hosts is interrupted by the undesirable presence of humans in the cycle. A total of 26 species of Argasid and Ixodid ticks have been recorded in Iran; including nine Hyalomma, two Rhipicephalus, two Dermacentor, five Haemaphysalis, two Boophilus, one Ixodes and two Argas as well as three Ornithodoros species as blood sucking ectoparasites of livestock and poultries. The present paper reviews tick vectors of CCHF virus in Iran, focusing on the role of ticks in different provinces of Iran using reverse transcription polymerase chain reaction (RT-PCR assay.Methods: During ten years study, 1054 tick specimens; including two species of Argasidae and 17 species of Ixodidae were examined for their infection to CCHF virus genome. The output of all studies as well as related publications were discussed in the current paper.Results: The results show that Rhipicephalus sanguineus, Hyalomma marginatum, H. anatolicum, H. asiaticum and H. dromedarii were known as the most frequent species which were positive for CCHF virus.Conclusion: The status of ticks which were positive for CCHF virus revealed that unlike the most common idea that Hyalomma species are the most important vectors of CCHF virus, other ticks including Rhipicephalus,Haemaphysalis and Dermacentor can be reservoir of this virus; thus, considering geographical distribution, type of host and environmental conditions, different tick control measurements should be carried out in areas with high incidence of CCHF disease.

  13. Modification of Non-Vector Aphid Feeding Behavior on Virus-Infected Host Plant

    OpenAIRE

    Hu, Zuqing; Zhao, Huiyan; Thieme, Thomas

    2013-01-01

    Virus-infected host plants can have positive, neutral or negative effects on vector aphids. Even though the proportion of non-vector aphids associated with a plant far exceeds that of vector species, little is known about the effect of virus-infected plants on non-vector aphids. In the present study, the English grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphididae), a non-vector of Wheat dwarf virus (WDV) and Cereal yellow dwarf virus-RPV (CYDV-RPV), was monitored on, virus-infected,...

  14. Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1

    Directory of Open Access Journals (Sweden)

    Shuo Li

    2015-03-01

    Full Text Available The small brown planthopper (SBPH is the main vector for rice stripe virus (RSV, which causes serious rice stripe disease in East Asia. To characterize the virus-vector interactions, the SBPH cDNA library was screened with RSV ribonucleoprotein (RNP as bait using a GAL4-based yeast two-hybrid system. The interaction between RSV-RNP and the Himetobi P virus (HiPV, an insect picorna-like virus VP1 protein was identified. The relationships between HiPV and RSV in SBPH were further investigated, and the results showed that the titer of RSV was commonly higher in single insect that exhibited more VP1 expression. After the VP1 gene was repressed by RNA silencing, the accumulation of RSV decreased significantly in the insect, whereas the virus acquisition ability of SBPH was unaffected, which suggests that HiPV VP1 potentially facilitates the accumulation of RSV in SBPH.

  15. [Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus].

    Science.gov (United States)

    Li, Taiming; Pan, Junjie; Qi, Jing; Zhang, Chun

    2015-08-01

    AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.

  16. Simian virus 40 vectors for pulmonary gene therapy

    Directory of Open Access Journals (Sweden)

    Oppenheim Ariella

    2007-10-01

    Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

  17. Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors.

    Science.gov (United States)

    Trobridge, Grant; Russell, David W

    2004-03-01

    Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

  18. Gene therapy for haemophilia...yes, but...with non-viral vectors?

    Science.gov (United States)

    Liras, A; Olmedillas, S

    2009-05-01

    High-purity plasma-derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid-term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half-life. In the long-term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno-associated viruses and non-viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno-associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non-viral vectors. There have been in recent years relevant advances in non-viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the

  19. Status and prospects of plant virus control through interference with vector transmission

    NARCIS (Netherlands)

    Bragard, C.; Caciagli, P.; Lemaire, O.; Lopez-Moya, J.J.; MacFarlane, S.; Peters, D.; Susi, P.; Torrance, L.

    2013-01-01

    Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus–vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, pla

  20. [Effects of plant viruses on vector and non-vector herbivorous arthropods and their natural enemies: a mini review].

    Science.gov (United States)

    He, Xiao-Chan; Xu, Hong-Xing; Zhou, Xiao-Jun; Zheng, Xu-Song; Sun, Yu-Jian; Yang, Ya-Jun; Tian, Jun-Ce; Lü, Zhong-Xian

    2014-05-01

    Plant viruses transmitted by arthropods, as an important biotic factor, may not only directly affect the yield and quality of host plants, and development, physiological characteristics and ecological performances of their vector arthropods, but also directly or indirectly affect the non-vector herbivorous arthropods and their natural enemies in the same ecosystem, thereby causing influences to the whole agro-ecosystem. This paper reviewed the progress on the effects of plant viruses on herbivorous arthropods, including vector and non-vector, and their natural enemies, and on their ecological mechanisms to provide a reference for optimizing the management of vector and non-vector arthropod populations and sustainable control of plant viruses in agro-ecosystem.

  1. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    Directory of Open Access Journals (Sweden)

    Sang-Ho Park

    2016-08-01

    Full Text Available Grapevine Algerian latent virus (GALV is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP, but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  2. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    Science.gov (United States)

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  3. Semliki Forest Virus: a viral vector with multiple applications.

    Directory of Open Access Journals (Sweden)

    Luis Felipe Henao

    2009-11-01

    Full Text Available Se han utilizado los alfavirus como vectores de expresión, entre estos se encuentra el Semliki Forest virus (SFV, que es un virus envuelto, el cual, además de replicarse en el citoplasma, tiene la propiedad de expresar por separado las proteínas estructurales de las no estructurales, permitiendo un mayor control de la expresión. Los vectores derivados del SFV pueden tener una gama amplia de aplicaciones. Se pueden obtener altos títulos virales para la expresión eficiente de proteínas en diferentes líneas celulares. Pueden infectar un espectro amplio de células de mamíferos, así como de tejidos. Son prometedores para ser usados en la terapia génica como vehículos para el envío de genes específicos in vivo o in vitro, tanto en la terapia contra el cáncer como en la neuronal, especialmente cuando sólo sea necesaria una expresión a corto plazo. Sus aplicaciones en la producción de vacunas profilácticas o terapéuticas, es otro aspecto estudiado; se ha demostrado la generación de respuestas inmunes importantes contra diferentes enfermedades virales y tumorales. El desarrollo de nuevos vectores no citopáticos, de otros regulados por temperatura, así como también de otros con replicación persistente; permitirán la prolongación de la expresión. Debido a estas nuevas ventajas y a las ya conocidas, gradualmente se podrían ampliar los usos para los vectores derivados del SFV a medida que se controlen sus efectos no deseados.

  4. Vector dynamics and transmission of dengue virus: implications for dengue surveillance and prevention strategies: vector dynamics and dengue prevention.

    Science.gov (United States)

    Scott, Thomas W; Morrison, Amy C

    2010-01-01

    Accounting for variation in mosquito vector populations will improve dengue surveillance and prevention. Because Aedes aegypti, the principle dengue virus (DENV) vector, transmit the virus with remarkable efficiency, entomological thresholds are especially low. Assessing risk of human infection based on immature mosquito indices has proven difficult. Greater emphasis should be placed on relative abundance of adult vectors in relation to human serotype-specific herd immunity, introduction of unique viruses, mosquito-human contact and weather. The most appropriate spatial scale for assessing entomological risk is the individual household. The scale for measuring DENV transmission risk has yet to be determined but is clearly larger than the household and likely to exceed several city blocks. Because households are expected to be a primary site for human DENV infection, intradomicile vector control strategies should be a priority, especially when the force of transmission is high. The most effective intervention strategy will combine vector control with vaccine delivery for rapid and sustained disease prevention.

  5. Production of immunogenic West Nile virus-like particles using a herpes simplex virus 1 recombinant vector.

    Science.gov (United States)

    Taylor, Travis J; Diaz, Fernando; Colgrove, Robert C; Bernard, Kristen A; DeLuca, Neal A; Whelan, Sean P J; Knipe, David M

    2016-09-01

    West Nile virus (WNV) is a flavivirus that swept rapidly across North America in 1999, declined in prevalence, and then resurged in 2012. To date, no vaccine is available to prevent infection in the human population. Herpes simplex virus (HSV) replication-defective vaccine vectors induce a durable immunity characterized by strong antibody and CD8(+) T cell responses even in HSV-immune animals. In this study, a WNV protein expression cassette was optimized for virus-like particle (VLP) production in transfection studies, and the cassette was recombined into an HSV-1 d106-WNV virus vector, which produced extracellular VLPs, as confirmed by immunoelectron microscopy. Immunization of mice with the d106-WNV recombinant vector elicited a specific anti-WNV IgG response. This study highlights the flavivirus coding sequences needed for efficient assembly of virus-like particles. This information will facilitate generation of additional vaccine vectors against other flaviviruses including the recently emerged Zika virus.

  6. Modification of non-vector aphid feeding behavior on virus-infected host plant.

    Science.gov (United States)

    Hu, Zuqing; Zhao, Huiyan; Thieme, Thomas

    2013-01-01

    Virus-infected host plants can have positive, neutral or negative effects on vector aphids. Even though the proportion of non-vector aphids associated with a plant far exceeds that of vector species, little is known about the effect of virus-infected plants on non-vector aphids. In the present study, the English grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphididae), a non-vector of Wheat dwarf virus (WDV) and Cereal yellow dwarf virus-RPV (CYDV-RPV), was monitored on, virus-infected, virus-free and leafhopper/aphid-infested, and virus- and insect-free (control) barley, Hordeum vulgare L. (Poales: Poaceae), plants. Electrical penetration graph recordings were performed. Compared with the control plants, S. avenae on infected plants exhibited reduced non-probing and pathway phase, and increased phloem sap ingestion phase, and more aphids reached sustained phloem ingestion. However, the electrical penetration graph parameters described above showed no significant differences in aphid feeding behavior on virus-free and vector pre-infested plants and the control barley plants during S. avenae feeding. The results suggest that WDV/CYDV-RPV-infected host plants positively affected the feeding behavior of the non-vector aphid S. avenae. Based on these results, the reasons and trends among the virus-infected host plants' effects on the feeding behavior of non-vector aphids are discussed.

  7. Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Kaoru Hida

    Full Text Available Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF must similarly penetrate purulent lung airway mucus (sputum to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d∼80 nm and adeno-associated virus (AAV serotype 5; d∼20 nm, leading to average effective diffusivities that are ∼3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ∼40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date.

  8. 转化生长因子-β3与基质金属蛋白酶抑制剂-2联合转染兔骨髓间充质干细胞复合丝素蛋白/壳聚糖生物支架修复兔软骨缺损%Resurfacing rabbit's cartilage defects using mesenchymal stem cells co-transfected with transforming growth factor-β3 and matrix metalloproteinases inhibitors-2 by adeno-associated virus embedded in Silk fibroin/chitosan scaffolds

    Institute of Scientific and Technical Information of China (English)

    孟飞; 吕成昱; 张海宁; 申成凯; 冯尚祥

    2015-01-01

    conditions,we took the third generation of the logarithmic growth phase of rabbit bone marrow mesenchymal stem cells (BMSCs).And we used recombinant adeno-associated virus which carrying each group' s purpose gene to transfected them.After two months we killed the rabbits,as well as visually assessed of cartilage defect repair situation by hematoxylin-eosin staining (HE) staining.And conduct characteristic of cartilage cells that stained with toluidine blue staining and type Ⅱ collagen immunohistochemical staining.Results We observed that:after two months,cartilage-like substance formed in each expermental group' s cariliage defect area,and the co-transfection group induced cartilage were closer to hyaline cartilage.We found the Moran score of difference groups were:concrol group:0.600 ± 0.548,non-transfection group:1.800 ± 0.447,pure TGF-β3 transfected group:3.800 ± 0.837,co-transfection group:5.400 ± 0.548.And the difference of score between co-transfection group and pure TGF-β3 transfected group was statistically significant (P < 0.01).HE staining results suggested that co-transfected cartilage repair group' s result was better than pure TGF-β3 transfected group.Conclusion In animal experiments,pure TGF-β3 transfected rabbit bone marrow mesenchymal stem cells can repair rabbit articular cartilage defects,TGF-β3 and matrix metalloproteinases inhibitors-2 (TIMP-2) co-transfection group was more effective.

  9. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus.

    OpenAIRE

    Sardelis, M. R.; Turell, M.J.; Dohm, D. J.; O'Guinn, M. L.

    2001-01-01

    To control West Nile virus (WNV), it is necessary to know which mosquitoes are able to transmit this virus. Therefore, we evaluated the WNV vector potential of several North American mosquito species. Culex restuans and Cx. salinarius, two species from which WNV was isolated in New York in 2000, were efficient laboratory vectors. Cx. quinquefasciatus and Cx. nigripalpus from Florida were competent but only moderately efficient vectors. Coquillettidia perturbans was an inefficient laboratory v...

  10. Highly efficient virus-induced gene silencing in apple and soybean by apple latent spherical virus vector and biolistic inoculation.

    Science.gov (United States)

    Yamagishi, Noriko; Yoshikawa, Nobuyuki

    2013-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the analysis of the gene function in plants within a short time. However, in woody fruit tree like apple, some of Solanum crops, and soybean, it is generally difficult to inoculate virus vector by conventional inoculation methods. Here, we show efficient VIGS in apple and soybean by Apple latent spherical virus (ALSV) vector and biolistic inoculation. The plants inoculated with ALSV vectors by particle bombardment showed uniform silenced phenotypes of target genes within 2-3 weeks post inoculation.

  11. Status and prospects of plant virus control through interference with vector transmission.

    Science.gov (United States)

    Bragard, C; Caciagli, P; Lemaire, O; Lopez-Moya, J J; MacFarlane, S; Peters, D; Susi, P; Torrance, L

    2013-01-01

    Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

  12. Designing Efficacious Vesicular Stomatitis Virus-Vectored Vaccines Against Ebola Virus.

    Science.gov (United States)

    Wong, Gary; Qiu, Xiangguo

    2016-01-01

    Infection with the Ebola virus (EBOV) causes an aggressive hemorrhagic disease in humans and nonhuman primates. Traditional approaches, such as vaccination with inactivated virion preparations, have had limited efficacy, whereas immunization with live-attenuated EBOV is not feasible due to the highly lethal nature of the pathogen. This has necessitated the development of other approaches towards an effective EBOV vaccine. Over the past decade, recombinant viruses expressing the EBOV glycoprotein (GP) have constituted the most promising platforms, as evidenced by their ability to protect naïve nonhuman primates from a lethal EBOV challenge. The vesicular stomatitis virus (VSV) is one such vector and is currently progressing through the clinical pipeline. This chapter presents methodologies for the design, cloning, rescue, and preparation of live, recombinant VSV vaccines expressing GP for research purposes.

  13. West Nile virus vector Culex modestus established in southern England

    Directory of Open Access Journals (Sweden)

    Golding Nick

    2012-02-01

    Full Text Available Abstract Background The risk posed to the United Kingdom by West Nile virus (WNV has previously been considered low, due to the absence or scarcity of the main Culex sp. bridge vectors. The mosquito Culex modestus is widespread in southern Europe, where it acts as the principle bridge vector of WNV. This species was not previously thought to be present in the United Kingdom. Findings Mosquito larval surveys carried out in 2010 identified substantial populations of Cx. modestus at two sites in marshland in southeast England. Host-seeking-adult traps placed at a third site indicate that the relative seasonal abundance of Cx. modestus peaks in early August. DNA barcoding of these specimens from the United Kingdom and material from southern France confirmed the morphological identification. Conclusions Cx. modestus appears to be established in the North Kent Marshes, possibly as the result of a recent introduction. The addition of this species to the United Kingdom's mosquito fauna may increase the risk posed to the United Kingdom by WNV.

  14. Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

    Science.gov (United States)

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

    2015-10-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans.

  15. Tubular structure induced by a plant virus facilitates viral spread in its vector insect.

    Directory of Open Access Journals (Sweden)

    Qian Chen

    Full Text Available Rice dwarf virus (RDV replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.

  16. Plant Virus-Insect Vector Interactions: Current and Potential Future Research Directions.

    Science.gov (United States)

    Dietzgen, Ralf G; Mann, Krin S; Johnson, Karyn N

    2016-11-09

    Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus-insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.

  17. Varroa destructor, a potential vector of Israeli Acute Paralysis Virus in honey bees, Apis mellifera

    Science.gov (United States)

    Although the role of the parasitic mite, Varroa destructor, as a vector in transmission of viruses between honey bees is well established, no study has shown that it can similarly transmit Israeli Acute Paralysis Virus (IAPV), a virus that was found to be associated with Colony Collapse Disorder (CC...

  18. Effect of Grapevine Fanleaf Virus on the Reproduction and Survival of its Nematode Vector, Xiphinema index Thorne &Allen.

    Science.gov (United States)

    Das, S; Raski, D J

    1969-04-01

    Studies on the virus-vector interaction between the grapevine fanleaf virus (GFV) and its nematode vector, Xiphinema index, indicate the virus had no measurable effect on the rate of reproduction of its vector, but significantly influenced survNal of the nematodes.

  19. Aedes hensilli as a Potential Vector of Chikungunya and Zika Viruses

    OpenAIRE

    Ledermann, Jeremy P.; Laurent Guillaumot; Lawrence Yug; Saweyog, Steven C.; Mary Tided; Paul Machieng; Moses Pretrick; Maria Marfel; Anne Griggs; Martin Bel; Duffy, Mark R.; W Thane Hancock; Tai Ho-Chen; Powers, Ann M.

    2014-01-01

    An epidemic of Zika virus (ZIKV) illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s) involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s). Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at...

  20. Recombination of replicon and helper RNAs and the emergence of propagation-competent vectors upon Sindbis virus vector production.

    Science.gov (United States)

    Hyvärinen, Anna; Yongabi, Felicitas; Mäkinen, Kimmo; Wahlfors, Jarmo; Pellinen, Riikka

    2013-08-01

    Sindbis vectors have shown remarkable antitumor efficacy and tumor-targeting capacity in animal models and hold promise for cancer therapy. Different packaging systems are used to produce propagation-incompetent Sindbis vectors. However, the vectors produced using either DH-BB single helper RNA or split helper RNA can spread in permissive cell cultures. We investigated the mechanisms of vector spreading and show, here, that recombination occurs between the replicon and DH-BB helper RNA, leading to formation of the full-length virus genome. Split helper RNA may not completely prevent wild-type reversion, although the frequency is greatly reduced. Contrary to propagation of Sindbis DH-BB vectors, Sindbis split helper vectors were frequently able to spread without cytopathic effect (CPE), a feature that was linked to wild-type reversion. Our results support the hypothesis that the non-cytopathic local spreading constantly observed with Sindbis split helper vector results from unspecific packaging of helper RNAs into vector particles and co-infection with particles containing replicon and helper RNAs. Several malignant cell lines with defective interferon responses were found to be permissive for non-cytopathic spreading of the Sindbis split helper vector. Interferon-α suppressed the spreading providing a possible option to control the vector.

  1. Efficient Strategy to Generate a Vectored Duck Enteritis Virus Delivering Envelope of Duck Tembusu Virus

    Directory of Open Access Journals (Sweden)

    Zhong Zou

    2014-06-01

    Full Text Available Duck Tembusu virus (DTMUV is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV was examined as a candidate vaccine vector to deliver the envelope (E of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC of C-KCE (vBAC-C-KCE. The envelope (E gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs. Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.

  2. Transfection of brain-derived neurotrophic factor gene by recombinant adeno-associated virus vector in retinal ganglion cells in vitro%腺伴随病毒介导的脑源性神经营养因子对体外培养的鼠视网膜神经节细胞转染及生长特性的影响

    Institute of Scientific and Technical Information of China (English)

    李海燕; 赵家良; 张华

    2008-01-01

    目的 探讨重组腺伴随病毒载体介导的脑源性神经营养因子(rAAV-BDNF)对体外培养的鼠视网膜神经节细胞(RGCs)转染及其生长活性的影响.方法 实验研究.(1)应用rAAV-BDNF对体外培养2 d的RGCs进行转染;(2)应用逆转录聚合酶链反应(RT-PCR)技术,检测外源性BDNF基因在RGCs细胞mRNA水平的表达情况;(3)应用酶联免疫吸附测定(ELISA)法,对细胞培养液中BDNF含量进行检测;(4)对rAAV-BDNF转染细胞、未转染细胞及加入BDNF的培养细胞进行MTT比色分析;(5)应用Annexin V-FITC凋亡检测试剂盒和流式细胞仪,检测rAAV-BDNF转染细胞、未转染细胞及加入BDNF培养细胞的凋亡比率.结果 (1)RT-PCR检测结果:转染细胞表达外源性BDNF基因,而未转染细胞不表达BDNF基因.(2)ELISA法检测结果:rAAV-BDNF转染细胞的培养液中BDNF含量:转染7 d后为(616.1±40.0)ng/L,转染14 d后为(1075.1±48.7)ng/L.(3)MTT比色结果:转染3和6 d后,rAAV-BDNF转染细胞与未转染细胞间的吸光度(A)值差异无统计学意义(t=1.084,1.582;P=0.284,0.120);转染9 d后,转染细胞的A值高于未转染细胞(t=4.854,P=0.000).(4)流式细胞仪检测结果:rAAV-BDNF转染细胞和加入BDNF培养细胞的凋亡率明显低于未转染细胞的凋亡率,差异有统计学意义(P=0.015,0.017).结论 rAAV-BDNF可有效转染体外培养的鼠RGCs,转染细胞可在转录水平和翻译水平表达外源性BDNF基因,且生长活性改善,凋亡细胞减少.这为青光眼视神经保护的基因治疗提供了理论和技术支持.%Objective To determine whether rat retinal ganglion cells(RGCs)could be infected by rAAV-BDNF in vitro and to evaluate the influence of rAAV-BDNF transfection on the survival and apoptosis of rat RGCs.Methods It Was a experimental study.(1)RGCs were isolated from neonatal Sprague-Dawley rats(postnatal within 24 h).(2)Two days after the cultivation,the RGCs were transfected with rAAV- BDNF at a dosage of MOI=103 and then incubated for 7 days.Total RNA were extracted from rAAV-BDNF transfected cells using Trizol reagent.The gene expression of BDNF gene in RGCs waft analyzed by reverse transcription polymerase-chain reaction(RT-PCR).(3)Supernatant of the rAAV-BDNF transfected cells was collected at 7 days and 14 days after transfection.The protein expression of BDNF in the cell supernatant Was examined with ELISA assay.(4)The survival and apoptosis of rAAV-BDNF transfected cells, untransfeeted cells and the cells with addition of BDNF in culture medium were evaluated bv MTT colorimetric assay and flow cytometry with Annexin V-FITC staining.respectively.Results (1)RT-PCR analysis showed that mRNA expression of BDNF gene could be detected in transfected ceHs but not in untransfected ceHs.(2)The concentrations of BDNF protein in the conditioned medium of the rAAV-BDNF transfected cells were(616.1±40.0)ng/L and(1075.1±48.7)ng/L 7 days and 14 days after the transfection.respectively.(3)MTF colorimetric assay showed that the OD values of rAAV.BDNF transfected cells and untransfected cells were similar at the time of 3 and 6 days after transfection(t=1.084 and 1.582. P=0.284 and 0.120).The OD value of transfected cells was higher than that of untransfected ceHs 9 days after the transfection(t=4.854,P=0.001).(4)The apoptosis rate in rAAV-BDNF transfected cells and the cells with BDNF exposure was lower than that of the untransfected cells(P=0.015,0.017).Conclusions Rat RGCs are abie to be transfected by rAAV-BDNF in vitro.The transfected ceHs Can express BDNF gene at the level of beth mRNA and protein.Apoptosis rate is lOW in the transfected cells.This study indicates that rAAV-BDNF transfection Can be used for the potential gene tllerapy in glaucoma neuroprotection.

  3. 晚期糖化终产物受体反义 RNA 腺相关病毒载体的构建及在肾脏系膜细胞中表达%Construction of recombinant RAGE antisense RNA adeno-associated virus vector and its expression in rat mesangial cell

    Institute of Scientific and Technical Information of China (English)

    游捷; 赵蓉; 刘礼斌; 林建银

    2007-01-01

    目的 构建晚期糖化终产物受体(RAGE)反义RNA腺相关病毒载体,并在大鼠肾脏系膜细胞中表达. 方法 构建腺相关病毒介导的RAGE反义RNA载体,3个质粒共转染293细胞,获得病毒原液,感染大鼠肾脏系膜细胞,流式细胞术、RT-PCR、ELISA检测重组病毒感染的细胞RAGE的表达和分泌细胞外基质的情况.结果 经酶切鉴定、序列分析显示RAGE基因片段正确完整反向插入pAAV-MCS.利用293细胞包装获得病毒原液的滴度为8.7×107VP/mL.感染重组病毒的细胞与正常细胞比较RAGE表达被抑制(48.2±6.1)%,分泌Ⅳ型胶原(ColⅣ)水平明显下降(P<0.05).结论 成功构建具有抑制功能的RAGE反义RNA腺相关病毒载体,为进一步研究RAGE的作用机制,以及基因治疗RAGE相关疾病提供一个重要工具.

  4. Feline Foamy Virus-Based Vectors: Advantages of an Authentic Animal Model

    Directory of Open Access Journals (Sweden)

    Martin Löchelt

    2013-07-01

    Full Text Available New-generation retroviral vectors have potential applications in vaccination and gene therapy. Foamy viruses are particularly interesting as vectors, because they are not associated to any disease. Vector research is mainly based on primate foamy viruses (PFV, but cats are an alternative animal model, due to their smaller size and the existence of a cognate feline foamy virus (FFV. The potential of replication-competent (RC FFV vectors for vaccination and replication-deficient (RD FFV-based vectors for gene delivery purposes has been studied over the past years. In this review, the key achievements and functional evaluation of the existing vectors from in vitro cell culture systems to out-bred cats will be described. The data presented here demonstrate the broad application spectrum of FFV-based vectors, especially in pathogen-specific prophylactic and therapeutic vaccination using RD vectors in cats and in classical gene delivery. In the cat-based system, FFV-based vectors provide an advantageous platform to evaluate and optimize the applicability, efficacy and safety of foamy virus (FV vectors, especially the understudied aspect of FV cell and organ tropism.

  5. Vectores recombinantes basados en el virus Vaccinia modificado de Ankara (MVA) como vacunas contra la leishmaniasis

    OpenAIRE

    Pérez Jiménez, Eva; Larraga, Vicente; Esteban, Mariano

    2005-01-01

    Vectores recombinantes basados en el virus vaccinia modificado de Ankara (MVA) como vacunas contra la leishmaniasis. Los vectores de la invención contienen secuencias codificantes de la proteína LACK, preferentemente insertadas en el locus de hemaglutinina del virus y bajo el control de un promotor que permite su expresión a lo largo del ciclo de infección del virus. Son vectores seguros, estables, que dan lugar a una potente respuesta inmune que confiere protección frente a la leishmaniasis,...

  6. Recombinant vesicular stomatitis virus vector mediates postexposure protection against Sudan Ebola hemorrhagic fever in nonhuman primates.

    Science.gov (United States)

    Geisbert, Thomas W; Daddario-DiCaprio, Kathleen M; Williams, Kinola J N; Geisbert, Joan B; Leung, Anders; Feldmann, Friederike; Hensley, Lisa E; Feldmann, Heinz; Jones, Steven M

    2008-06-01

    Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.

  7. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus.

    Science.gov (United States)

    Sardelis, M R; Turell, M J; Dohm, D J; O'Guinn, M L

    2001-01-01

    To control West Nile virus (WNV), it is necessary to know which mosquitoes are able to transmit this virus. Therefore, we evaluated the WNV vector potential of several North American mosquito species. Culex restuans and Cx. salinarius, two species from which WNV was isolated in New York in 2000, were efficient laboratory vectors. Cx. quinquefasciatus and Cx. nigripalpus from Florida were competent but only moderately efficient vectors. Coquillettidia perturbans was an inefficient laboratory vector. As WNV extends its range, exposure of additional mosquito species may alter its epidemiology.

  8. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Stoker, A.W.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1988-03-01

    The authors have constructed an avian leukosis virus derivative with a 5{prime} deletion extending from within the tRNA primer binding site to a SacI site in the leader region. The aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1{Psi}{sup {minus}}, to develop vector-packaging cell lines. They show that RAV-1{Psi}{sup {minus}} can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, they have demonstrated that QT6-derived lines expressing RAV-1{Psi}{sup {minus}} can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-{Psi}{sup {minus}}-expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. They show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.

  9. Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells.

    Science.gov (United States)

    Price, Mary A; Case, Scott S; Carbonaro, Denise A; Yu, Xiao-Jin; Petersen, Denise; Sabo, Kathleen M; Curran, Michael A; Engel, Barbara C; Margarian, Hovanes; Abkowitz, Janis L; Nolan, Garry P; Kohn, Donald B; Crooks, Gay M

    2002-11-01

    Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

  10. Recombinant adeno-associated virus-mediated inhibiting of interleukin-4 expression in rat model of asthma

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Asthma is a chronic disease characterized by reversible airway obstruction, airway hyper- responsiveness, and inflammation of airways. Th2 cells, one sort of CD4+ T lymphocytes, are currently considered to play an important role in the chronic airway inflammation of asthma. Meanwhile, a number of laboratories have clearly established the importance of the Th2-derived cytokine interleukin-4 (IL-4) in mediating the airway inflammatory response. Anti-IL-4 therapy might be beneficial in treatment of chronic asthma.

  11. Differential profiles of direct and indirect modification of vector feeding behaviour by a plant virus.

    Science.gov (United States)

    He, Wen-Bo; Li, Jie; Liu, Shu-Sheng

    2015-01-08

    Plant viruses interact with their insect vectors directly and indirectly via host plants, and this tripartite interaction may produce fitness benefits to both the vectors and the viruses. Our previous studies show that the Middle East-Asia Minor 1 (MEAM1) species of the whitefly Bemisia tabaci complex improved its performance on tobacco plants infected by the Tomato yellow leaf curl China virus (TYLCCNV), which it transmits, although virus infection of the whitefly per se reduced its performance. Here, we use electrical penetration graph recording to investigate the direct and indirect effects of TYLCCNV on the feeding behaviour of MEAM1. When feeding on either cotton, a non-host of TYLCCNV, or uninfected tobacco, a host of TYLCCNV, virus-infection of the whiteflies impeded their feeding. Interestingly, when viruliferous whiteflies fed on virus-infected tobacco, their feeding activities were no longer negatively affected; instead, the virus promoted whitefly behaviour related to rapid and effective sap ingestion. Our findings show differential profiles of direct and indirect modification of vector feeding behaviour by a plant virus, and help to unravel the behavioural mechanisms underlying a mutualistic relationship between an insect vector and a plant virus that also has features reminiscent of an insect pathogen.

  12. Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

    Science.gov (United States)

    Roy, Avijit; Hartung, John S; Schneider, William L; Shao, Jonathan; Leon, Guillermo; Melzer, Michael J; Beard, Jennifer J; Otero-Colina, Gabriel; Bauchan, Gary R; Ochoa, Ronald; Brlansky, Ronald H

    2015-07-01

    Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

  13. Aedes hensilli as a potential vector of Chikungunya and Zika viruses.

    Directory of Open Access Journals (Sweden)

    Jeremy P Ledermann

    2014-10-01

    Full Text Available An epidemic of Zika virus (ZIKV illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s. Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at select sites around the capital city. The predominant species found on the island was Aedes (Stegomyia hensilli. No virus isolates were obtained from the adult field material collected, nor did any of the immature mosquitoes that were allowed to emerge to adulthood contain viable virus or nucleic acid. Therefore, laboratory studies of the probable vector, Ae. hensilli, were undertaken to determine the likelihood of this species serving as a vector for Zika virus and other arboviruses. Infection rates of up to 86%, 62%, and 20% and dissemination rates of 23%, 80%, and 17% for Zika, chikungunya, and dengue-2 viruses respectively, were found supporting the possibility that this species served as a vector during the Zika outbreak and that it could play a role in transmitting other medically important arboviruses.

  14. Aedes hensilli as a potential vector of Chikungunya and Zika viruses.

    Science.gov (United States)

    Ledermann, Jeremy P; Guillaumot, Laurent; Yug, Lawrence; Saweyog, Steven C; Tided, Mary; Machieng, Paul; Pretrick, Moses; Marfel, Maria; Griggs, Anne; Bel, Martin; Duffy, Mark R; Hancock, W Thane; Ho-Chen, Tai; Powers, Ann M

    2014-10-01

    An epidemic of Zika virus (ZIKV) illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s) involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s). Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at select sites around the capital city. The predominant species found on the island was Aedes (Stegomyia) hensilli. No virus isolates were obtained from the adult field material collected, nor did any of the immature mosquitoes that were allowed to emerge to adulthood contain viable virus or nucleic acid. Therefore, laboratory studies of the probable vector, Ae. hensilli, were undertaken to determine the likelihood of this species serving as a vector for Zika virus and other arboviruses. Infection rates of up to 86%, 62%, and 20% and dissemination rates of 23%, 80%, and 17% for Zika, chikungunya, and dengue-2 viruses respectively, were found supporting the possibility that this species served as a vector during the Zika outbreak and that it could play a role in transmitting other medically important arboviruses.

  15. Regional and seasonal response of a West Nile virus vector to climate change

    OpenAIRE

    Cory W Morin; Comrie, Andrew C.

    2013-01-01

    The potential impacts of climate change on human health are possibly large and not yet well understood, especially for vector-borne diseases. This study provides projections of how climate change may affect the population of a West Nile virus mosquito vector across the southern United States. Using a climate-driven mosquito population model, we simulate vector abundance under base and future climate. Under future climate, many locations exhibit a lengthening of the mosquito season with a decr...

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

    Science.gov (United States)

    2010-04-01

    Zaire Ebola virus . Virology 346:394–401. 21. Leffel, E. K., and D. S. Reed. 2004. Marburg and Ebola viruses as aerosol threats. Biosecur. Bioterror. 2...the event of a natural virus outbreak or biological threat. The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe...vaccine efficacy are justifiable. While the combination of the two CAdVax-based vectors for Ebola virus (EBO7) and Marburg virus (M8) GPs constitute the

  17. Newcastle disease virus as a vaccine vector for infectious laryngotracheitis

    Science.gov (United States)

    Effective, safe, and incapable of reverting to virulence are characteristics desirable for infectious laryngotracheitis virus (ILTV) vaccines. Recombinant Newcastle disease virus (NDV) expressing foreign antigens of avian and mammalian pathogens have been demonstrated to elicit protective immunity....

  18. Patterns of scAAV vector insertion associated with oncogenic events in a mouse model for genotoxicity.

    Science.gov (United States)

    Rosas, Lucia E; Grieves, Jessica L; Zaraspe, Kimberly; La Perle, Krista Md; Fu, Haiyan; McCarty, Douglas M

    2012-11-01

    Recombinant adeno-associated virus (rAAV) vectors have gained an extensive record of safety and efficacy in animal models of human disease. Infrequent reports of genotoxicity have been limited to specific vectors associated with excess hepatocellular carcinomas (HCC) in mice. In order to understand potential mechanisms of genotoxicity, and identify patterns of insertion that could promote tumor formation, we compared a self-complementary AAV (scAAV) vector designed to promote insertional activation (scAAV-CBA-null) to a conventional scAAV-CMV-GFP vector. HCC-prone C3H/HeJ mice and severe combined immunodeficiency (SCID) mice were infected with vector plus secondary treatments including partial hepatectomy (HPX) and camptothecin (CPT) to determine the effects of cell cycling and DNA damage on tumor incidence. Infection with either vector led to a significant increase in HCC incidence in male C3H/HeJ mice. Partial HPX after infection reduced HCC incidence in the cytomegalovirus-green fluorescent protein (CMV-GFP)-infected mice, but not in the cognate chicken β-actin (CBA)-null infected group. Tumors from CBA-null infected, hepatectomized mice were more likely to contain significant levels of vector DNA than tumors from the corresponding CMV-GFP-infected group. Most CBA-null vector insertions recovered from tumors were associated with known proto-oncogenes or tumor suppressors. Specific patterns of insertion suggested read-through transcription, enhancer effects, and disruption of tumor suppressors as likely mechanisms for genotoxicity.

  19. Preventive and curative effects of Apple latent spherical virus vectors harboring part of the target virus genome against potyvirus and cucumovirus infections.

    Science.gov (United States)

    Tamura, Akihiro; Kato, Takahiro; Taki, Ayano; Sone, Mikako; Satoh, Nozomi; Yamagishi, Noriko; Takahashi, Tsubasa; Ryo, Bo-Song; Natsuaki, Tomohide; Yoshikawa, Nobuyuki

    2013-11-01

    Apple latent spherical virus (ALSV)-based vectors experimentally infect a broad range of plant species without causing symptoms and can effectively induce stable virus-induced gene silencing in plants. Here, we show that pre-infection of ALSV vectors harboring part of a target viral genome (we called ALSV vector vaccines here) inhibits the multiplication and spread of the corresponding challenge viruses [Bean yellow mosaic virus, Zucchini yellow mosaic virus (ZYMV), and Cucumber mosaic virus (CMV)] by a homology-dependent resistance. Further, the plants pre-infected with an ALSV vector having genome sequences of both ZYMV and CMV were protected against double inoculation of ZYMV and CMV. More interestingly, a curative effect of an ALSV vector vaccine could also be expected in ZYMV-infected cucumber plants, because the symptoms subsided on subsequent inoculation with an ALSV vector vaccine. This may be due to the invasion of ALSV, but not ZYMV, in the shoot apical meristem of cucumber.

  20. rAAV vector-mediated gene therapy for experimental ischemic stroke

    Directory of Open Access Journals (Sweden)

    Li Zhao-Jian

    2008-01-01

    Full Text Available The safest viral vector system for gene therapy is based on recombinant adeno-associated virus (rAAV up to date in Phase I clinical trials, which has been developed rapidly and applied for ischemic stroke gene therapy in animal experiments since the past seven years. rAAV vector has made great progress in improving gene delivery by modification of the capsid and increasing transgene expression by encapsidation of double-stranded rAAV genome. And in all, nine therapeutic genes in 12 animal studies were successfully delivered using rAAV vector to ischemic brain via different approaches in rat or mice stroke models for gene therapy and the results suggested that rAAV could mediate genes′ expression efficiently; most of them displayed evidently therapeutic efficacy with satisfactory biological safety. Gene therapy involving rAAV vector seems effective in attenuation of ischemic damage in stroke and has greatly promising potential use for patients in the future. In this review, we will focus on the basic biology and development of rAAV vector itself as well as the recent progress in the use of this vector for ischemic stroke gene therapy in animal experiments.

  1. Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

    Institute of Scientific and Technical Information of China (English)

    Michael A van Geer; Koert FD Kuhlmann; Conny T Bakker; Fibo JW ten Kate; Ronald PJ Oude Elferink; Piter J Bosma

    2009-01-01

    AIM: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions.METHODS: Human pancreatic tissue samples (malignant and normal) were obtained from surgical specimens and processed immediately to tissue slices.Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% O2. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the repor ter genes GFP or DsRed.Expression of these reporter genes was measured at 72 h after infection.RESULTS: With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants.CONCLUSION: The presented sys tem al lows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

  2. Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2010-01-01

    Full Text Available Abstract Background The ability to efficiently and selectively target gene delivery vectors to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. We pursued two different strategies to target lentiviral vector delivery to specific cell types. In one of the strategies, vector particles bearing a membrane-bound stem cell factor sequence plus a separate fusion protein based either on Sindbis virus strain TR339 glycoproteins or the vesicular stomatitis virus G glycoprotein were used to selectively transduce cells expressing the corresponding stem cell factor receptor (c-kit. An alternative approach involved soluble avian sarcoma/leukosis virus receptors fused to cell-specific ligands including stem cell factor and erythropoietin for targeting lentiviral vectors pseudotyped with avian sarcoma/leukosis virus envelope proteins to cells that express the corresponding receptors. Results The titers of unconcentrated vector particles bearing Sindbis virus strain TR339 or vesicular stomatitis virus G fusion proteins plus stem cell factor in the context of c-kit expressing cells were up to 3.2 × 105 transducing units per ml while vector particles lacking the stem cell factor ligand displayed titers that were approximately 80 fold lower. On cells that lacked the c-kit receptor, the titers of stem cell factor-containing vectors were approximately 40 times lower compared to c-kit-expressing cells. Lentiviral vectors pseudotyped with avian sarcoma/leukosis virus subgroup A or B envelope proteins and bearing bi-functional bridge proteins encoding erythropoietin or stem cell factor fused to the soluble extracellular domains of the avian sarcoma/leukosis virus subgroup A or B receptors resulted in efficient transduction of erythropoietin receptor or c-kit-expressing cells. Transduction of erythropoietin receptor-expressing cells mediated by bi-functional bridge proteins was found to be dependent on the dose, the

  3. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    Science.gov (United States)

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  4. The Balance between CD8(+) T Cell-Mediated Clearance of AAV-Encoded Antigen in the Liver and Tolerance Is Dependent on the Vector Dose.

    Science.gov (United States)

    Kumar, Sandeep R P; Hoffman, Brad E; Terhorst, Cox; de Jong, Ype P; Herzog, Roland W

    2017-04-05

    The liver continuously receives antigens from circulation and the gastrointestinal tract. A complex immune regulatory system has evolved in order to both limit inflammation and promote tolerance in the liver. Although in situ immune tolerance mechanisms enable successful gene therapy and liver transplantation, at the same time they facilitate chronic infections by pathogens such as hepatitis viruses. It is, however, poorly understood why hepatocytes infected with hepatitis viruses or transduced with adeno-associated virus (AAV)-based vectors may be rejected by CD8(+) T cells several months later. We found that hepatic transfer of limited doses of an AAV-ovalbumin vector rapidly induced antigen-specific CD8(+) T cells that only became functionally competent after >2 months. At this time, CD8(+) T cells had downregulated negative checkpoint markers, e.g., the programmed death 1 [PD-1] receptor, and upregulated expression of relevant cytokines. At further reduced vector dose, only intrahepatic rather than systemic CD8(+) T cell responses occurred, showing identical delay in antigen clearance. In contrast, PD-1-deficient mice rapidly cleared ovalbumin. Interestingly, higher vector dose directed sustained transgene expression without CD8(+) T cell responses. Regulatory T cells, IL-10 expression, and Fas-L contributed to high-dose tolerance. Thus, viral vector doses profoundly impact CD8(+) T cell responses.

  5. Sequential infection of Rice dwarf virus in the internal organs of its insect vector after ingestion of virus.

    Science.gov (United States)

    Chen, Hongyan; Chen, Qian; Omura, Toshihiro; Uehara-Ichiki, Tamaki; Wei, Taiyun

    2011-09-01

    Confocal microscopy revealed that Rice dwarf virus (RDV) initially accumulated in epithelial cells of the filter chamber of leafhopper vector Nephotettix cincticeps 2 days after acquisition access feeding on diseased plants. Subsequently, RDV accumulation progressed to the anterior midgut, and then spread to the nervous system before infection of other organs. Furthermore, RDV accumulation progressed to the visceral muscles surrounding the anterior midgut. Later, RDV accumulation was detected in other parts of the alimentary canal, salivary glands and the follicular cells of the ovarioles in viruliferous insect vector. Our results suggest that RDV may use the muscle or neural tissues for viral dissemination from the infected vector's midgut into other tissues.

  6. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    Science.gov (United States)

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong

    2013-07-01

    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  7. The Use of Viral Vectors in Gene Transfer Therapy

    Directory of Open Access Journals (Sweden)

    A. Dziaková

    2016-05-01

    Full Text Available Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA, single-stranded DNA (ssDNA, plasmid DNA and antisense oligodeoxynucleotides (ASON. The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%, retroviruses (344 clinical studies; 20.5%, unenveloped/plasmid DNA (304 clinical studies, 17.7%, adeno-associated viruses (75 clinical studies; 4.5% and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.

  8. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

    Science.gov (United States)

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75-98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing.

  9. RNA Interference in Insect Vectors for Plant Viruses

    Directory of Open Access Journals (Sweden)

    Surapathrudu Kanakala

    2016-12-01

    Full Text Available Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

  10. RNA Interference in Insect Vectors for Plant Viruses.

    Science.gov (United States)

    Kanakala, Surapathrudu; Ghanim, Murad

    2016-12-12

    Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

  11. Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector

    Science.gov (United States)

    Liu, Si; Xie, Jiatao; Cheng, Jiasen; Li, Bo; Chen, Tao; Fu, Yanping; Li, Guoqing; Wang, Manqun; Jin, Huanan; Wan, Hu; Jiang, Daohong

    2016-01-01

    Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a nonhost fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1–like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage. PMID:27791095

  12. Newcastle Disease Virus as a Vaccine Vector for Development of Human and Veterinary Vaccines.

    Science.gov (United States)

    Kim, Shin-Hee; Samal, Siba K

    2016-07-04

    Viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. Avirulent NDV strains LaSota and B1 have long track records of safety and efficacy. Therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. NDV replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. As a vaccine vector, NDV can accommodate foreign sequences with a good degree of stability and as a RNA virus, there is limited possibility for recombination with host cell DNA. Using NDV as a vaccine vector in humans offers several advantages over other viral vaccine vectors. NDV is safe in humans due to host range restriction and there is no pre-existing antibody to NDV in the human population. NDV is antigenically distinct from common human pathogens. NDV replicates to high titer in a cell line acceptable for human vaccine development. Therefore, NDV is an attractive vaccine vector for human pathogens for which vaccines are currently not available. NDV is also an attractive vaccine vector for animal pathogens.

  13. Vectores

    OpenAIRE

    2016-01-01

    Documento que contiene la explicación sobre las temáticas de Sistemas coordenados, Cantidades vectoriales y escalares, Algunas propiedades de los vectores, Componentes de un vector y vectores unitarios

  14. Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

    Institute of Scientific and Technical Information of China (English)

    DAI Chunming; ZHANG Xiaoyan; WANG Shuhui; LIU Ying; DUAN Danli; SHEN Rongxian; SHAO Yiming

    2004-01-01

    In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.

  15. Glycoprotein Exchange Vectors Based on Vesicular Stomatitis Virus Allow Effective Boosting and Generation of Neutralizing Antibodies to a Primary Isolate of Human Immunodeficiency Virus Type 1

    OpenAIRE

    Rose, Nina F.; Roberts, Anjeanette; Buonocore, Linda; Rose, John K.

    2000-01-01

    Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with ...

  16. Ecological Niche Modeling of Potential West Nile Virus Vector Mosquito Species in Iowa

    OpenAIRE

    2010-01-01

    Ecological niche modeling (ENM) algorithms, Maximum Entropy Species Distribution Modeling (Maxent) and Genetic Algorithm for Rule-set Prediction (GARP), were used to develop models in Iowa for three species of mosquito — two significant, extant West Nile virus (WNV) vectors (Culex pipiens L and Culex tarsalis Coquillett (Diptera: Culicidae)), and the nuisance mosquito, Aedes vexans Meigen (Diptera: Culicidae), a potential WNV bridge vector. Occurrence data for the three mosquito species from ...

  17. Projection of Climate Change Influences on U.S. West Nile Virus Vectors

    OpenAIRE

    Brown, Heidi E.; Young, Alex; Lega, Joceline; Theodore G Andreadis; Schurich, Jessica; Comrie, Andrew

    2015-01-01

    While estimates of the impact of climate change on health are necessary for health care planners and climate change policy makers, models to produce quantitative estimates remain scarce. We describe a freely available dynamic simulation model parameterized for three West Nile virus vectors, which provides an effective tool for studying vector-borne disease risk due to climate change. The Dynamic Mosquito Simulation Model is parameterized with species specific temperature-dependent development...

  18. Immunogenic Subviral Particles Displaying Domain III of Dengue 2 Envelope Protein Vectored by Measles Virus

    Directory of Open Access Journals (Sweden)

    Indira S. Harahap-Carrillo

    2015-07-01

    Full Text Available Vaccines against dengue virus (DV are commercially nonexistent. A subunit vaccination strategy may be of value, especially if a safe viral vector acts as biologically active adjuvant. In this paper, we focus on an immunoglobulin-like, independently folded domain III (DIII from DV 2 envelope protein (E, which contains epitopes that elicits highly specific neutralizing antibodies. We modified the hepatitis B small surface antigen (HBsAg, S in order to display DV 2 DIII on a virus-like particle (VLP, thus generating the hybrid antigen DIII-S. Two varieties of measles virus (MV vectors were developed to express DIII-S. The first expresses the hybrid antigen from an additional transcription unit (ATU and the second additionally expresses HBsAg from a separate ATU. We found that this second MV vectoring the hybrid VLPs displaying DIII-S on an unmodified HBsAg scaffold were immunogenic in MV-susceptible mice (HuCD46Ge-IFNarko, eliciting robust neutralizing responses (averages against MV (1:1280 NT90, hepatitis B virus (787 mIU/mL, and DV2 (1:160 NT50 in all of the tested animals. Conversely, the MV vector expressing only DIII-S induced immunity against MV alone. In summary, DV2 neutralizing responses can be generated by displaying E DIII on a scaffold of HBsAg-based VLPs, vectored by MV.

  19. Semliki Forest Virus: un vector viral con múltiples aplicaciones

    OpenAIRE

    2007-01-01

    Se han utilizado los alfavirus como vectores de expresión, entre estos se encuentra el Semliki Forest virus (SFV), que es un virus envuelto, el cual, además de replicarse en el citoplasma, tiene la propiedad de expresar por separado las proteínas estructurales de las no estructurales, permitiendo un mayor control de la expresión. Los vectores derivados del SFV pueden tener una gama amplia de aplicaciones. Se pueden obtener altos títulos virales para la expresión eficiente de proteínas en dife...

  20. Gene therapy model of X-linked severe combined immunodeficiency using a modified foamy virus vector.

    Science.gov (United States)

    Horino, Satoshi; Uchiyama, Toru; So, Takanori; Nagashima, Hiroyuki; Sun, Shu-Lan; Sato, Miki; Asao, Atsuko; Haji, Yoichi; Sasahara, Yoji; Candotti, Fabio; Tsuchiya, Shigeru; Kure, Shigeo; Sugamura, Kazuo; Ishii, Naoto

    2013-01-01

    X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.

  1. Gene therapy model of X-linked severe combined immunodeficiency using a modified foamy virus vector.

    Directory of Open Access Journals (Sweden)

    Satoshi Horino

    Full Text Available X-linked severe combined immunodeficiency (SCID-X1 is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc gene, and characterized by a complete defect of T and natural killer (NK cells. Gene therapy for SCID-X1 using conventional retroviral (RV vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.

  2. Gene Therapy Model of X-linked Severe Combined Immunodeficiency Using a Modified Foamy Virus Vector

    Science.gov (United States)

    Horino, Satoshi; Uchiyama, Toru; So, Takanori; Nagashima, Hiroyuki; Sun, Shu-lan; Sato, Miki; Asao, Atsuko; Haji, Yoichi; Sasahara, Yoji; Candotti, Fabio; Tsuchiya, Shigeru; Kure, Shigeo; Sugamura, Kazuo; Ishii, Naoto

    2013-01-01

    X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1. PMID:23990961

  3. Optimization of vesicular stomatitis virus-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer.

    Science.gov (United States)

    Song, Jae Jin; Lee, Boyoung; Chang, Jin Woo; Kim, Joo-Hang; Kwon, Yunhee Kim; Lee, Heuiran

    2003-05-01

    FIV-based lentiviral vector has shown a unique opportunity as an efficient gene delivery vehicle, especially to nondividing human cells. Here, we genetically reconstructed the FIV-based vector by serially deleting residual virus genes of gag and vif, leading to minimized cytotoxicity together with efficient virus production and gene transfer. The modified FIV- based vector was generated by transiently transfecting 293T cells with three plasmids of the gene transfer vector with minimal gag region, the packaging plasmid without vif and the VSV-G-expressing plasmid. The vector was routinely generated as many as 1 x 10(7) transducing particles per ml and easily concentrated by simple centrifugation. The cytotoxic effect significantly decreased in sensitive cells to FIV infection even at high multiplicity of infection (MOI), such as 500. Moreover, the transduction efficiency was consistently retained after cell cycle was arrested in a variety of human cells. Taken together, our results suggest that the modified VSV-G pseudotyped FIV-based vector efficiently transduce dividing and nondividing human cells with minimal cytotoxicity.

  4. Spread of Zika virus: The key role of mosquito vector control

    OpenAIRE

    Giovanni Benelli

    2016-01-01

    Mosquitoes (Diptera: Culicidae) represent a key threat for millions of humans and animals worldwide, since they act as vectors for important parasites and pathogens, including malaria, filariasis and a wide number of arboviruses. The recent outbreaks of Zika virus infections occurring in South America, Central America, and the Caribbean, represent the most recent four arrivals of important arboviruses in the western hemisphere, over the last 20 years, namely dengue, West Nile virus, and chiku...

  5. Aedes (Stegomyia albopictus (Skuse: a potential vector of Zika virus in Singapore.

    Directory of Open Access Journals (Sweden)

    Pei-Sze Jeslyn Wong

    Full Text Available Zika virus (ZIKV is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80-85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi. Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose50 assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.

  6. Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Chad E Mire

    Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

  7. Virus infection mediates the effects of elevated CO2 on plants and vectors

    Science.gov (United States)

    Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

    2016-03-01

    Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production.

  8. Preparing the United States for Zika Virus: Pre-emptive Vector Control and Personal Protection.

    Science.gov (United States)

    Diaz, James H

    2016-12-01

    Discovered in 1947 in a monkey in the Zika forest of Uganda, Zika virus was dismissed as a cause of a mild illness that was confined to Africa and Southeast Asia and transmitted by Aedes mosquitoes. In 2007, Zika virus appeared outside of its endemic borders in an outbreak on the South Pacific Island of Yap. In 2013, Zika virus was associated with a major neurological complication, Guillain-Barré syndrome, in a larger outbreak in the French Polynesian Islands. From the South Pacific, Zika invaded Brazil in 2015 and caused another severe neurological complication, fetal microcephaly. The mosquito-borne transmission of Zika virus can be propagated by sexual transmission and, possibly, by blood transfusions, close personal contacts, and organ transplants, like other flaviviruses. Since these combined mechanisms of infectious disease transmission could result in catastrophic incidences of severe neurological diseases in adults and children, the public should know what to expect from Zika virus, how to prevent infection, and what the most likely failures in preventive measures will be. With federal research funding stalled, a Zika vaccine is far away. The only national strategies to prepare the United States for Zika virus invasion now are effective vector control measures and personal protection from mosquito bites. In addition to a basic knowledge of Aedes mosquito vectors and their biting behaviors, an understanding of simple household vector control measures, and the selection of the best chemical and physical mosquito repellents will be required to repel the Zika threat.

  9. A cryptic promoter in potato virus X vector interrupted plasmid construction

    Directory of Open Access Journals (Sweden)

    Schultz Ronald D

    2007-03-01

    Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.

  10. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations

    NARCIS (Netherlands)

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, J.; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-01-01

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression

  11. High density recombinant AAV particles are competent vectors for in vivo transduction

    Science.gov (United States)

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  12. New Paradigms for Virus Detection, Surveillance and Control of Zika Virus Vectors in the Settings of Southeast Asia

    Science.gov (United States)

    Vythilingam, Indra; Sam, Jamal I-C.; Chan, Yoke F.; Khaw, Loke T.; Sulaiman, Wan Y. Wan

    2016-01-01

    Zika virus (ZIKV) has now become a global public health concern. The vectors for ZIKV are Aedes aegypti and A. albopictus. Both these mosquitoes are predominant in Southeast Asia and are also responsible for the spread of other arboviral diseases like dengue virus and chikungunya virus. The incidence of dengue has been increasing over the years and this is of concern to public health workers. Simple laboratory tools for the detection of ZIKV is also lacking. In the absence of drugs and vaccine for these arboviral diseases, vector control is the main option for surveillance and control. Aedes larval surveys have been the hallmark of dengue control along with larviciding and fogging when cases are reported. However, we need new paradigms and options for control of these vectors. The current situation in Southeast Asia clearly proves that effective strategies for vector control need to be proactive and not reactive. This will be the way forward to control epidemics of these diseases inclusive of ZIKV until a vaccine becomes available. PMID:27679623

  13. New Paradigms for Virus Detection, Surveillance and Control of Zika Virus Vectors in the Settings of Southeast Asia

    Directory of Open Access Journals (Sweden)

    Indra Vythilingam

    2016-09-01

    Full Text Available Zika virus (ZIKV has now become a global public health concern. The vectors for ZIKV are Aedes aegypti and Aedes albopictus. Both these mosquitoes are predominant in Southeast Asia and are also responsible for the spread of other arboviral diseases like dengue virus (DENV and chikungunya virus (CHIKV. The incidence of dengue has been increasing over the years and this is of concern to public health workers. Simple laboratory tools for the detection of ZIKV is also lacking. In the absence of drugs and vaccine for these arboviral diseases, vector control is the main option for surveillance and control. Aedes larval surveys have been the hallmark of dengue control along with larviciding and fogging when cases are reported. However, we need new paradigms and options for control of these vectors. The current situation in Southeast Asia clearly proves that effective strategies for vector control need to be proactive and not reactive. This will be the way forward to control epidemics of these diseases inclusive of ZIKV until a vaccine becomes available.

  14. Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus

    Science.gov (United States)

    Ljungberg, Karl; Kümmerer, Beate M.; Gosse, Leslie; Dereuddre-Bosquet, Nathalie; Tchitchek, Nicolas; Hallengärd, David; García-Arriaza, Juan; Meinke, Andreas; Esteban, Mariano; Merits, Andres

    2017-01-01

    Chikungunya virus (CHIKV) is rapidly spreading across the globe, and millions are infected. Morbidity due to this virus is a serious threat to public health, but at present, there is no vaccine against this debilitating disease. We have recently developed a number of vaccine candidates, and here we have evaluated 3 of them in a nonhuman primate model. A single immunization with an attenuated strain of CHIKV (Δ5nsP3), a homologous prime-boost immunization with a DNA-launched RNA replicon encoding CHIKV envelope proteins (DREP-E), and a DREP-E prime followed by a recombinant modified vaccinia virus Ankara encoding CHIKV capsid and envelope (MVA-CE) boost all induced protection against WT CHIKV infection. The attenuated Δ5nsP3 virus proved to be safe and did not show any clinical signs typically associated with WT CHIKV infections such as fever, skin rash, lymphopenia, or joint swelling. These vaccines are based on an East/Central/South African strain of Indian Ocean lineage, but they also generated neutralizing antibodies against an isolate of the Asian genotype that now is rapidly spreading across the Americas. These results form the basis for clinical development of an efficacious CHIKV vaccine that generates both humoral and cellular immunity with long-term immunological memory. PMID:28352649

  15. A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

    Directory of Open Access Journals (Sweden)

    Liu Yi

    2012-10-01

    Full Text Available Abstract Background Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. Results We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116 and HDAC2 KO cells from the colorectal cancer cell line (DLD1. Conclusions Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.

  16. Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene

    Science.gov (United States)

    The thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of the thermostable NDV as a vaccine vector, an infectious clone of the...

  17. Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes.

    Science.gov (United States)

    Fortuna, C; Remoli, M E; Severini, F; Di Luca, M; Toma, L; Fois, F; Bucci, P; Boccolini, D; Romi, R; Ciufolini, M G

    2015-12-01

    West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.

  18. Engineering Cowpea Mosaic Virus RNA-2 into a vector to express heterologous proteins in plants

    NARCIS (Netherlands)

    Kodetham Gopinath,; Wellink, J.; Porta, C.; Taylor, K.M.; Lomonossoff, G.P.; Kammen, van A.

    2000-01-01

    series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP

  19. Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector

    OpenAIRE

    Haller, Aurelia A.; Miller, Tessa; Mitiku, Misrach; Coelingh, Kathleen

    2000-01-01

    Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive...

  20. Vector-virus mutualism accelerates population increase of an invasive whitefly.

    Directory of Open Access Journals (Sweden)

    Min Jiu

    Full Text Available The relationships between plant viruses, their herbivore vectors and host plants can be beneficial, neutral, or antagonistic, depending on the species involved. This variation in relationships may affect the process of biological invasion and the displacement of indigenous species by invaders when the invasive and indigenous organisms occur with niche overlap but differ in the interactions. The notorious invasive B biotype of the whitefly complex Bemisia tabaci entered China in the late 1990s and is now the predominant or only biotype in many regions of the country. Tobacco curly shoot virus (TbCSV and Tomato yellow leaf curl China virus (TYLCCNV are two whitefly-transmitted begomoviruses that have become widespread recently in south China. We compared the performance of the invasive B and indigenous ZHJ1 whitefly biotypes on healthy, TbCSV-infected and TYLCCNV-infected tobacco plants. Compared to its performance on healthy plants, the invasive B biotype increased its fecundity and longevity by 12 and 6 fold when feeding on TbCSV-infected plants, and by 18 and 7 fold when feeding on TYLCCNV-infected plants. Population density of the B biotype on TbCSV- and TYLCCNV-infected plants reached 2 and 13 times that on healthy plants respectively in 56 days. In contrast, the indigenous ZHJ1 performed similarly on healthy and virus-infected plants. Virus-infection status of the whiteflies per se of both biotypes showed limited effects on performance of vectors on cotton, a nonhost plant of the viruses. The indirect mutualism between the B biotype whitefly and these viruses via their host plants, and the apparent lack of such mutualism for the indigenous whitefly, may contribute to the ability of the B whitefly biotype to invade, the displacement of indigenous whiteflies, and the disease pandemics of the viruses associated with this vector.

  1. Vector competence of Culex tarsalis from Orange County, California, for West Nile virus.

    Science.gov (United States)

    Turell, Michael J; O'Guinn, Monica L; Dohm, David J; Webb, James P; Sardelis, Michael R

    2002-01-01

    To evaluate the vector competence of Culex tarsalis Coquillett for West Nile virus (WN), females reared from larvae collected in Huntington Beach, Orange County, CA, were fed on 2-3-day-old chickens previously inoculated with a New York strain (Crow 397-99) of WN. The Cx. tarsalis mosquitoes were efficient laboratory vectors of WN, with estimated transmission rates of 81% and 91% for mosquitoes that ingested 10(6.5) or 10(7.3) plaque-forming units of WN/mL of blood, respectively. Based on efficiency of viral transmission and the role of this species in the transmission of the closely related St. Louis encephalitis virus, Cx. tarsalis should be considered a potentially important vector of WN in the western United States.

  2. Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus.

    Science.gov (United States)

    Turell, M J; O'Guinn, M L; Dohm, D J; Jones, J W

    2001-03-01

    We evaluated the potential for several North American mosquito species to transmit the newly introduced West Nile (WN) virus. Mosquitoes collected in the New York City metropolitan area during the recent WN virus outbreak, at the Assateague Island Wildlife Refuge, VA, or from established colonies were allowed to feed on chickens infected with WN virus isolated from a crow that died during the 1999 outbreak. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus (Theobald) were highly susceptible to infection, and nearly all individuals with a disseminated infection transmitted virus by bite. Culex pipiens L. and Aedes sollicitans (Walker) were moderately susceptible. In contrast, Aedes vexans (Meigen), Aedes aegypti (L.), and Aedes taeniorhynchus (Wiedemann) were relatively refractory to infection, but individual mosquitoes inoculated with WN virus did transmit virus by bite. Infected female Cx. pipiens transmitted WN virus to one of 1,618 F1 progeny, indicating the potential for vertical transmission of this virus. In addition to laboratory vector competence, host-feeding preferences, relative abundance, and season of activity also determine the role that these species could play in transmitting WN virus.

  3. Exploring the limits of vector construction based on Citrus tristeza virus.

    Science.gov (United States)

    El-Mohtar, Choaa; Dawson, William O

    2014-01-05

    We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees.

  4. DNA Virus Vectors for Vaccine Production in Plants: Spotlight on Geminiviruses

    Directory of Open Access Journals (Sweden)

    Kathleen L. Hefferon

    2014-08-01

    Full Text Available Plants represent a safe, efficacious and inexpensive production platform by which to provide vaccines and other therapeutic proteins to the world’s poor. Plant virus expression vector technology has rapidly become one of the most popular methods to express pharmaceutical proteins in plants. This review discusses several of the state-of-the-art plant expression systems based upon geminiviruses that have been engineered for vaccine production. An overview of the advantages of these small, single-stranded DNA viruses is provided and comparisons are made with other virus expression systems. Advances in the design of several different geminivirus vectors are presented in this review, and examples of vaccines and other biologics generated from each are described.

  5. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes.

    Science.gov (United States)

    Zhu, Wuyang; Li, Jiangjiao; Tang, Li; Wang, Huanqin; Li, Jia; Fu, Juanjuan; Liang, Guodong

    2011-07-10

    To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  6. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  7. Enhancing the clinical potential of AAV vectors by capsid engineering to evade pre-existing immunity

    Directory of Open Access Journals (Sweden)

    Melissa eBartel

    2011-10-01

    Full Text Available Vectors based on adeno-associated viruses have shown considerable promise in both preclinical models and increasingly in clinical trials. However, one formidable challenge is pre-existing immunity due to widespread exposure to numerous AAV variants and serotypes within the human population, which affect efficacy of clinical trials due to the accompanying high levels of anti-capsid neutralizing antibodies. Transient immunosuppression has promise in mitigating cellular and humoral responses induced by vector application in naïve hosts, but cannot overcome the problem that pre-existing neutralizing antibodies pose towards the goal of safe and efficient gene delivery. Shielding of AAV from antibodies, however, may be possible by covalent attachment of polymers to the viral capsid or by encapsulation of vectors inside biomaterials. In addition, there has been considerable progress in using rational mutagenesis, combinatorial libraries, and directed evolution approaches to engineer capsid variants that are not recognized by anti-AAV antibodies generally present in the human population. While additional progress must be made, such strategies, alone or in combination with immunosuppression to avoid de novo induction of antibodies, have strong potential to significantly enhance the clinical efficacy of AAV vectors.

  8. Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

    Directory of Open Access Journals (Sweden)

    Juliette Doumayrou

    2016-11-01

    Full Text Available Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

  9. High expression of hepatitis B virus based vector with reporter gene in hepatitis B virus infection system

    Institute of Scientific and Technical Information of China (English)

    Shi-Hong Li; Wen-Ge Huang; Bing Huang; Xi-Gu Chen

    2007-01-01

    AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector.METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA.RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media.CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.

  10. Interleukin 10 Mediated By Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 in Rats

    Science.gov (United States)

    Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C.; Candiotti, Keith A.; Lubarsky, David A.; Hao, Shuanglin

    2014-01-01

    Background Human Immunodeficiency Virus (HIV)-associated sensory neuropathy is a common neurological complication of HIV infection affecting up to 30% of HIV-positive individuals. However, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments for HIV-neuropathic pain (NP). In this study, we tested the hypothesis that inhibition of proinflammatory factors with overexpression of interleukin (IL)-10 reduces HIV-related NP in a rat model. Methods NP was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. The hindpaws of rats were inoculated with nonreplicating herpes simplex virus (HSV) vectors expressing antiinflammatory cytokine IL-10 or control vector. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The mechanical threshold response was assessed over time using the area under curves (AUC). The expression of phosphorylated p38 mitogen-activated kinase, as tumor necrosis factor alpha, stromal cell-derived factor-1α (SDF-1α), and C-X-C chemokine receptor type 4 in both the lumbar spinal cord and the L4/5 dorsal root ganglia (DRG) was examined at 14 and 28 days after vector inoculation using Western blots. Results We found that in the gp120-induced NP model, IL-10 overexpression mediated by the HSV vector resulted in a significant elevation of the mechanical threshold that was apparent on day 3 after vector inoculation compared with the control vector (PIL-10 lasted more than 28 days. The AUC in the HSV vector expressing IL-10 was increased compared with that in the control vector (PIL-10 reversed the upregulation of phosphorylated p38 mitogen-activated kinase, as tumor necrosis factor alpha, stromal cell-derived factor-1α (SDF-1α), and C-X-C chemokine receptor type 4 expression at 14 and/or 28 days in the DRG and/or the spinal dorsal horn. Conclusions Our studies demonstrate that blocking the signaling of these

  11. Zika virus infection: Past and present of another emerging vector-borne disease

    Directory of Open Access Journals (Sweden)

    Hercules Sakkas

    2016-01-01

    Full Text Available Zika virus infection is an emerging mosquito-borne disease, first identified in Uganda in 1947. It is caused by the Zika arbovirus, and transmitted by the bites of infected mosquitoes of the genus Aedes. For almost half a century, the Zika virus was reported as the causative agent of sporadic human infections. In 2007, the Zika virus emerged outside Asia and Africa causing an epidemic on the Island of Yap in Micronesia. The manifestation of the newly acquired human infection varies from asymptomatic to self-limiting acute febrile illness with symptoms and clinical features similar to those caused by the Dengue virus (′Dengue-like syndrome′. The real-time PCR and serological methods have been successfully applied for the diagnosis of the disease. The treatment is symptomatic, since there is no specific antiviral treatment or a vaccine. During the recent outbreaks in French Polynesia and Brazil, incidents of Guillain-Barrι syndrome and microcephaly were associated with Zika virus infection, giving rise to fears of further global spread of the virus. Prevention and vector control strategies have to be urgently implemented by national health authorities in order to contain future outbreaks in vulnerable populations. This review summarizes the existing information on Zika virus characteristics, pathogenesis and epidemiology, the available methods for the diagnosis of Zika virus infection and recent approaches for prevention and control.

  12. Zika virus infection: Past and present of another emerging vector-borne disease.

    Science.gov (United States)

    Sakkas, Hercules; Economou, Vangelis; Papadopoulou, Chrissanthy

    2016-01-01

    Zika virus infection is an emerging mosquito-borne disease, first identified in Uganda in 1947. It is caused by the Zika arbovirus, and transmitted by the bites of infected mosquitoes of the genus Aedes. For almost half a century, the Zika virus was reported as the causative agent of sporadic human infections. In 2007, the Zika virus emerged outside Asia and Africa causing an epidemic on the Island of Yap in Micronesia. The manifestation of the newly acquired human infection varies from asymptomatic to self-limiting acute febrile illness with symptoms and clinical features similar to those caused by the Dengue virus ('Dengue-like syndrome'). The real-time PCR and serological methods have been successfully applied for the diagnosis of the disease. The treatment is symptomatic, since there is no specific antiviral treatment or a vaccine. During the recent outbreaks in French Polynesia and Brazil, incidents of Guillain-Barrι syndrome and microcephaly were associated with Zika virus infection, giving rise to fears of further global spread of the virus. Prevention and vector control strategies have to be urgently implemented by national health authorities in order to contain future outbreaks in vulnerable populations. This review summarizes the existing information on Zika virus characteristics, pathogenesis and epidemiology, the available methods for the diagnosis of Zika virus infection and recent approaches for prevention and control.

  13. Rationale for developing new virus vectors to analyze gene function in grasses through virus-induced gene silencing.

    Science.gov (United States)

    Ramanna, Hema; Ding, Xin Shun; Nelson, Richard S

    2013-01-01

    The exploding availability of genome and EST-based sequences from grasses requires a technology that allows rapid functional analysis of the multitude of genes that these resources provide. There are several techniques available to determine a gene's function. For gene knockdown studies, silencing through RNAi is a powerful tool. Gene silencing can be accomplished through stable transformation or transient expression of a fragment of a target gene sequence. Stable transformation in rice, maize, and a few other species, although routine, remains a relatively low-throughput process. Transformation in other grass species is difficult and labor-intensive. Therefore, transient gene silencing methods including Agrobacterium-mediated and virus-induced gene silencing (VIGS) have great potential for researchers studying gene function in grasses. VIGS in grasses already has been used to determine the function of genes during pathogen challenge and plant development. It also can be used in moderate-throughput reverse genetics screens to determine gene function. However, the number of viruses modified to serve as silencing vectors in grasses is limited, and the silencing phenotype induced by these vectors is not optimal: the phenotype being transient and with moderate penetration throughout the tissue. Here, we review the most recent information available for VIGS in grasses and summarize the strengths and weaknesses in current virus-grass host systems. We describe ways to improve current virus vectors and the potential of other grass-infecting viruses for VIGS studies. This work is necessary because VIGS for the foreseeable future remains a higher throughput and more rapid system to evaluate gene function than stable transformation.

  14. Expression of IMP1 enhances production of murine leukemia virus vector by facilitating viral genomic RNA packaging.

    Directory of Open Access Journals (Sweden)

    Yun Mai

    Full Text Available Murine leukemia virus (MLV-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1 enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

  15. Expression of IMP1 enhances production of murine leukemia virus vector by facilitating viral genomic RNA packaging.

    Science.gov (United States)

    Mai, Yun; Gao, Guangxia

    2010-12-29

    Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

  16. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  17. Intravenous injection of a foamy virus vector to correct canine SCID-X1.

    Science.gov (United States)

    Burtner, Christopher R; Beard, Brian C; Kennedy, Douglas R; Wohlfahrt, Martin E; Adair, Jennifer E; Trobridge, Grant D; Scharenberg, Andrew M; Torgerson, Troy R; Rawlings, David J; Felsburg, Peter J; Kiem, Hans-Peter

    2014-06-05

    Current approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3(+) T lymphocytes. CD3(+) cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

  18. Oncolytic herpes simplex virus vectors for the treatment of human breast cancer

    Institute of Scientific and Technical Information of China (English)

    LIU Ren-bin; Samuel D.Rabkin

    2005-01-01

    Background Oncolytic herpes simplex virus (HSV) vectors can be used for cancer therapy as direct cytotoxic agents, inducers of anti-tumor immune responses, and as expressers of anti-cancer genes. In this study, the efficacy of HSV vectors, G47Δ and NV1023 were examined for the treatment of the human breast cancer.Methods Human breast cancer MDA-MB-435 cells were cultured or implanted subcutaneously in BALB/c nude mice. The cells or tumors were inoculated with G47Δ or NV1023, and cell killing or inhibition of tumor growth determined. Both viruses contained the LacZ gene and expression in infected cells was detected with X-gal histochemistry. Results G47Δ and NV1023 were highly cytotoxic to MDA-MB-435 cells in vitro at very low multiplicities of infection. X-gal staining of infected tumor cells in vitro and in vivo illustrated the replication and spread of both viruses. G47Δ and NV1023 inoculation inhibited tumor growth and prolonged mouse survival. Both vectors behaved similarly.Conclusions Oncolytic HSV vectors, G47Δ and NV1023, were extremely effective at killing human breast cancer cells in vitro and in tumor xenografts in vivo. This novel form of cancer therapy warrants further investigation and consideration of clinical application.

  19. Varroa destructor is an effective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera.

    Science.gov (United States)

    Di Prisco, Gennaro; Pennacchio, Francesco; Caprio, Emilio; Boncristiani, Humberto F; Evans, Jay D; Chen, Yanping

    2011-01-01

    The Israeli acute paralysis virus (IAPV) is a significant marker of honeybee colony collapse disorder (CCD). In the present work, we provide the first evidence that Varroa destructor is IAPV replication-competent and capable of vectoring IAPV in honeybees. The honeybees became infected with IAPV after exposure to Varroa mites that carried the virus. The copy number of IAPV in bees was positively correlated with the density of Varroa mites and time period of exposure to Varroa mites. Further, we showed that the mite-virus association could possibly reduce host immunity and therefore promote elevated levels of virus replication. This study defines an active role of Varroa mites in IAPV transmission and sheds light on the epidemiology of IAPV infection in honeybees.

  20. Aedes albopictus may not be vector of dengue virus in human epidemics in Brazil

    Directory of Open Access Journals (Sweden)

    Degallier Nicolas

    2003-01-01

    Full Text Available Over 60,500 dengue cases were reported in the state of Espírito Santo (ES, Brazil, between 1995 and 1998. The study's purpose was to identify whether Aedes albopictus was transmitting the dengue virus during an epidemic in the locality of Vila Bethânia (Viana County,Vitória, ES. From April 3 to 9, 1998, blood and serum samples were collected daily for virus isolation and serological testing. Four autochthonous cases were confirmed through DEN 1 virus isolation and two autochthonous cases through MAC ELISA testing. Of 37 Ae. aegypti and 200 Ae. albopictus adult mosquitoes collected and inoculated, DEN1 virus was isolated only from a pool of two Ae. aegypti female mosquitoes. The study results suggest that Ae. albopictus still cannot be considered an inter-human vector in dengue epidemics in Brazil.

  1. Avirulent Marek's disease virus type 1 strain 814 vectored vaccine expressing avian influenza (AI virus H5 haemagglutinin induced better protection than turkey herpesvirus vectored AI vaccine.

    Directory of Open Access Journals (Sweden)

    Hongyu Cui

    Full Text Available BACKGROUND: Herpesvirus of turkey (HVT as a vector to express the haemagglutinin (HA of avian influenza virus (AIV H5 was developed and its protection against lethal Marek's disease virus (MDV and highly pathogenic AIV (HPAIV challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1. METHODOLOGY/PRINCIPAL FINDINGS: A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%. In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose. CONCLUSIONS/SIGNIFICANCE: The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.

  2. Properties and use of novel replication-competent vectors based on Semliki Forest virus

    Directory of Open Access Journals (Sweden)

    Lulla Valeria

    2009-03-01

    Full Text Available Abstract Background Semliki Forest virus (SFV has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. Results We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. Conclusion The properties of replication-competent vectors

  3. Ecological niche modelling of Rift Valley fever virus vectors in Baringo, Kenya

    Directory of Open Access Journals (Sweden)

    Alfred O. Ochieng

    2016-11-01

    Full Text Available Background: Rift Valley fever (RVF is a vector-borne zoonotic disease that has an impact on human health and animal productivity. Here, we explore the use of vector presence modelling to predict the distribution of RVF vector species under climate change scenario to demonstrate the potential for geographic spread of Rift Valley fever virus (RVFV. Objectives: To evaluate the effect of climate change on RVF vector distribution in Baringo County, Kenya, with an aim of developing a risk map for spatial prediction of RVF outbreaks. Methodology: The study used data on vector presence and ecological niche modelling (MaxEnt algorithm to predict the effect of climatic change on habitat suitability and the spatial distribution of RVF vectors in Baringo County. Data on species occurrence were obtained from longitudinal sampling of adult mosquitoes and larvae in the study area. We used present (2000 and future (2050 Bioclim climate databases to model the vector distribution. Results: Model results predicted potential suitable areas with high success rates for Culex quinquefasciatus, Culex univitattus, Mansonia africana, and Mansonia uniformis. Under the present climatic conditions, the lowlands were found to be highly suitable for all the species. Future climatic conditions indicate an increase in the spatial distribution of Cx. quinquefasciatus and M. africana. Model performance was statistically significant. Conclusion: Soil types, precipitation in the driest quarter, precipitation seasonality, and isothermality showed the highest predictive potential for the four species.

  4. Localization of Transmissible and Nontransmissible Viruses in the Vector Nematode Xiphinema americanum.

    Science.gov (United States)

    Wang, Shouhua; Gergerich, Rose C; Wickizer, Sandra L; Kim, Kyung S

    2002-06-01

    ABSTRACT The inner lining of the food canal of nematodes that transmit plantinfecting viruses is regarded as the retention region of viruses. To characterize the location of transmissible and nontransmissible viruses in the vector nematode Xiphinema americanum, three nepoviruses, Tobacco ringspot virus (TRSV), Tomato ringspot virus(TomRSV), and Cherry leaf roll virus(CLRV), and one non-nematode-transmissible virus, Squash mosaic virus (SqMV), were evaluated for transmission efficiency and localization sites in the nematode. Transmission trials showed highest transmission efficiency for TomRSV (38% with 1 and 100% with 10 nematodes, respectively), intermediate efficiency for TRSV (27% with 1 and 65% with 10 nematodes, respectively), and no transmission for CLRV and SqMV. Electron microscopy and immunofluorescent labeling revealed that TRSV was primarily localized to the lining of the lumen of the stylet extension and the anterior esophagus, but only rarely in the triradiate lumen. Within a nematode population, particles of TRSV were no longer observed in these three regions 10 weeks after acquisition, and it is assumed that there was gradual and random loss of the virus from these areas. The percentage of nematodes that were labeled by virus-specific immunofluorescent labeling in a population of viruliferous nematodes decreased gradually after TRSV acquisition when the nematodes were placed on a nonhost of the virus, and the loss of immunofluorescent labeling paralleled the decrease in the ability of the nematode population to transmit the virus. TomRSV was localized only in the triradiate lumen based on thin-section electron microscopy. No virus-like particles were observed in any part of the food canal of nematodes that had fed on CLRV-infected plants. Virus-like particles that appeared to be partially degraded were observed only in the triradiate lumen of nematodes that had fed on SqMV-infected plants. These results clarified the status of localization of two

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

    Science.gov (United States)

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

    2008-03-01

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

  6. Evidence of local adaptation in plant virus effects on host-vector interactions.

    Science.gov (United States)

    Mauck, K E; De Moraes, C M; Mescher, M C

    2014-07-01

    Recent research suggests that plant viruses, and other pathogens, frequently alter host-plant phenotypes in ways that facilitate transmission by arthropod vectors. However, many viruses infect multiple hosts, raising questions about whether these pathogens are capable of inducing transmission-facilitating phenotypes in phylogenetically divergent host plants and the extent to which evolutionary history with a given host or plant community influences such effects. To explore these issues, we worked with two newly acquired field isolates of cucumber mosaic virus (CMV)-a widespread multi-host plant pathogen transmitted in a non-persistent manner by aphids-and explored effects on the phenotypes of different host plants and on their subsequent interactions with aphid vectors. An isolate collected from cultivated squash fields (KVPG2-CMV) induced in the native squash host (Cucurbita pepo) a suite of effects on host-vector interactions suggested by previous work to be conducive to transmission (including reduced host-plant quality for aphids, rapid aphid dispersal from infected to healthy plants, and enhanced aphid attraction to the elevated emission of a volatile blend similar to that of healthy plants). A second isolate (P1-CMV) collected from cultivated pepper (Capsicum annuum) induced more neutral effects in its native host (largely exhibiting non-significant trends in the direction of effects seen for KVPG2-CMV in squash). When we attempted cross-host inoculations of these two CMV isolates (KVPG2-CMV in pepper and P1-CMV in squash), P1-CMV was only sporadically able to infect the novel host; KVPG2-CMV infected the novel pepper host with somewhat reduced success compared with its native host and reached virus titers significantly lower than those observed for either strain in its native host. Furthermore, KVPG2-CMV induced changes in the phenotype of the novel host, and consequently in host-vector interactions, dramatically different than those observed in the native

  7. Weather, host and vector--their interplay in the spread of insect-borne animal virus diseases.

    Science.gov (United States)

    Sellers, R F

    1980-08-01

    The spread of insect-borne animal virus diseases is influenced by a number of factors. Hosts migrate, move or are conveyed over long distances: vectors are carried on the wind for varying distances in search of hosts and breeding sites; weather and climate affect hosts and vectors through temperature, moisture and wind. As parasites of host and vector, viruses are carried by animals, birds and insects, and their spread can be correlated with the migration of hosts and the carriage of vectors on winds associated with the movements of the Intertropical Convergence Zone (ITCZ) and warm winds to the north and south of the limits of the ITCZ. The virus is often transmitted from a local cycle to a migratory cycle and back again.Examples of insect-borne virus diseases and their spread are analysed. Japanese, Murray Valley, Western equine, Eastern equine and St Louis encephalitis represent viruses transmitted by mosquito-bird or pig cycles.THE AREAS EXPERIENCING INFECTION WITH THESE VIRUSES CAN BE DIVIDED INTO A NUMBER OF ZONES: A, B, C, D, E and F. In zone A there is a continuous cycle of virus in host and vector throughout the year; in zone B, there is an upsurge in the cycle during the wet season, but the cycle continues during the dry season; there is movement of infected vectors between and within zones A and B on the ITCZ and the virus is introduced to zone C by infected vectors on warm winds; persistence may occur in zone C if conditions are right. In zone D, virus is introduced each year by infected vectors on warm winds and the arrival of the virus coincides with the presence of susceptible nestling birds and susceptible piglets. The disappearance of virus occurs at the time when migrating mosquitoes and birds are returning to warmer climates. The virus is introduced to zone E only on occasions every 5-10 years when conditions are suitable. Infected hosts introduced to zone F do not lead to circulation of virus, since the climate is unsuitable for vectors. Zones A

  8. Construction and Application of Newcastle Disease Virus-Based Vector Vaccines.

    Science.gov (United States)

    Wichgers Schreur, Paul J

    2016-01-01

    Paramyxoviruses are able to stably express a wide-variety of heterologous antigens at relatively high levels in various species and are consequently considered as potent gene delivery vehicles. A single vaccination is frequently sufficient for the induction of robust humoral and cellular immune responses. Here we provide detailed methods for the construction and application of Newcastle disease virus (NDV)-based vector vaccines. The in silico design and in vitro rescue as well as the in vivo evaluation of NDV based vectors are described in this chapter.

  9. Wolbachia Occurrence in Planthopper (Hemiptera: Delphacidae) Vectors of Cereal Viruses in Argentina.

    Science.gov (United States)

    Mattio, M F; Argüello Caro, E B; Rodriguero, M S; Dumón, A D; Alemandri, V M; Truol, G

    2015-08-01

    Maize (Zea mays L.) and wheat (Triticum aestivum L.) are the most important cereal crops for the Argentinean economy and are affected by several diseases. Different planthopper species transmit causal agents of some of those diseases, including Mal de Río Cuarto virus, barley yellow striate mosaic virus, and the recently proposed maize yellow striate virus. Many planthopper species are sap feeders and therefore are expected to host bacteria providing essential nutrients lacking in the diet. Previous studies have evidenced that some of these bacterial symbionts are involved in the virus transmission. Wolbachia is a group of obligate intracellular bacteria infecting numerous arthropod species and causing reproductive alterations in their hosts. These bacteria have been detected in planthopper species, considered rice pests in various regions of the world. To date, Wolbachia infection status of planthopper species of Argentina is unknown. Amplification by PCR and sequencing of 16S rDNA, wsp- and ftsZ-specific genes demonstrated Wolbachia infection in Caenodelphax teapae (Fowler), Delphacodes kuscheli Fennah, Pyrophagus tigrinus Remes Lenicov & Varela, Tagosodes orizicolus (Muir), and Toya propinqua (Fieber). This is the first report of Wolbachia in delphacid vectors of viruses affecting maize and wheat. An understanding of the bacterial diversity harbored by these insect vectors could lead to new options for future management of diseases of economically important crops in a developing country.

  10. Dengue and Zika viruses: lessons learned from the similarities between these Aedes mosquito-vectored arboviruses.

    Science.gov (United States)

    Suwanmanee, San; Luplertlop, Natthanej

    2017-02-01

    The currently spreading arbovirus epidemic is having a severe impact on human health worldwide. The two most common flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), are transmitted through the same viral vector, Aedes spp. mosquitoes. Since the discovery of DENV in 1943, this virus has been reported to cause around 390 million human infections per year, approximately 500,000 of which require hospitalization and over 20,000 of which are lethal. The present DENV epidemic is primarily concentrated in Southeast Asia. ZIKV, which was discovered in 1952, is another important arthropod-borne flavivirus. The neurotropic role of ZIKV has been reported in infected newborns with microcephaly and in adults with Guillain-Barre syndrome. Despite DENV and ZIKV sharing the same viral vector, their complex pathogenic natures are poorly understood, and the infections they cause do not have specific treatments or effective vaccines. Therefore, this review will describe what is currently known about the clinical characteristics, pathogenesis mechanisms, and transmission of these two viruses. Better understanding of the interrelationships between DENV and ZIKV will provide a useful perspective for developing an effective strategy for controlling both viruses in the future.

  11. Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

    Directory of Open Access Journals (Sweden)

    Eutimio Gustavo Fernández Núñez

    2013-10-01

    Full Text Available The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

  12. Viral vectors for cystic fibrosis gene therapy: What does the future hold?

    Directory of Open Access Journals (Sweden)

    Uta Griesenbach

    2010-12-01

    Full Text Available Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

  13. Genomic approaches for understanding dengue: insights from the virus, vector, and host.

    Science.gov (United States)

    Sim, Shuzhen; Hibberd, Martin L

    2016-03-02

    The incidence and geographic range of dengue have increased dramatically in recent decades. Climate change, rapid urbanization and increased global travel have facilitated the spread of both efficient mosquito vectors and the four dengue virus serotypes between population centers. At the same time, significant advances in genomics approaches have provided insights into host-pathogen interactions, immunogenetics, and viral evolution in both humans and mosquitoes. Here, we review these advances and the innovative treatment and control strategies that they are inspiring.

  14. Haemaphysalis longicornis Ticks as Reservoir and Vector of Severe Fever with Thrombocytopenia Syndrome Virus in China

    OpenAIRE

    Luo, Li-mei; Zhao, Li; Wen, Hong-Ling; Zhang, Zhen-Tang; Liu, Jian-Wei; Fang, Li-Zhu; Xue, Zai-Feng; Ma, Dong-Qiang; Zhang, Xiao-Shuang; Ding, Shu-Jun; Lei, Xiao-Ying; Yu, Xue-jie

    2015-01-01

    Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia caused by SFTS virus (SFTSV), a newly discovered phlebovirus. The Haemaphysalis longicornis tick has been suspected to be the vector of SFTSV. To determine whether SFTSV can be transmitted among ticks, from ticks to animals, and from animals to ticks, we conducted transmission studies between developmental stages of H. longicornis ticks and between ticks and mice. Using reverse transcription PCR, ...

  15. Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

  16. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

  17. Gene therapy as a therapeutic approach for the treatment of rheumatoid arthritis: innovative vectors and therapeutic genes.

    Science.gov (United States)

    Adriaansen, J; Vervoordeldonk, M J B M; Tak, P P

    2006-06-01

    In recent years, significant progress has been made in the treatment of rheumatoid arthritis (RA). In addition to conventional therapy, novel biologicals targeting tumour necrosis factor-alpha have successfully entered the clinic. However, the majority of the patients still has some actively inflamed joints and some patients suffer from side-effects associated with the high systemic dosages needed to achieve therapeutic levels in the joints. In addition, due to of the short half-life of these proteins there is a need for continuous, multiple injections of the recombinant protein. An alternative approach might be the use of gene transfer to deliver therapeutic genes locally at the site of inflammation. Several viral and non-viral vectors are being used in animal models of RA. The first gene therapy trials for RA have already entered the clinic. New vectors inducing long-term and regulated gene expression in specific tissue are under development, resulting in more efficient gene transfer, for example by using distinct serotypes of viral vectors such as adeno-associated virus. This review gives an overview of some promising vectors used in RA research. Furthermore, several therapeutic genes are discussed that could be used for gene therapy in RA patients.

  18. Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

    Science.gov (United States)

    Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

    2013-09-17

    Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

  19. Paracoccus burnerae (HOMOPTERA; PLANOCOCCIDAE AS A VECTOR OF Banana streak virus

    Directory of Open Access Journals (Sweden)

    Muturi S M

    2013-11-01

    Full Text Available The Banana streak virus ( BSV is a causative agent of the banana streak disease (BSD which causes considerable damage to banana production in tropical countries. The virus is vectored by several mealy bug species. However, the competence of the oleander mealy bug ( Paracoccus burnerae , in the transmission of BSV is unknown. Rolling Circle Amplification (RCA technique was used to select both diseased and healthy plantlets fo r transmission experiments. RCA was conducted on viruliferous instars of P . burnerae and virus - inoculated plantlet DNA samples. The results revealed that P . burnerae is a vector of BSV . However, during hot conditions (24 - 30ºC, the insect was unable to acq uire and transmit BSV . Under cool conditions (9 - 20ºC, a minimum of 6 h of feeding time was necessary for P . burnerae instars to become viruliferous. These results indicate that P . burnerae is a vector of BSV and transmission efficiency depends on the ambi ent temperature and the feeding time.

  20. Evolution of codon usage in Zika virus genomes is host and vector specific.

    Science.gov (United States)

    Butt, Azeem Mehmood; Nasrullah, Izza; Qamar, Raheel; Tong, Yigang

    2016-10-12

    The codon usage patterns of viruses reflect the evolutionary changes that allow them to optimize their survival and adapt their fitness to the external environment and, most importantly, their hosts. Here we report the genotype-specific codon usage patterns of Zika virus (ZIKV) strains from the current and previous outbreaks. Several genotype-specific and common codon usage traits were noted in the ZIKV coding sequences, indicating their independent evolutionary origins from a common ancestor. The overall influence of natural selection was more profound than that of mutation pressure, acting on a specific set of viral genes in the Asian-genotype ZIKV strains from the recent outbreak. An interplay between codon adaptation and deoptimization may have allowed the virus to adapt to multiple host and vectors and is reported for the first time in ZIKV genomes. Combining our codon analysis with geographical data on Aedes populations in the Americas suggested that ZIKV has evolved host- and vector-specific codon usage patterns to maintain successful replication and transmission chains within multiple hosts and vectors.

  1. Tracking of specific integrant clones in dogs treated with foamy virus vectors.

    Science.gov (United States)

    Ohmine, Ken; Li, Yi; Bauer, Thomas R; Hickstein, Dennis D; Russell, David W

    2011-02-01

    Vector integration can lead to proto-oncogene activation and malignancies during hematopoietic stem cell gene therapy. We previously used foamy virus vectors to deliver the CD18 gene under the control of an internal murine stem cell virus promoter and successfully treated dogs with canine leukocyte adhesion deficiency. Here we have tracked the copy numbers of 11 specific proviruses found in these animals for 36-42 months after transplantation, including examples within or near proto-oncogenes, tumor suppressor genes, and genes unrelated to cancer. We found no evidence for clonal expansion of any of the clones, including those with proviruses in the MECOM gene (MDS1-EVI1 complex). These results suggest that although foamy virus vectors may integrate near proto-oncogenes, this does not necessarily lead to clonal expansion and malignancies. Additionally, we show that copy number estimates of these specific proviruses based on linker-mediated PCR results are different from those obtained by quantitative PCR, but can provide a qualitative assessment of provirus levels.

  2. Seasonal activity, vector relationships and genetic analysis of mosquito-borne Stratford virus

    Science.gov (United States)

    Toi, Cheryl S.; Webb, Cameron E.; Haniotis, John; Clancy, John; Doggett, Stephen L.

    2017-01-01

    There are many gaps to be filled in our understanding of mosquito-borne viruses, their relationships with vectors and reservoir hosts, and the environmental drivers of seasonal activity. Stratford virus (STRV) belongs to the genus Flavivirus and has been isolated from mosquitoes and infected humans in Australia but little is known of its vector and reservoir host associations. A total of 43 isolates of STRV from mosquitoes collected in New South Wales between 1995 and 2013 was examined to determine the genetic diversity between virus isolates and their relationship with mosquito species. The virus was isolated from six mosquito species; Aedes aculeatus, Aedes alternans, Aedes notoscriptus, Aedes procax, Aedes vigilax, and Anopheles annulipes. While there were distinct differences in temporal and spatial activity of STRV, with peaks of activity in 2006, 2010 and 2013, a sequence homology of 95.9%–98.4% was found between isolates and the 1961 STRV prototype with 96.2%–100% identified among isolates. Temporal differences but no apparent nucleotide divergence by mosquito species or geographic location was evident. The result suggests the virus is geographically widespread in NSW (albeit only from coastal regions) and increased local STRV activity is likely to be driven by reservoir host factors and local environmental conditions influencing vector abundance. While STRV may not currently be associated with major outbreaks of human disease, with the potential for urbanisation and climate change to increase mosquito-borne disease risks, and the possibility of genomic changes which could produce pathogenic strains, understanding the drivers of STRV activity may assist the development of strategic response to public health risks posed by zoonotic flaviviruses in Australia. PMID:28253306

  3. Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables.

    Science.gov (United States)

    Deichmeister, Jayne M; Telang, Aparna

    2011-06-01

    It is currently unclear if the potential for West Nile virus transmission by mosquito vectors in the eastern United States is related to landscape or climate factors or both. We compared abundance of vector species between urban and suburban neighborhoods of Henrico County, VA, in relation to the following factors: temperature, precipitation, canopy cover, building footprint, and proximity to drainage infrastructure. Mosquitoes were collected throughout the 2005, 2006, and 2007 seasons and tested for West Nile virus (WNV) in pools of 10-50. Test results of mosquito pools were compared to average site abundance from 37 sites in Henrico County, VA; abundance was then examined in relation to ecological variables. Urban infrastructure was positively correlated with the abundance of Culex pipiens L./Cx. restuans, and our findings implicate combined sewer overflow systems as large contributors to Culex vector populations. No measure of urbanization examined in our study was correlated with Aedes albopictus abundance. Our study showed that certain landscape variables identified using Geographic Information Systems are valuable for predicting primary WNV vector abundance in Virginia, and that temperature along with low precipitation are strong predictors of population growth. Our results support other regional studies that found WNV proliferates under drought conditions.

  4. In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

    Institute of Scientific and Technical Information of China (English)

    Dennis Hoffmann; Wibke Bayer; Oliver Wildner

    2007-01-01

    AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and "F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/ F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector-treated tumor. We assessed the induction of a tumor-specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay.RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

  5. The vector competence of colonized Aedes (Stegomyia) katherinensis for dengue-2 virus.

    Science.gov (United States)

    Leake, C J

    1984-01-01

    Colonized Aedes (Stegomyia) katherinensis mosquitoes from Australia were infected with the PR-159 strain of dengue-2 virus using a membrane feeding technique and by intrathoracic inoculation. Virus replication to low levels was detected when mosquitoes infected by both routes were assayed using the virus-sensitive Ae. pseudoscutellaris (LSTM-AP-61) mosquito cell line in a microculture system. Analysis by indirect immunofluorescence revealed the expected 100% infection rates in inoculated mosquitoes compared with only 45% in orally infected mosquitoes. Few of the orally infected mosquitoes showed any viral antigen associated with the head and no virus transmission was detected. Preliminary studies also demonstrated that Ae. (S.) katherinesis was refractory to oral infection with Japanese encephalitis virus but was readily infected by intrathoracic inoculation. On the basis of this data, it is concluded that there is a high threshold of infection in this mosquito and that it is unlikely that Ae. (S.) katherinensis could be important as a vector of dengue-2 virus in Australia.

  6. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

    Science.gov (United States)

    Falkner, F G; Moss, B

    1988-06-01

    Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

  7. Tick-borne encephalitis virus subtypes emerged through rapid vector switches rather than gradual evolution.

    Science.gov (United States)

    Kovalev, Sergey Y; Mukhacheva, Tatyana A

    2014-11-01

    Tick-borne encephalitis is the most important human arthropod-borne virus disease in Europe and Russia, with an annual incidence of about 13 thousand people. Tick-borne encephalitis virus (TBEV) is distributed in the natural foci of forest and taiga zones of Eurasia, from the Pacific to the Atlantic coast. Currently, there are three mutually exclusive hypotheses about the origin and distribution of TBEV subtypes, although they are based on the same assumption of gradual evolution. Recently, we have described the structure of TBEV populations in terms of a clusteron approach, a clusteron being a structural unit of viral population [Kovalev and Mukhacheva (2013) Infect. Genet. Evol., 14, 22-28]. This approach allowed us to investigate questions of TBEV evolution in a new way and to propose a hypothesis of quantum evolution due to a vector switch. We also consider a possible mechanism for this switch occurring in interspecific hybrids of ticks. It is necessarily accompanied by a rapid accumulation of mutations in the virus genome, which is contrary to the generally accepted view of gradual evolution in assessing the ages of TBEV populations. The proposed hypothesis could explain and predict not only the formation of new subtypes, but also the emergence of new vector-borne viruses.

  8. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

    Science.gov (United States)

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-10-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

  9. Artemia franciscana as a vector for infectious myonecrosis virus (IMNV) to Litopenaeus vannamei juvenile.

    Science.gov (United States)

    da Silva, Suzianny Maria Bezerra Cabral; Lavander, Henrique David; de Santana Luna, Manuella Maria; de Melo Eloi da Silva, Ana Odete; Gálvez, Alfredo Olivera; Coimbra, Maria Raquel Moura

    2015-03-01

    In 2004, the infectious myonecrosis virus (IMNV) was recognized as the main cause of Litopenaeusvannamei shrimp culture's drop in Brazil. In health animal control programs, in order to reduce virus prevalence in production units it is necessary to screen live feed used. Among live diets used in aquaculture, the brine shrimp Artemia sp. is essential in crustacean larviculture and maturation. The aim of the present study was to investigate the susceptibility of Artemiafranciscana to IMNV through an immersion challenge and virus-phytoplankton adhesion route and to elucidate its role as a vector for IMNV transmission to L.vannamei. A. franciscana adults were infected with IMNV through both routes, as demonstrated by PCR-positive reactions. However, infected A. franciscana showed no signs of infection. More than 40% of L. vannamei juveniles fed with IMNV-infected A. franciscana by virus-phytoplankton adhesion route were positive by real-time PCR, whereas only a 10% infection rate was found among shrimp fed with IMNV-infected brine shrimp using the immersion challenge. Significant differences were found in mean viral load between immersion and virus-phytoplankton adhesion shrimp treatments (p ⩽ 0.05). Moreover, the mean viral loads were 1.34 × 10(2) and 1.48 × 10(4) copies/μg(-1) of total RNA for virus-phytoplankton adhesion and IMNV-infected tissue treatments, respectively, and the difference was not significant (p ⩾ 0.05). The results indicated that A. franciscana act as a vector for IMNV transmission under the experimental conditions examined. Although no mass mortalities were detected in L. vannamei fed with IMNV-infected brine shrimp, these infected shrimp should not be disregarded as a source of IMNV in grow-out units.

  10. Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Juan YIN; Yuan-yuan ZHANG; Shu-ya JIA; Zuo-jia CHEN; Jiang ZHONG

    2012-01-01

    Aim:To compare the specific immune responses elicited by different baculovirus vectors in immunized mice.Methods:We constructed and characterized two recombinant baculoviruses carrying the expression cassette for the H5N1 influenza virus hemagglutinin (HA) gene driven by either an insect cell promoter (vAc-HA) or a dual-promoter active both in insect and mammalian cells (vAc-HA-DUAL).Virus without the HA gene (vAc-EGFP) was used as a control.These viruses were used to immunize mice subcutaneously and intraperitoneally.The production of total and specific antibodies was determined by ELISA and competitive ELISA.Cytokine production by the spleen cells of immunized mice was studied using the ELISPOT assay.Results:Both the vAc-HA and vAc-HA-DUAL vectors expressed HA proteins in insect Sf9 cells,and HA antigen was present in progeny virions.The vAc-HA-DUAL vector also mediated HA expression in virus-transduced mammalian cell lines (BHK and A547).Both vAo-HA and vAc-HA-DUAL exhibited higher transduction efficiencies than vAc-EGFP in mammalian cells,as shown by the expression of the reporter gene egfp.Additionally,both vAc-HA and vAc-HA-DUAL induced high levels of HA-specific antibody production in immunized mice; vAc-HA-DUAL was more efficient in inducing IFN-Y and IL-2 upon stimulation with specific antigen,whereas vAc-HA was more efficient in inducing IL-4 and IL-6.Conclusion:Baculovirus vectors elicited efficient,specific immune responses in immunized mice.The vector displaying the HA antigen on the virion surface (vAc-HA) elicited a Th2-biased immune response,whereas the vector displaying HA and mediating HA expression in the cell (vAc-HA-DUAL) elicited a Th1-biased immune response.

  11. Transcriptome of the plant virus vector Graminella nigrifrons, and the molecular interactions of Maize fine streak rhabdovirus transmission

    Science.gov (United States)

    Background: Leafhoppers (Hemiptera:Cicadellidae) are plant-phloem feeders that are known for their ability to vector plant pathogens. The black-faced leafhopper (Graminella nigrifrons) has been identified as the only known vector for the Maize fine streak virus (MFSV), an emerging plant pathogen in...

  12. Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS v...

  13. Quantification of vector and host competence and abundance for Japanese Encephalitis Virus: a systematic review of the literature.

    Science.gov (United States)

    Japanese encephalitis (JE) is a vector-borne disease caused by the Japanese encephalitis virus (JEV) that affects humans in Eastern and Southeastern Asia. Although it could be prevented by a vaccine, JE has no treatment and the inadvertent introduction of the virus into JEV-free countries, such as t...

  14. A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling.

    Directory of Open Access Journals (Sweden)

    Estanislao Nistal-Villan

    Full Text Available Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV. In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.

  15. Targeting the central nervous system with herpes simplex virus / Sleeping Beauty hybrid amplicon vectors.

    Science.gov (United States)

    de Silva, Suresh; Bowers, William J

    2011-10-01

    The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies.

  16. European Aedes albopictus and Culex pipiens Are Competent Vectors for Japanese Encephalitis Virus

    Science.gov (United States)

    Desprès, Philippe

    2017-01-01

    Background Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis, the leading cause of viral encephalitis in Asia. JEV transmission cycle involves mosquitoes and vertebrate hosts. The detection of JEV RNA in a pool of Culex pipiens caught in 2010 in Italy raised the concern of a putative emergence of the virus in Europe. We aimed to study the vector competence of European mosquito populations, such as Cx. pipiens and Aedes albopictus for JEV genotypes 3 and 5. Findings After oral feeding on an infectious blood meal, mosquitoes were dissected at various times post-virus exposure. We found that the peak for JEV infection and transmission was between 11 and 13 days post-virus exposure. We observed a faster dissemination of both JEV genotypes in Ae. albopictus mosquitoes, when compared with Cx. pipiens mosquitoes. We also dissected salivary glands and collected saliva from infected mosquitoes and showed that Ae. albopictus mosquitoes transmitted JEV earlier than Cx. pipiens. The virus collected from Ae. albopictus and Cx. pipiens saliva was competent at causing pathogenesis in a mouse model for JEV infection. Using this model, we found that mosquito saliva or salivary glands did not enhance the severity of the disease. Conclusions In this study, we demonstrated that European populations of Ae. albopictus and Cx. pipiens were efficient vectors for JEV transmission. Susceptible vertebrate species that develop high viremia are an obligatory part of the JEV transmission cycle. This study highlights the need to investigate the susceptibility of potential JEV reservoir hosts in Europe, notably amongst swine populations and local water birds. PMID:28085881

  17. Regional and seasonal response of a West Nile virus vector to climate change.

    Science.gov (United States)

    Morin, Cory W; Comrie, Andrew C

    2013-09-24

    Climate change will affect the abundance and seasonality of West Nile virus (WNV) vectors, altering the risk of virus transmission to humans. Using downscaled general circulation model output, we calculate a WNV vector's response to climate change across the southern United States using process-based modeling. In the eastern United States, Culex quinquefasciatus response to projected climate change displays a latitudinal and elevational gradient. Projected summer population depressions as a result of increased immature mortality and habitat drying are most severe in the south and almost absent further north; extended spring and fall survival is ubiquitous. Much of California also exhibits a bimodal pattern. Projected onset of mosquito season is delayed in the southwestern United States because of extremely dry and hot spring and summers; however, increased temperature and late summer and fall rains extend the mosquito season. These results are unique in being a broad-scale calculation of the projected impacts of climate change on a WNV vector. The results show that, despite projected widespread future warming, the future seasonal response of C. quinquefasciatus populations across the southern United States will not be homogeneous, and will depend on specific combinations of local and regional conditions.

  18. Projection of Climate Change Influences on U.S. West Nile Virus Vectors.

    Science.gov (United States)

    Brown, Heidi E; Young, Alex; Lega, Joceline; Andreadis, Theodore G; Schurich, Jessica; Comrie, Andrew

    2015-12-01

    While estimates of the impact of climate change on health are necessary for health care planners and climate change policy makers, models to produce quantitative estimates remain scarce. We describe a freely available dynamic simulation model parameterized for three West Nile virus vectors, which provides an effective tool for studying vector-borne disease risk due to climate change. The Dynamic Mosquito Simulation Model is parameterized with species specific temperature-dependent development and mortality rates. Using downscaled daily weather data, we estimate mosquito population dynamics under current and projected future climate scenarios for multiple locations across the country. Trends in mosquito abundance were variable by location, however, an extension of the vector activity periods, and by extension disease risk, was almost uniformly observed. Importantly, mid-summer decreases in abundance may be off-set by shorter extrinsic incubation periods resulting in a greater proportion of infective mosquitoes. Quantitative descriptions of the effect of temperature on the virus and mosquito are critical to developing models of future disease risk.

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

    Science.gov (United States)

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

    2013-12-01

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

  20. A top-down approach for construction of hybrid polymer-virus gene delivery vectors.

    Science.gov (United States)

    Ramsey, Joshua D; Vu, Halong N; Pack, Daniel W

    2010-05-21

    Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

  1. Spread of Zika virus:The key role of mosquito vector control

    Institute of Scientific and Technical Information of China (English)

    Giovanni Benelli

    2016-01-01

    Mosquitoes (Diptera: Culicidae) represent a key threat for millions of humans and ani-mals worldwide, since they act as vectors for important parasites and pathogens, including malaria, filariasis and a wide number of arboviruses. The recent outbreaks of Zika virus infections occurring in South America, Central America, and the Caribbean, represent the most recent four arrivals of important arboviruses in the western hemi-sphere, over the last 20 years, namely dengue, West Nile virus, and chikungunya. Since there are no specific treatments for Zika virus and the other arboviruses mentioned above, it should be highlighted that the eco-friendly and effective control of mosquito vectors is of pivotal importance. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in presence of ultra-low quantities of green-synthesized nano-particles, which magnify their predation efficiency. Furthermore, behaviour-based control tools relying on the employ of swarming behaviour manipulation (i.e. the“lure and kill”approach), pheromone traps, sound traps need further research attention. In particular, detailed basic information on the physical and chemical cues routing mosquito swarming and mating dynamics is urgently required.

  2. Spread of Zika virus:The key role of mosquito vector control

    Institute of Scientific and Technical Information of China (English)

    Giovanni Benelli

    2016-01-01

    Mosquitoes(Diptera: Culicidae) represent a key threat for millions of humans and animals worldwide, since they act as vectors for important parasites and pathogens,including malaria, filariasis and a wide number of arboviruses. The recent outbreaks of Zika virus infections occurring in South America, Central America, and the Caribbean,represent the most recent four arrivals of important arboviruses in the western hemisphere, over the last 20 years, namely dengue, West Nile virus, and chikungunya. Since there are no specific treatments for Zika virus and the other arboviruses mentioned above,it should be highlighted that the eco-friendly and effective control of mosquito vectors is of pivotal importance. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in presence of ultra-low quantities of green-synthesized nanoparticles, which magnify their predation efficiency. Furthermore, behaviour-based control tools relying on the employ of swarming behaviour manipulation(i.e. the "lure and kill"approach), pheromone traps, sound traps need further research attention. In particular,detailed basic information on the physical and chemical cues routing mosquito swarming and mating dynamics is urgently required.

  3. Haemaphysalis longicornis Ticks as Reservoir and Vector of Severe Fever with Thrombocytopenia Syndrome Virus in China.

    Science.gov (United States)

    Luo, Li-Mei; Zhao, Li; Wen, Hong-Ling; Zhang, Zhen-Tang; Liu, Jian-Wei; Fang, Li-Zhu; Xue, Zai-Feng; Ma, Dong-Qiang; Zhang, Xiao-Shuang; Ding, Shu-Jun; Lei, Xiao-Ying; Yu, Xue-jie

    2015-10-01

    Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia caused by SFTS virus (SFTSV), a newly discovered phlebovirus. The Haemaphysalis longicornis tick has been suspected to be the vector of SFTSV. To determine whether SFTSV can be transmitted among ticks, from ticks to animals, and from animals to ticks, we conducted transmission studies between developmental stages of H. longicornis ticks and between ticks and mice. Using reverse transcription PCR, we also analyzed the prevalence of SFTSV infection among H. longicornis ticks collected from vegetation in Shandong Province, China. Our results showed a low prevalence of SFTSV among collected ticks (0.2%, 8/3,300 ticks), and we showed that ticks fed on SFTSV-infected mice could acquire the virus and transstadially and transovarially transmit it to other developmental stages of ticks. Furthermore, SFTSV-infected ticks could transmit the virus to mice during feeding. Our findings indicate ticks could serve as a vector and reservoir of SFTSV.

  4. Spread of Zika virus: The key role of mosquito vector control

    Directory of Open Access Journals (Sweden)

    Giovanni Benelli

    2016-06-01

    Full Text Available Mosquitoes (Diptera: Culicidae represent a key threat for millions of humans and animals worldwide, since they act as vectors for important parasites and pathogens, including malaria, filariasis and a wide number of arboviruses. The recent outbreaks of Zika virus infections occurring in South America, Central America, and the Caribbean, represent the most recent four arrivals of important arboviruses in the western hemisphere, over the last 20 years, namely dengue, West Nile virus, and chikungunya. Since there are no specific treatments for Zika virus and the other arboviruses mentioned above, it should be highlighted that the eco-friendly and effective control of mosquito vectors is of pivotal importance. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in presence of ultra-low quantities of green-synthesized nanoparticles, which magnify their predation efficiency. Furthermore, behaviour-based control tools relying on the employ of swarming behaviour manipulation (i.e. the “lure and kill” approach, pheromone traps, sound traps need further research attention. In particular, detailed basic information on the physical and chemical cues routing mosquito swarming and mating dynamics is urgently required.

  5. Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean.

    Science.gov (United States)

    Zhang, Chunquan; Ghabrial, Said A

    2006-01-20

    Plant virus-based vectors provide valuable tools for expression of foreign proteins in plants and for gene function studies. None of the presently available virus vectors is suitable for use in soybean. In the present study, we produced Bean pod mottle virus (BPMV)-based vectors that are appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The genes of interest were inserted into the RNA2-encoded polyprotein open reading frame between the movement protein (MP) and the large coat protein (L-CP) coding regions. Additional proteinase cleavage sites were created to flank the foreign protein by duplicating the MP/L-CP cleavage site. To minimize the chances of homologous recombination and thus insert instability, we took advantage of the genetic code degeneracy and altered the nucleotide sequence of the duplicated regions without affecting amino acid sequences. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. These included the reporter proteins GFP and DsRed, phosphinothricin acetyltransferase (encoded by the herbicide resistance bar gene), and the RNA silencing suppressors encoded by Tomato bushy stunt virus, Turnip crinkle virus, Tobacco etch virus, and Soybean mosaic virus. The possible use of BPMV as a VIGS vector to study gene function in soybean was also demonstrated with the phytoene desaturase gene. Our results suggest that the BPMV-based vectors are suitable for expression of foreign proteins in soybean and for functional genomics applications.

  6. Multifunctional Virus-Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer

    Science.gov (United States)

    2012-06-01

    cancer cells in synergy with gene therapy. We proposed to develop virus- nanoshell assemblies by attaching adeno-associated virus (AAV) to gold... nanoshells (Au NS) through chemical bonds. We have successfully completed majority of tasks 1 and 2 of our Statement of Work. Specifically, we have...therapy, virus, Au nanoshell Multifunctional Virus- Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer Dr. Fang Wei

  7. Effects of larval rearing temperature on immature development and West Nile virus vector competence of Culex tarsalis

    Directory of Open Access Journals (Sweden)

    Dodson Brittany L

    2012-09-01

    Full Text Available Abstract Background Temperature is known to induce changes in mosquito physiology, development, ecology, and in some species, vector competence for arboviruses. Since colonized mosquitoes are reared under laboratory conditions that can be significantly different from their field counterparts, laboratory vector competence experiments may not accurately reflect natural vector-virus interactions. Methods We evaluated the effects of larval rearing temperature on immature development parameters and vector competence of two Culex tarsalis strains for West Nile virus (WNV. Results Rearing temperature had a significant effect on mosquito developmental parameters, including shorter time to pupation and emergence and smaller female body size as temperature increased. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days post infectious feeding were not consistently affected. Conclusions These results suggest that varying constant larval rearing temperature does not significantly affect laboratory estimates of vector competence for WNV in Culex tarsalis mosquitoes.

  8. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

    Science.gov (United States)

    Zhang, Chunquan; Bradshaw, Jeffrey D; Whitham, Steven A; Hill, John H

    2010-05-01

    Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

  9. VAPA, an innovative "virus-acquisition phenotyping assay" opens new horizons in research into the vector-transmission of plant viruses.

    Directory of Open Access Journals (Sweden)

    Alexandre Martinière

    Full Text Available Host-to-host transmission--a key step in plant virus infection cycles--is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vector-transmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membrane-feeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin--a microtubule-depolymerising drug. The example presented here demonstrates that our technically simple "virus-acquisition phenotyping assay" (VAPA provides a first opportunity to implement correlative studies relating the physiological state of infected plant cells to vector-transmission efficiency.

  10. VAPA, an innovative "virus-acquisition phenotyping assay" opens new horizons in research into the vector-transmission of plant viruses.

    Science.gov (United States)

    Martinière, Alexandre; Macia, Jean-Luc; Bagnolini, Guillaume; Jridi, Chiraz; Bak, Aurélie; Blanc, Stéphane; Drucker, Martin

    2011-01-01

    Host-to-host transmission--a key step in plant virus infection cycles--is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vector-transmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membrane-feeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV) and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin--a microtubule-depolymerising drug. The example presented here demonstrates that our technically simple "virus-acquisition phenotyping assay" (VAPA) provides a first opportunity to implement correlative studies relating the physiological state of infected plant cells to vector-transmission efficiency.

  11. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection

    Science.gov (United States)

    Wassmer, Sarah J.; Carvalho, Livia S.; György, Bence; Vandenberghe, Luk H.; Maguire, Casey A.

    2017-01-01

    Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye. PMID:28361998

  12. In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis.

    Science.gov (United States)

    Rezvani, Milad; Español-Suñer, Regina; Malato, Yann; Dumont, Laure; Grimm, Andrew A; Kienle, Eike; Bindman, Julia G; Wiedtke, Ellen; Hsu, Bernadette Y; Naqvi, Syed J; Schwabe, Robert F; Corvera, Carlos U; Grimm, Dirk; Willenbring, Holger

    2016-06-02

    Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.

  13. Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants.

    Science.gov (United States)

    Pignolet, Béatrice; Boullier, Séverine; Gelfi, Jacqueline; Bozzetti, Marjorie; Russo, Pierre; Foulon, Eliane; Meyer, Gilles; Delverdier, Maxence; Foucras, Gilles; Bertagnoli, Stéphane

    2008-06-01

    Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

  14. Vector competence of three North American strains of Aedes albopictus for West Nile virus.

    Science.gov (United States)

    Sardelis, Michael R; Turell, Michael J; O'Guinn, Monica L; Andre, Richard G; Roberts, Donald R

    2002-12-01

    To evaluate the potential for North American (NA) Aedes albopictus to transmit West Nile virus (WN), mosquito strains derived from 3 NA sources (Frederick County, Maryland, FRED strain; Cheverly, MD, CHEV strain; Chambers and Liberty counties, Texas, TAMU strain) were tested. These strains were tested along with a previously tested strain from a Hawaiian source (OAHU strain). Mosquitoes were fed on 2- to 3-day-old chickens previously inoculated with a New York strain (Crow 397-99) of WN. All of the NA strains were competent laboratory vectors of WN, with transmission rates of 36, 50, 83, and 92% for the FRED, CHEV, OAHU, and TAMU strains, respectively. The extrinsic incubation period for WN in Ae. albopictus held at 26 degrees C was estimated to be 10 days. Based on efficiency of viral transmission, evidence of natural infection, bionomics, and distribution, Ae. albopictus could be an important bridge vector of WN in the southeastern USA.

  15. Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

    Science.gov (United States)

    Moore, Lauren; Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

  16. Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization.

    Directory of Open Access Journals (Sweden)

    Robyn Manley

    Full Text Available Schmallenberg virus (SBV, an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR.Two colony lines of Cx. pipiens were created in the UK ('Brookwood' and 'Caldbeck' from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.

  17. Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors.

    Science.gov (United States)

    Lotery, Andrew J; Derksen, Todd A; Russell, Stephen R; Mullins, Robert F; Sauter, Sybille; Affatigato, Louisa M; Stone, Edwin M; Davidson, Beverly L

    2002-04-10

    We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina. We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys. Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes. Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes. rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given. After surgery the animals were clinically evaluated by retinal photography and electroretinography. beta-Galactosidase expression was evaluated, at a final end point, in histological sections. We found photoreceptor and Müller cells to have the greatest transgene expression. Focal inflammatory responses localized to the injection site were seen histologically in all eyes. No difference in transduction efficiency was seen between injections near the macula and more peripheral injections. Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections. We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina. rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye.

  18. Preclinical potency and safety studies of an AAV2-mediated gene therapy vector for the treatment of MERTK associated retinitis pigmentosa.

    Science.gov (United States)

    Conlon, Thomas J; Deng, Wen-Tao; Erger, Kirsten; Cossette, Travis; Pang, Ji-jing; Ryals, Renee; Clément, Nathalie; Cleaver, Brian; McDoom, Issam; Boye, Shannon E; Peden, Marc C; Sherwood, Mark B; Abernathy, Corinne R; Alkuraya, Fowzan S; Boye, Sanford L; Hauswirth, William W

    2013-03-01

    Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

  19. Correction of Murine Diabetic Hyperglycaemia With A Single Systemic Administration of An AAV2/8 Vector Containing A Novel Codon Optimized Human Insulin Gene.

    Science.gov (United States)

    Gan, Shu Uin; Notaridou, Maria; Fu, Zhen Ying; Lee, Kok Onn; Sia, Kian Chuan; Nathwani, Amit Chunilal; Della Peruta, Marco; Calne, Roy Yorke

    2016-01-01

    We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector. The constitutive secretion of insulin achieved with a single administration of the vector could be of therapeutic value for some diabetic patients.

  20. Insect-specific viruses detected in laboratory mosquito colonies and their potential implications for experiments evaluating arbovirus vector competence.

    Science.gov (United States)

    Bolling, Bethany G; Vasilakis, Nikos; Guzman, Hilda; Widen, Steven G; Wood, Thomas G; Popov, Vsevolod L; Thangamani, Saravanan; Tesh, Robert B

    2015-02-01

    Recently, there has been a dramatic increase in the detection and characterization of insect-specific viruses in field-collected mosquitoes. Evidence suggests that these viruses are ubiquitous in nature and that many are maintained by vertical transmission in mosquito populations. Some studies suggest that the presence of insect-specific viruses may inhibit replication of a super-infecting arbovirus, thus altering vector competence of the mosquito host. Accordingly, we screened our laboratory mosquito colonies for insect-specific viruses. Pools of colony mosquitoes were homogenized and inoculated into cultures of Aedes albopictus (C6/36) cells. The infected cells were examined by electron microscopy and deep sequencing was performed on RNA extracts. Electron micrograph images indicated the presence of three different viruses in three of our laboratory mosquito colonies. Potential implications of these findings for vector competence studies are discussed.

  1. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo

    Science.gov (United States)

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-01-01

    Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain. PMID:27775045

  2. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.

    Science.gov (United States)

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-10-24

    Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

  3. Candidate Vectors and Rodent Hosts of Venezuelan Equine Encephalitis Virus, Chiapas, 2006–2007

    Science.gov (United States)

    Deardorff, Eleanor R.; Estrada-Franco, Jose G.; Freier, Jerome E.; Navarro-Lopez, Roberto; Da Rosa, Amelia Travassos; Tesh, Robert B.; Weaver, Scott C.

    2011-01-01

    Enzootic Venezuelan equine encephalitis virus (VEEV) has been known to occur in Mexico since the 1960s. The first natural equine epizootic was recognized in Chiapas in 1993 and since then, numerous studies have characterized the etiologic strains, including reverse genetic studies that incriminated a specific mutation that enhanced infection of epizootic mosquito vectors. The aim of this study was to determine the mosquito and rodent species involved in enzootic maintenance of subtype IE VEEV in coastal Chiapas. A longitudinal study was conducted over a year to discern which species and habitats could be associated with VEEV circulation. Antibody was rarely detected in mammals and virus was not isolated from mosquitoes. Additionally, Culex (Melanoconion) taeniopus populations were found to be spatially related to high levels of human and bovine seroprevalence. These mosquito populations were concentrated in areas that appear to represent foci of stable, enzootic VEEV circulation. PMID:22144461

  4. Vector Competence of Aedes aegypti and Aedes polynesiensis Populations from French Polynesia for Chikungunya Virus.

    Directory of Open Access Journals (Sweden)

    Vaea Richard

    2016-05-01

    Full Text Available From October 2014 to March 2015, French Polynesia experienced for the first time a chikungunya outbreak. Two Aedes mosquitoes may have contributed to chikungunya virus (CHIKV transmission in French Polynesia: the worldwide distributed Ae. aegypti and the Polynesian islands-endemic Ae. polynesiensis mosquito.To investigate the vector competence of French Polynesian populations of Ae. aegypti and Ae. polynesiensis for CHIKV, mosquitoes were exposed per os at viral titers of 7 logs tissue culture infectious dose 50%. At 2, 6, 9, 14 and 21 days post-infection (dpi, saliva was collected from each mosquito and inoculated onto C6/36 mosquito cells to check for the presence of CHIKV infectious particles. Legs and body (thorax and abdomen of each mosquito were also collected at the different dpi and submitted separately to viral RNA extraction and CHIKV real-time RT-PCR.CHIKV infection rate, dissemination and transmission efficiencies ranged from 7-90%, 18-78% and 5-53% respectively for Ae. aegypti and from 39-41%, 3-17% and 0-14% respectively for Ae. polynesiensis, depending on the dpi. Infectious saliva was found as early as 2 dpi for Ae. aegypti and from 6 dpi for Ae. polynesiensis. Our laboratory results confirm that the French Polynesian population of Ae. aegypti is highly competent for CHIKV and they provide clear evidence for Ae. polynesiensis to act as an efficient CHIKV vector.As supported by our findings, the presence of two CHIKV competent vectors in French Polynesia certainly contributed to enabling this virus to quickly disseminate from the urban/peri-urban areas colonized by Ae. aegypti to the most remote atolls where Ae. polynesiensis is predominating. Ae. polynesiensis was probably involved in the recent chikungunya outbreaks in Samoa and the Cook Islands. Moreover, this vector may contribute to the risk for CHIKV to emerge in other Polynesian islands like Fiji, and more particularly Wallis where there is no Ae. aegypti.

  5. Semliki Forest Virus: un vector viral con múltiples aplicaciones

    Directory of Open Access Journals (Sweden)

    Luis Felipe Henao

    2007-06-01

    Full Text Available Se han utilizado los alfavirus como vectores de expresión, entre estos se encuentra el Semliki Forest virus (SFV, que es un virus envuelto, el cual, además de replicarse en el citoplasma, tiene la propiedad de expresar por separado las proteínas estructurales de las no estructurales, permitiendo un mayor control de la expresión. Los vectores derivados del SFV pueden tener una gama amplia de aplicaciones. Se pueden obtener altos títulos virales para la expresión eficiente de proteínas en diferentes líneas celulares. Pueden infectar un espectro amplio de células de mamíferos, así como de tejidos. Son prometedores para ser usados en la terapia génica como vehículos para el envío de genes específicos in vivo o in vitro, tanto en la terapia contra el cáncer como en la neuronal, especialmente cuando sólo sea necesaria una expresión a corto plazo. Sus aplicaciones en la producción de vacunas profilácticas o terapéuticas, es otro aspecto estudiado; se ha demostrado la generación de respuestas inmunes importantes contra diferentes enfermedades virales y tumorales. El desarrollo de nuevos vectores no citopáticos, de otros regulados por temperatura, así como también de otros con replicación persistente; permitirán la prolongación de la expresión. Debido a estas nuevas ventajas y a las ya conocidas, gradualmente se podrían ampliar los usos para los vectores derivados del SFV a medida que se controlen sus efectos no deseados.

  6. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Michael R. Strand

    2012-01-01

    Full Text Available Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1 they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2 they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution.

  7. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    Science.gov (United States)

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  8. Vector competence of Aedes albopictus from Houston, Texas, for dengue serotypes 1 to 4, yellow fever and Ross River viruses.

    Science.gov (United States)

    Mitchell, C J; Miller, B R; Gubler, D J

    1987-09-01

    A combination of virus infection and transmission experiments showed that a Houston, Texas strain of Aedes albopictus is a competent vector for dengue (DEN), yellow fever (YF) and Ross River (RR) viruses. However, at 14 days incubation, DEN virus infection rates in a Puerto Rican strain of Aedes aegypti were significantly higher for each of the four DEN serotypes, except DEN-1, than in Houston Ae. albopictus fed simultaneously on the same virus suspensions. The degree of correlation between disseminated DEN infection rates in Houston Ae. albopictus and transmission to an in vitro system ranged from 42 to 88% for the four DEN serotypes. No significant difference was noted in YF virus infection rates or transmission rates in the two mosquito species fed on the same virus suspensions and incubated for the same time period. Also, RR virus infection and transmission rates in Houston and Hawaiian strains of Ae. albopictus were generally comparable.

  9. [Post-translational ligation and function of dual-vector transferred split CFTR gene].

    Science.gov (United States)

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-01-01

    The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

  10. Multiplex Reverse Transcription-PCR for Simultaneous Detection of Beet Necrotic Yellow Vein Virus, Beet Soilborne Virus, and Beet Virus Q and Their Vector Polymyxa betae KESKIN on Sugar Beet

    OpenAIRE

    Meunier, Alexandre; Schmit, Jean-François; Stas, Arnaud; Kutluk, Nazli; Bragard, Claude

    2003-01-01

    Three soilborne viruses transmitted by Polymyxa betae KESKIN in sugar beet have been described: Beet necrotic yellow vein virus (BNYVV), the agent of rhizomania, Beet soilborne virus (BSBV), and Beet virus Q (BVQ). A multiplex reverse transcription-PCR technique was developed to simultaneously detect BNYVV, BSBV, and BVQ, together with their vector, P. betae. The detection threshold of the test was up to 128 times greater than that of an enzyme-linked immunosorbent assay. Systematic associati...

  11. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Directory of Open Access Journals (Sweden)

    Zihua Wang

    Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

  12. A novel non-toxic combined CTA1-DD and ISCOMS adjuvant vector for effective mucosal immunization against influenza virus.

    Science.gov (United States)

    Eliasson, Dubravka Grdic; Helgeby, Anja; Schön, Karin; Nygren, Caroline; El-Bakkouri, Karim; Fiers, Walter; Saelens, Xavier; Lövgren, Karin Bengtsson; Nyström, Ida; Lycke, Nils Y

    2011-05-23

    Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation.

  13. Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference

    Science.gov (United States)

    Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a ne...

  14. Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology: proof of concept in an avian cell model.

    Science.gov (United States)

    Linke, Lyndsey M; Wilusz, Jeffrey; Pabilonia, Kristy L; Fruehauf, Johannes; Magnuson, Roberta; Olea-Popelka, Francisco; Triantis, Joni; Landolt, Gabriele; Salman, Mo

    2016-03-01

    Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

  15. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    Science.gov (United States)

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  16. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Science.gov (United States)

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  17. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Directory of Open Access Journals (Sweden)

    Javier López-Vidal

    Full Text Available Vaccines based on virus-like particles (VLPs have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60 were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  18. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Directory of Open Access Journals (Sweden)

    Chiara Falcicchia

    Full Text Available Brain-derived neurotrophic factor (BDNF has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1 derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  19. Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco

    Directory of Open Access Journals (Sweden)

    Nargys Es-Sette

    2014-04-01

    Full Text Available Background & objectives: An entomological study using CDC miniature light-traps was performed in El Hanchane locality, where cutaneous leishmaniasis (CL was emerging during the summer of 2011. The aim of this study is to identify the vectors of Leishmania and of phleboviruses. Methods: In the field, a total of 643 sandfly specimens were collected, identified by morphological keys and categorized by sex and species. A total of nine distinct species were morphologically identified where seven belonged to the Phlebotomus genus and two species to the Sergentomyia genus. Phlebotomus sergenti was the most abundant species (76%. Phleboviruses were detected by nested RT-PCR using 30 pooled sandflies while P. sergenti females were tested individually for infections of Leishmania species. Results: By using ITS1-PCR-RFLP approach, Leishmania tropica DNA was detected in 10 females, caught in this emerging focus, and provide additional evidence in favour of the role of P. sergenti as vector of L. tropica in Morocco. Real-time PCR screening for phlebovirus RNA, using an assay targeting the polymerase gene, showed positive result in one pool of male P. sergenti. Interpretation & conclusion: In this study, P. sergenti were infected by L. tropica and Toscana virus. To our knowledge, actually this is the first time that Toscana virus has been detected in P. sergenti.

  20. A Compartmental Model for Zika Virus with Dynamic Human and Vector Populations

    Science.gov (United States)

    Lee, Eva K; Liu, Yifan; Pietz, Ferdinand H

    2016-01-01

    The Zika virus (ZIKV) outbreak in South American countries and its potential association with microcephaly in newborns and Guillain-Barré Syndrome led the World Health Organization to declare a Public Health Emergency of International Concern. To understand the ZIKV disease dynamics and evaluate the effectiveness of different containment strategies, we propose a compartmental model with a vector-host structure for ZIKV. The model utilizes logistic growth in human population and dynamic growth in vector population. Using this model, we derive the basic reproduction number to gain insight on containment strategies. We contrast the impact and influence of different parameters on the virus trend and outbreak spread. We also evaluate different containment strategies and their combination effects to achieve early containment by minimizing total infections. This result can help decision makers select and invest in the strategies most effective to combat the infection spread. The decision-support tool demonstrates the importance of “digital disease surveillance” in response to waves of epidemics including ZIKV, Dengue, Ebola and cholera. PMID:28269870

  1. Vector Competence of French Polynesian Aedes aegypti and Aedes polynesiensis for Zika Virus

    Science.gov (United States)

    Richard, Vaea; Paoaafaite, Tuterarii; Cao-Lormeau, Van-Mai

    2016-01-01

    Background In 2013–2014, French Polynesia experienced for the first time a Zika outbreak. Two Aedes mosquitoes may have contributed to Zika virus (ZIKV) transmission in French Polynesia: the worldwide distributed Ae. aegypti and the Polynesian islands-endemic Ae. polynesiensis mosquito. Methodology/Principal Findings To evaluate their vector competence for ZIKV, mosquitoes were infected per os at viral titers of 7 logs tissue culture infectious dose 50%. At several days post-infection (dpi), saliva was collected from each mosquito and inoculated onto C6/36 mosquito cells to check for the presence of ZIKV infectious particles. Legs and body of each mosquito were also collected and submitted separately to RNA extraction and ZIKV RT-PCR. In Ae. aegypti the infection rate was high as early as 6 dpi and the dissemination efficiency get substantial from 9 dpi while the both rates remained quite low in Ae. polynesiensis. The transmission efficiency was poor in Ae. aegypti until 14 dpi and no infectious saliva was found in Ae. polynesiensis at the time points studied. Conclusions/Significance In our experimental conditions, the late ability of the French Polynesian Ae. aegypti to transmit ZIKV added by the poor competence of Ae. polynesiensis for this virus suggest the possible contribution of another vector for the propagation of ZIKV during the outbreak, in particular in remote islands where Ae. polynesiensis is predominating. PMID:27654962

  2. Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

    Science.gov (United States)

    Top, Sokunthea; Foucras, Gilles; Deplanche, Martine; Rives, Germain; Calvalido, Jérôme; Comtet, Loic; Bertagnoli, Stéphane; Meyer, Gilles

    2012-02-21

    Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

  3. Vaccinia Virus LC16m8∆ as a Vaccine Vector for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Minoru Kidokoro

    2014-10-01

    Full Text Available The LC16m8 strain of vaccinia virus, the active ingredient in the Japanese smallpox vaccine, was derived from the Lister/Elstree strain. LC16m8 is replication-competent and has been administered to over 100,000 infants and 3,000 adults with no serious adverse reactions. Despite this outstanding safety profile, the occurrence of spontaneously-generated large plaque-forming virulent LC16m8 revertants following passage in cell culture is a major drawback. We identified the gene responsible for the reversion and deleted the gene (B5R from LC16m8 to derive LC16m8Δ. LC16m8∆ is non-pathogenic in immunodeficient severe combined immunodeficiency (SCID mice, genetically-stable and does not reverse to a large-plaque phenotype upon passage in cell culture, even under conditions in which most LC16m8 populations are replaced by revertants. Moreover, LC16m8∆ is >500-fold more effective than the non-replicating vaccinia virus (VV, Modified Vaccinia Ankara (MVA, at inducing murine immune responses against pathogenic VV. LC16m8∆, which expresses the SIV gag gene, also induced anti-Gag CD8+ T-cells more efficiently than MVA and another non-replicating VV, Dairen I minute-pock variants (DIs. Moreover, LC16m8∆ expressing HIV-1 Env in combination with a Sendai virus vector induced the production of anti-Env antibodies and CD8+ T-cells. Thus, the safety and efficacy of LC16m8∆ mean that it represents an outstanding platform for the development of human vaccine vectors.

  4. The effects of monitoring the abundance and species composition of aphids as virus vectors on seed potato production in Serbia

    Directory of Open Access Journals (Sweden)

    Drago Milošević

    2014-03-01

    Full Text Available Aphids are the most important vectors of potato viruses during the crop’s growing season. The most widespread and damaging viruses, the potato virus Y and potato leaf roll virus, are transmitted by aphids in non-persistent and persistent manner, respectively. The two viruses cause the greatest concern of potato producers and a great constraint to seed potato production in Serbia, the region and across the world. Potato virus Y is particularly harmful, given its distribution and spreading rate. Seed potato production systems under well-managed conditions involve a series of virus control measures, including the monitoring of outbreaks of winged aphids, their abundance and species composition, in order to forecast virosis, i.e. potential plant and tuber infection periods. Monitoring the aphid vectors of potato viruses enables determination of optimum dates for haulm destruction when higher than normal numbers of winged aphids as vectors of economically harmful diseases have been observed. Haulm destruction in a potato crop reduces the risk of plant infection and virus translocation from the aboveground parts to tubers, thus keeping the proportion of infected tubers within tolerance limits allowed for certain categories of seed potatoes. This practice has positive effects if used in combination with other viral disease control measures; otherwise, it becomes ineffective. This paper provides an integral analysis of the effects and role of monitoring outbreaks of aphids, their abundance and species composition in timing haulm growth termination to prevent plant infection, virus translocation and tuber infestation in potato crops in Serbia and the wider region.

  5. Recognition for avian influenza virus proteins based on support vector machine and linear discriminant analysis

    Institute of Scientific and Technical Information of China (English)

    LIANG GuiZhao; LIAO ChunYang; WU ShiRong; LI GenRong; HE Liu; GAO JianKun; Gan MengYu; LI DeJing; CHEN GuoPing; WANG GuiXue; LONG Sha; CHEN ZeCong; JING JuHua; ZHENG XiaoLin; ZENG Hui; ZHANG QiaoXia; ZHANG MengJun; YANG Qi; TIAN FeiFei; TONG JianBo; WANG JiaoNa; LIU YongHong; YANG ShanBin; LI Bo; QIU LiangJia; CAI ShaoXi; ZHAO Na; YANG Yan; SU XiaLi; SONG Jian; CHEN MeiXia; ZHANG XueJiao; SUN JiaYing; MEI Hu; LI JingWei; CHEN GuoHua; CHEN Gang; DENG Jie; PENG ChuanYou; ZHU WanPing; XU LuoNan; WU YuQuan; LIAO LiMin; LI Zhi; ZHOU Yuan; LI Jun; LU DaJun; SU QinLiang; HUANG ZhengHu; ZHOU Ping; LI ZhiLiang; YANG Li; ZHOU Peng; YANG ShengXi; SHU Mao

    2008-01-01

    Total 200 properties related to structural characteristics were employed to represent structures of 400 HA coded proteins of influenza virus as training samples.Some recognition models for HA proteins of avian influenza virus (AIV) were developed using support vector machine (SVM) and linear discriminant analysis (LDA).The results obtained from LDA are as follows: the identification accuracy (Ria) for training samples is 99.8% and Ria by leave one out cross validation is 99.5%.Both Ria of 99.8% for training samples and Ria of 99.3% by leave one out cross validation are obtained using SVM model, respectively.External 200 HA proteins of influenza virus were used to validate the external predictive power of the resulting model.The external Ria for them is 95.5% by LDA and 96.5% by SVM, respectively, which shows that HA proteins of AIVs are preferably recognized by SVM and LDA, and the performances by SVM are superior to those by LDA.

  6. Recognition for avian influenza virus proteins based on support vector machine and linear discriminant analysis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Total 200 properties related to structural characteristics were employed to represent structures of 400 HA coded proteins of influenza virus as training samples. Some recognition models for HA proteins of avian influenza virus (AIV) were developed using support vector machine (SVM) and linear discriminant analysis (LDA). The results obtained from LDA are as follows: the identification accuracy (Ria) for training samples is 99.8% and Ria by leave one out cross validation is 99.5%. Both Ria of 99.8% for training samples and Ria of 99.3% by leave one out cross validation are obtained using SVM model, respectively. External 200 HA proteins of influenza virus were used to validate the external predictive power of the resulting model. The external Ria for them is 95.5% by LDA and 96.5% by SVM, respectively, which shows that HA proteins of AIVs are preferably recognized by SVM and LDA, and the performances by SVM are superior to those by LDA.

  7. Biology and management of Bemisia whitefly vectors of cassava virus pandemics in Africa.

    Science.gov (United States)

    Legg, James P; Shirima, Rudolph; Tajebe, Lensa S; Guastella, Devid; Boniface, Simon; Jeremiah, Simon; Nsami, Elibariki; Chikoti, Patrick; Rapisarda, Carmelo

    2014-10-01

    Cassava mosaic disease and cassava brown streak disease are caused by viruses transmitted by Bemisia tabaci and affect approximately half of all cassava plants in Africa, resulting in annual production losses of more than $US 1 billion. A historical and current bias towards virus rather than vector control means that these diseases continue to spread, and high Bemisia populations threaten future virus spread even if the extant strains and species are controlled. Progress has been made in parts of Africa in replicating some of the successes of integrated Bemisia control programmes in the south-western United States. However, these management efforts, which utilise chemical insecticides that conserve the Bemisia natural enemy fauna, are only suitable for commercial agriculture, which presently excludes most cassava cultivation in Africa. Initiatives to strengthen the control of B. tabaci on cassava in Africa need to be aware of this limitation, and to focus primarily on control methods that are cheap, effective, sustainable and readily disseminated, such as host-plant resistance and biological control. A framework based on the application of force multipliers is proposed as a means of prioritising elements of future Bemisia control strategies for cassava in Africa.

  8. Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

    Science.gov (United States)

    Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A.; Song, Juan

    2016-01-01

    Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design. PMID:27699236

  9. Control of aphid-vectored and thrips-borne virus spread in lily, tulip, iris and dahlia by sprays of mineral oil, polydimethylsiloxane and pyrethroid insecticide in the field

    NARCIS (Netherlands)

    Asjes, J.; Blom-Barnhoorn, G.J.

    2001-01-01

    In this study control of spread by insect vectors of non-persistent Lily symptomless virus and Lily mottle virus in lily, Tulip breaking virus in tulip, Iris mild mosaic virus, Narcissus latent virus and Iris severe mosaic virus in bulbous iris, and semi-persistent Dahlia mosaic virus and persistent

  10. Identification of multiple novel viruses, including a parvovirus and a hepevirus, in feces of red foxes.

    Science.gov (United States)

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

    2013-07-01

    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.

  11. The feasibility of rabies virus-vectored immunocontraception in a mouse model

    Directory of Open Access Journals (Sweden)

    Xianfu Wu

    2014-01-01

    Full Text Available Immunocontraceptive vaccines may be an alternative to surgical sterilization. Dual rabies vaccination and dog population management is a helpful tool for rabies prevention. A synthetic gonadotropin-releasing hormone (GnRH peptide coupled to a carrier protein or T cell epitope is efficacious in inducing immunocontraception in a variety of mammals. However, virus-vectored GnRH recombinant vaccines have advantages over the conjugation method. In a previous in vitro study, we were able to insert a GnRH-coding sequence into the rabies virus (RABV glycoprotein (G gene, and the recombinant viruses grew to high titers in cells. Here, we further focused on the RABV G in accepting various copy numbers of GnRH. We demonstrated although RABV G protein with up to 4 copies of GnRH was well expressed, the recombinant virus was recovered only when 2 copies of GnRH (20 amino acids were incorporated into the G, indicating a possible insertion limit in making a full infectious clone. The investigation provides insight into the utility of RABV G as a carrier for small peptides and its suitability for vaccine studies. Following our previous study, we selected ERAg3p/2GnRH and tested the construct in mice. The vaccine induced ⩾80% infertility after three doses without any adjuvant, in live (8 of 10 mice infertility or inactivated (13 of 14 mice infertility formulations; while the pregnancy rate was 100% (10 of 10 mice in the controls. This initial success of immunocontraception in mice is promising, and we are now optimizing the vaccine formulation by using adjuvants and exploring novel delivery methods to minimize the dosage.

  12. Adeno-Associated Virus Transfer of a Gene Encoding SNAP-25 Resistant to Botulinum Toxin A Attenuates Neuromuscular Paralysis Associated with Botulism

    Science.gov (United States)

    2008-04-02

    refinement of the technology for application to genetic disorders of motor neurons. 15. SUBJECT TERMS Clostridium botulinum, neurotoxiin, serotype...peripherally transported transgenic protein successfully substituted for native cleavage-susceptible S25, as demonstrated by enhanced retention of nerve

  13. 口服重组腺相关病毒基因药物%Oral recombinant adeno-associated virus gene medicine

    Institute of Scientific and Technical Information of China (English)

    刁勇; 许瑞安

    2009-01-01

    重组腺相关病毒(rAAV)载体介导的口服基冈药物引起业界广泛的重视.尽管经口服给药后转基因的有效表达面临许多障碍,但该技术的有效性已得到大量实验证实.本文总结了口服rAAV基冈药物的临床前研究结果,重点阐述了该类型药物的传递、吸收、分布和基冈转导等药动学特点.已证实rAAV基因药物对人体的安全性高,但口服rAAV基因药物的临床应用仍需对其作用机制和生物约剂学特征进行深入和广泛的研究.

  14. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    DEFF Research Database (Denmark)

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari;

    2010-01-01

    Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation...

  15. Vector Contact Rates on Eastern Bluebird Nestlings Do Not Indicate West Nile Virus Transmission in Henrico County, Virginia, USA

    Directory of Open Access Journals (Sweden)

    Kevin A. Caillouët

    2013-11-01

    Full Text Available Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV. Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.

  16. Vector contact rates on Eastern bluebird nestlings do not indicate West Nile virus transmission in Henrico County, Virginia, USA.

    Science.gov (United States)

    Caillouët, Kevin A; Robertson, Charles W; Wheeler, David C; Komar, Nicholas; Bulluck, Lesley P

    2013-11-27

    Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV). Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae) across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.

  17. Virus-derived DNA drives mosquito vector tolerance to arboviral infection

    Science.gov (United States)

    Goic, Bertsy; Stapleford, Kenneth A.; Frangeul, Lionel; Doucet, Aurélien J.; Gausson, Valérie; Blanc, Hervé; Schemmel-Jofre, Nidia; Cristofari, Gael; Lambrechts, Louis; Vignuzzi, Marco; Saleh, Maria-Carla

    2016-01-01

    Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes. PMID:27580708

  18. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

    Science.gov (United States)

    Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

  19. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site

    Science.gov (United States)

    Newcastle disease virus (NDV) has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU). Based on the well-accepted “stop-start” transcr...

  20. The role of environmental, virological and vector interactions in dictating biological transmission of arthropod-borne viruses by mosquitoes.

    Science.gov (United States)

    Kenney, Joan L; Brault, Aaron C

    2014-01-01

    Arthropod-borne viruses (arboviruses) are transmitted between vertebrate hosts and arthropod vectors. An inherently complex interaction among virus, vector, and the environment determines successful transmission of the virus. Once believed to be "flying syringes," recent advances in the field have demonstrated that mosquito genetics, microbiota, salivary components, and mosquito innate immune responses all play important roles in modulating arbovirus transmissibility. The literature on the interaction among virus, mosquito, and environment has expanded dramatically in the preceding decade and the utilization of next-generation sequencing and transgenic vector methodologies assuredly will increase the pace of knowledge acquisition in this field. This chapter outlines the interplay among the three factors in both direct physical and biochemical manners as well as indirectly through superinfection barriers and altered induction of innate immune responses in mosquito vectors. The culmination of the aforementioned interactions and the arms race between the mosquito innate immune response and the capacity of arboviruses to antagonize such a response ultimately results in the subjugation of mosquito cells for viral replication and subsequent transmission.

  1. Mapping the basic reproduction number (Ro) for vector-borne diseases: A case study on bluetongue virus.

    NARCIS (Netherlands)

    Hartemink, N.; Purse, B.V.; Meiswinkel, R.; Brown, H.E.; Koeijer, de A.A.; Elbers, A.R.W.; Boender, G.J.; Rogers, D.J.; Heesterbeek, J.A.P.

    2009-01-01

    Geographical maps indicating the value of the basic reproduction number, R0, can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool f

  2. P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression

    Science.gov (United States)

    Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic ...

  3. The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

    Directory of Open Access Journals (Sweden)

    Galler R.

    1997-01-01

    Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

  4. Zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently

    OpenAIRE

    Rengina Vorou

    2016-01-01

    Objectives: The widespread epidemic of Zika virus infection in South and Central America and the Caribbean in 2015, along with the increased incidence of microcephaly in fetuses born to mothers infected with Zika virus and the potential for worldwide spread, indicate the need to review the current literature regarding vectors, reservoirs, and amplification hosts. Vectors: The virus has been isolated in Africa in mosquitoes of the genera Aedes, Anopheles, and Mansonia, and in Southeast Asia...

  5. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

    Science.gov (United States)

    Ding, Xin Shun; Schneider, William L; Chaluvadi, Srinivasa Rao; Mian, M A Rouf; Nelson, Richard S

    2006-11-01

    Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

  6. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes.

    Science.gov (United States)

    Kang, W; Wang, L; Harrell, H; Liu, J; Thomas, D L; Mayfield, T L; Scotti, M M; Ye, G J; Veres, G; Knop, D R

    2009-02-01

    Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

  7. The Dengue Virus Mosquito Vector Aedes aegypti at High Elevation in México

    Science.gov (United States)

    Lozano-Fuentes, Saul; Hayden, Mary H.; Welsh-Rodriguez, Carlos; Ochoa-Martinez, Carolina; Tapia-Santos, Berenice; Kobylinski, Kevin C.; Uejio, Christopher K.; Zielinski-Gutierrez, Emily; Monache, Luca Delle; Monaghan, Andrew J.; Steinhoff, Daniel F.; Eisen, Lars

    2012-01-01

    México has cities (e.g., México City and Puebla City) located at elevations > 2,000 m and above the elevation ceiling below which local climates allow the dengue virus mosquito vector Aedes aegypti to proliferate. Climate warming could raise this ceiling and place high-elevation cities at risk for dengue virus transmission. To assess the elevation ceiling for Ae. aegypti and determine the potential for using weather/climate parameters to predict mosquito abundance, we surveyed 12 communities along an elevation/climate gradient from Veracruz City (sea level) to Puebla City (∼2,100 m). Ae. aegypti was commonly encountered up to 1,700 m and present but rare from 1,700 to 2,130 m. This finding extends the known elevation range in México by > 300 m. Mosquito abundance was correlated with weather parameters, including temperature indices. Potential larval development sites were abundant in Puebla City and other high-elevation communities, suggesting that Ae. aegypti could proliferate should the climate become warmer. PMID:22987656

  8. Intradermal delivery of recombinant vaccinia virus vector DIs induces gut-mucosal immunity.

    Science.gov (United States)

    Yoshino, N; Kanekiyo, M; Hagiwara, Y; Okamura, T; Someya, K; Matsuo, K; Ami, Y; Sato, S; Yamamoto, N; Honda, M

    2010-08-01

    Antigen-specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication-deficient vaccinia virus vector, DIs, generates antigen-specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27-specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody-forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag-specific IFN-gamma spot-forming CD8(+) T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat-inactivated rDIsSIVgag rarely induced antigen-specific humoral and T-helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen-positive (IA(+)) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen-specific humoral and cellular immunity in gut-mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.

  9. Genetic Drift, Purifying Selection and Vector Genotype Shape Dengue Virus Intra-host Genetic Diversity in Mosquitoes

    Science.gov (United States)

    Fontaine, Albin; Ar Gouilh, Meriadeg; Moltini-Conclois, Isabelle

    2016-01-01

    Due to their error-prone replication, RNA viruses typically exist as a diverse population of closely related genomes, which is considered critical for their fitness and adaptive potential. Intra-host demographic fluctuations that stochastically reduce the effective size of viral populations are a challenge to maintaining genetic diversity during systemic host infection. Arthropod-borne viruses (arboviruses) traverse several anatomical barriers during infection of their arthropod vectors that are believed to impose population bottlenecks. These anatomical barriers have been associated with both maintenance of arboviral genetic diversity and alteration of the variant repertoire. Whether these patterns result from stochastic sampling (genetic drift) rather than natural selection, and/or from the influence of vector genetic heterogeneity has not been elucidated. Here, we used deep sequencing of full-length viral genomes to monitor the intra-host evolution of a wild-type dengue virus isolate during infection of several mosquito genetic backgrounds. We estimated a bottleneck size ranging from 5 to 42 founding viral genomes at initial midgut infection, irrespective of mosquito genotype, resulting in stochastic reshuffling of the variant repertoire. The observed level of genetic diversity increased following initial midgut infection but significantly differed between mosquito genetic backgrounds despite a similar initial bottleneck size. Natural selection was predominantly negative (purifying) during viral population expansion. Taken together, our results indicate that dengue virus intra-host genetic diversity in the mosquito vector is shaped by genetic drift and purifying selection, and point to a novel role for vector genetic factors in the genetic breadth of virus populations during infection. Identifying the evolutionary forces acting on arboviral populations within their arthropod vector provides novel insights into arbovirus evolution. PMID:27304978

  10. Genetic Drift, Purifying Selection and Vector Genotype Shape Dengue Virus Intra-host Genetic Diversity in Mosquitoes.

    Science.gov (United States)

    Lequime, Sebastian; Fontaine, Albin; Ar Gouilh, Meriadeg; Moltini-Conclois, Isabelle; Lambrechts, Louis

    2016-06-01

    Due to their error-prone replication, RNA viruses typically exist as a diverse population of closely related genomes, which is considered critical for their fitness and adaptive potential. Intra-host demographic fluctuations that stochastically reduce the effective size of viral populations are a challenge to maintaining genetic diversity during systemic host infection. Arthropod-borne viruses (arboviruses) traverse several anatomical barriers during infection of their arthropod vectors that are believed to impose population bottlenecks. These anatomical barriers have been associated with both maintenance of arboviral genetic diversity and alteration of the variant repertoire. Whether these patterns result from stochastic sampling (genetic drift) rather than natural selection, and/or from the influence of vector genetic heterogeneity has not been elucidated. Here, we used deep sequencing of full-length viral genomes to monitor the intra-host evolution of a wild-type dengue virus isolate during infection of several mosquito genetic backgrounds. We estimated a bottleneck size ranging from 5 to 42 founding viral genomes at initial midgut infection, irrespective of mosquito genotype, resulting in stochastic reshuffling of the variant repertoire. The observed level of genetic diversity increased following initial midgut infection but significantly differed between mosquito genetic backgrounds despite a similar initial bottleneck size. Natural selection was predominantly negative (purifying) during viral population expansion. Taken together, our results indicate that dengue virus intra-host genetic diversity in the mosquito vector is shaped by genetic drift and purifying selection, and point to a novel role for vector genetic factors in the genetic breadth of virus populations during infection. Identifying the evolutionary forces acting on arboviral populations within their arthropod vector provides novel insights into arbovirus evolution.

  11. A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Xiqing Chai; Weina Kong; Lingyun Liu; Wenguo Yu; Zhenqing Zhang; Yimin Sun

    2014-01-01

    Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we construct-ed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1αgene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1αrepresses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results conifrmed that rAAV-HIF-1αsigniifcant-ly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1αadministration also induced robust and prolonged HIF-1αproduction in rat hippocampus. Single rAAV-HIF-1αadministration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer’s disease rat model established by intrace-rebroventricular injection of aggregated amyloid-beta protein (25-35). Our in vitro and in vivo ifndings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurode-generative diseases using gene therapy.

  12. Control of the Aedes vectors of the dengue viruses and Wuchereria bancrofti: the French Polynesian experience.

    Science.gov (United States)

    Lardeux, F; Rivière, F; Séchan, Y; Loncke, S

    2002-12-01

    In most of the 130 islands of French Polynesia, the stenotopic mosquitoes Aedes aegypti (the main local vector for the viruses causing dengue) and Aedes polynesiensis (the main local vector of Wuchereria bancrofti) share many breeding sites in water containers such as discarded cans, coconut shells, buckets and water-storage pots and drums. In addition to selective application of insecticides, non-polluting methods of controlling these mosquitoes have been evaluated during the last decade in two main ecological situations: (1) villages, where Aedes breeding sites are typically peridomestic; and (2) flooded burrows of land crabs, the major source of Ae. polynesiensis throughout the South Pacific region. Large-scale trials of biological control agents, such as mosquito fish (Gambusia affinis and Poecilia reticulata) and copepods (Mesocyclops aspericornis), and of integrated-control strategies have demonstrated the efficacy of certain techniques and control agents against the target Aedes populations in some village situations. Generally, mechanical methods (the use of layers of polystyrene beads against mosquito larvae and pupae, and screening against adult mosquitoes) were more efficient than use of the biological control agents. By integrating several methods of control, mosquito densities (as measured by human-bait collections and larval surveys) were reduced significantly compared with the results of concurrent sampling from untreated villages, and control remained effective for months after the interventions ceased. In land-crab burrows, the first attempts to control Aedes larvae used bacterial agents (Bacillus thuringiensis) and predatory copepods gave disappointing results. Mesocyclops aspericornis could be an effective control agent if the burrows were constantly flooded, but most burrows dry out and refill periodically, so copepod populations do not survive. As it proved difficult to reach all corners of the long sinuous burrows with any control agent

  13. Cross-protective immunity against multiple influenza virus subtypes by a novel modified vaccinia Ankara (MVA) vectored vaccine in mice.

    Science.gov (United States)

    Brewoo, Joseph N; Powell, Tim D; Jones, Jeremy C; Gundlach, Nancy A; Young, Ginger R; Chu, Haiyan; Das, Subash C; Partidos, Charalambos D; Stinchcomb, Dan T; Osorio, Jorge E

    2013-04-03

    Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA1/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (NP) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA1/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.

  14. Field detection of Tembusu virus in western Thailand by rt-PCR and vector competence determination of select culex mosquitoes for transmission of the virus.

    Science.gov (United States)

    O'Guinn, Monica L; Turell, Michael J; Kengluecha, Ampornpan; Jaichapor, Boonsong; Kankaew, Prasan; Miller, R Scott; Endy, Timothy P; Jones, James W; Coleman, Russell E; Lee, John S

    2013-11-01

    Tembusu virus (TMUV; Ntaya serocomplex) was detected in two pools of mosquitoes captured near Sangkhlaburi, Thailand, as well as from sera from sentinel ducks from the same area. Although TMUV has been isolated from several mosquito species in Asia, no studies have ever shown competent vectors for this virus. Therefore, we allowed mosquitoes captured near Sangkhlaburi to feed on young chickens that had been infected with TMUV. These mosquitoes were tested approximately 2 weeks later to determine infection, dissemination, and transmission rates. Culex vishnui developed high viral titers after feeding on TMUV-infected chicks and readily transmitted virus to naïve chickens. In contrast, Cx. fuscocephala seemed less susceptible to infection, and more importantly, zero of five fuscocephala with a disseminated infection transmitted virus by bite, indicating a salivary gland barrier. These results provide evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment.

  15. O'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Kali D Saxton-Shaw

    Full Text Available O'nyong nyong virus (ONNV and Chikungunya virus (CHIKV are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3, was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3 replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

  16. O'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3.

    Science.gov (United States)

    Saxton-Shaw, Kali D; Ledermann, Jeremy P; Borland, Erin M; Stovall, Janae L; Mossel, Eric C; Singh, Amber J; Wilusz, Jeffrey; Powers, Ann M

    2013-01-01

    O'nyong nyong virus (ONNV) and Chikungunya virus (CHIKV) are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3), was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

  17. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    Science.gov (United States)

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine.

  18. Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control.

    Science.gov (United States)

    Benelli, Giovanni; Mehlhorn, Heinz

    2016-05-01

    The fight against mosquito-borne diseases is a challenge of huge public health importance. To our mind, 2015 was an extraordinary year for malaria control, due to three hot news: the Nobel Prize to Youyou Tu for the discovery of artemisinin, the development of the first vaccine against Plasmodium falciparum malaria [i.e. RTS,S/AS01 (RTS,S)], and the fall of malaria infection rates worldwide, with special reference to sub-Saharan Africa. However, there are major challenges that still deserve attention, in order to boost malaria prevention and control. Indeed, parasite strains resistant to artemisinin have been detected, and RTS,S vaccine does not offer protection against Plasmodium vivax malaria, which predominates in many countries outside of Africa. Furthermore, the recent outbreaks of Zika virus infections, occurring in South America, Central America and the Caribbean, represent the most recent of four arrivals of important arboviruses in the Western Hemisphere, over the last 20 years. Zika virus follows dengue (which slyly arrived in the hemisphere over decades and became more aggressive in the 1990s), West Nile virus (emerged in 1999) and chikungunya (emerged in 2013). Notably, there are no specific treatments for these arboviruses. The emerging scenario highlights that the effective and eco-friendly control of mosquito vectors, with special reference to highly invasive species such as Aedes aegypti and Aedes albopictus, is crucial. The concrete potential of screening plant species as sources of metabolites for parasitological purposes is worthy of attention, as elucidated by the Y. Tu's example. Notably, plant-borne molecules are often effective at few parts per million against Aedes, Ochlerotatus, Anopheles and Culex young instars, can be used for the rapid synthesis of mosquitocidal nanoformulations and even employed to prepare cheap repellents with low human toxicity. In addition, behaviour-based control tools relying to the employ of sound traps and the

  19. Generation of induced pluripotent stem cells from human nasal epithelial cells using a Sendai virus vector.

    Directory of Open Access Journals (Sweden)

    Mizuho Ono

    Full Text Available The generation of induced pluripotent stem cells (iPSCs by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs-derived iPSCs by gene transduction with Sendai virus (SeV vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08-0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.

  20. Generation of Induced Pluripotent Stem Cells from Human Nasal Epithelial Cells Using a Sendai Virus Vector

    Science.gov (United States)

    Ono, Mizuho; Hamada, Yuko; Horiuchi, Yasue; Matsuo-Takasaki, Mami; Imoto, Yoshimasa; Satomi, Kaishi; Arinami, Tadao; Hasegawa, Mamoru; Fujioka, Tsuyoshi; Nakamura, Yukio; Noguchi, Emiko

    2012-01-01

    The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08–0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes. PMID:22912751

  1. Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development.

    Science.gov (United States)

    Johnston, J; Power, C

    1999-03-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 10(1.3) to 10(2.1) 50% tissue culture infective doses/10(6) cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy.

  2. Vector competence of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) for DEN2-43 and New Guinea C virus strains of dengue 2 virus.

    Science.gov (United States)

    Guo, Xiao-Xia; Zhu, Xiao-Juan; Li, Chun-Xiao; Dong, Yan-De; Zhang, Ying-Mei; Xing, Dan; Xue, Rui-De; Qin, Cheng-Feng; Zhao, Tong-Yan

    2013-12-01

    The vector competence of Aedes albopictus and Aedes aegypti with regard to DEN2-43 and New Guinea C (NGC) virus strains of Dengue 2 viruses was assessed and compared. The infection and dissemination rate and distribution of DEN2-43 antigens in orally infected Ae. albopictus was investigated using the reverse transcription polymerase chain reaction and an indirect immunofluorescence assay. To better understand the initial infection, dissemination and transmission of these viral strains in vector mosquitoes, Ae. albopoictus and Ae. aegypti were fed an artificial blood meal containing either the DEN2-43 or NGC strain. There was no significant difference in the infection and dissemination rates of DEN2-43 and NGC virus strains in Ae. albopictus, however, Ae. aegypti was more susceptible to infection by NGC than DEN2-43 vrius strain. Ae. albopictus mosquitoes infected with the NGC strain developed a higher percentage of midgut infections than those infected with the DEN2-43 strain (t=2.893, df=7, P=0.024). Approximately 26.7% of midgut samples were positive for the NGC antigen 5 days after infection, and 80% of mosquitoes had infected midgets after 15 days. The NGC antigen first became evident in mosquito salivary glands on Day 5, and 40% of mosquitoes had infected salivary by Day 9. In contrast, the DEN2-43 antigen first became evident in salivary glands on Day 7. The infection rate of NGC and DEN2-43 virus strains in salivary glands were similar. These results indicate that Ae. albopictus and Ae. aegypti are moderately competent vectors for the DEN2-43 virus, which could provide basic data for the epidemiology study of dengue fever in China.

  3. Tomato spotted wilt virus benefits a non-vector arthropod, Tetranychus urticae, by modulating different plant responses in tomato.

    Directory of Open Access Journals (Sweden)

    Punya Nachappa

    Full Text Available The interaction between plant viruses and non-vector arthropod herbivores is poorly understood. However, there is accumulating evidence that plant viruses can impact fitness of non-vector herbivores. In this study, we used oligonucleotide microarrays, phytohormone, and total free amino acid analyses to characterize the molecular mechanisms underlying the interaction between Tomato spotted wilt virus (TSWV and a non-vector arthropod, twospotted spider mite (Tetranychusurticae, on tomato plants, Solanumlycopersicum. Twospotted spider mites showed increased preference for and fecundity on TSWV-infected plants compared to mock-inoculated plants. Transcriptome profiles of TSWV-infected plants indicated significant up-regulation of salicylic acid (SA-related genes, but no apparent down-regulation of jasmonic acid (JA-related genes which could potentially confer induced resistance against TSM. This suggests that there was no antagonistic crosstalk between the signaling pathways to influence the interaction between TSWV and spider mites. In fact, SA- and JA-related genes were up-regulated when plants were challenged with both TSWV and the herbivore. TSWV infection resulted in down-regulation of cell wall-related genes and photosynthesis-associated genes, which may contribute to host plant susceptibility. There was a three-fold increase in total free amino acid content in virus-infected plants compared to mock-inoculated plants. Total free amino acid content is critical for arthropod nutrition and may, in part, explain the apparent positive indirect effect of TSWV on spider mites. Taken together, these data suggest that the mechanism(s of increased host suitability of TSWV-infected plants to non-vector herbivores is complex and likely involves several plant biochemical processes.

  4. Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors.

    Science.gov (United States)

    Yamagishi, Noriko; Yoshikawa, Nobuyuki

    2009-09-01

    Virus-induced gene silencing (VIGS) has great potential as a reverse-genetics tool in plant genomics. In this study, we examined the potential of VIGS in soybean seeds and the emergence stage of soybean plants using Apple latent spherical virus (ALSV) vectors. Inoculation of an ALSV vector (soyPDS-ALSV) carrying a fragment of the soybean phytoene desaturase (soyPDS) gene into soybean seedlings resulted in a highly uniform photo-bleached phenotype, typical of PDS inhibition, on the upper leaves throughout plant growth. The photo-bleached phenotype was also found on all immature pods, all seed coats, and about 50% embryos of seeds on soybean plants infected with soyPDS-ALSV. Infection with an ALSV vector (soyIFS2-ALSV) having a fragment of soybean isoflavone synthase 2 (soyIFS2) gene also led to a reduction of the levels of both soyIFS2- and soyIFS1- mRNAs and an isoflavone content in the cotyledons of about 36% mature seeds of infected soybean plants. Furthermore, VIGS of soyPDS was induced in the next generation plants by the seed transmission of soyPDS-ALSV. Thus ALSV vectors will be useful for studying gene functions in the reproductive stages and early growth stages, such as emergence and cotyledon stages, in addition to the vegetative stages of soybean plants.

  5. An ensemble distance measure of k-mer and Natural Vector for the phylogenetic analysis of multiple-segmented viruses.

    Science.gov (United States)

    Huang, Hsin-Hsiung

    2016-06-01

    The Natural Vector combined with Hausdorff distance has been successfully applied for classifying and clustering multiple-segmented viruses. Additionally, k-mer methods also yield promising results for global genome comparison. It is not known whether combining these two approaches can lead to more accurate results. The author proposes a method of combining the Hausdorff distances of the 5-mer counting vectors and natural vectors which achieves the best classification without cutting off any sample. Using the proposed method to predict the taxonomic labels for the 2363 NCBI reference viral genomes dataset, the accuracy rates are 96.95%, 94.37%, 99.41% and 93.82% for the Baltimore, family, subfamily, and genus labels, respectively. We further applied the proposed method to 48 isolates of the influenza A H7N9 viruses which have eight complete segments of nucleotide sequences. The single-linkage clustering trees and the statistical hypothesis testing results all indicate that the proposed ensemble distance measure can cluster viruses well using all of their segments of genome sequences.

  6. Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia

    Science.gov (United States)

    Hall-Mendelin, Sonja; Pyke, Alyssa T.; Moore, Peter R.; Mackay, Ian M.; McMahon, Jamie L.; Ritchie, Scott A.; Taylor, Carmel T.; Moore, Frederick A.J.; van den Hurk, Andrew F.

    2016-01-01

    Background Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. Methodology/Principal Findings Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. After 14 days incubation at 28°C and high relative humidity, infection, dissemination and transmission rates were assessed. Infection in Culex annulirostris and Cx. sitiens could not be detected. 8% of Cx. quinquefasciatus were infected, but the virus did not disseminate in this species. Despite having infection rates > 50%, Aedes notoscriptus and Ae. vigilax did not transmit ZIKV. In contrast, Ae. aegypti had infection and transmission rates of 57% and 27%, respectively. In susceptibility trials, the virus dose required to infect 50% (ID50) of Ae. aegypti was106.4 tissue culture infectious dose50 (TCID50)/mL. Additionally, a threshold viral load within the mosquito of at least 105.1 TCID50 equivalents/mL had to be reached before virus transmission occurred. Conclusions/Significance We confirmed Ae. aegypti to be the most likely mosquito vector of ZIKV in Australia, although the restricted distribution of this species will limit the receptive zone to northern Queensland where this species occurs. Importantly, the role in ZIKV transmission of Culex and other Aedes spp. tested will be negligible. Despite being the implicated vector, the relatively high ID50 and need for a high titer disseminated infection in Ae. aegypti suggest that high mosquito population densities will be required to facilitate epidemic ZIKV transmission among the currently immunologically naïve human population in Australia. PMID:27643685

  7. Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

    Directory of Open Access Journals (Sweden)

    Junmin Li

    Full Text Available Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi which generates viral-derived small interfering RNAs (siRNAs. However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus was infected by Rice black-streaked dwarf virus (RBSDV (Reoviridae; Fijivirus, more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV, a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

  8. Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

    Directory of Open Access Journals (Sweden)

    Katsuyuki Kaneda

    Full Text Available The superficial layer of the superior colliculus (sSC receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR, a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

  9. [Influence of Japanese enciphalitis virus capsid protein on the self-replicate ability of JEV replicon vectors].

    Science.gov (United States)

    Huang, Ying; Liu, Shan; Yang, Peng; Wang, Chao; Du, Yun; Sun, Zhiwei; Yu, Weiyuan

    2010-08-01

    To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.

  10. Laboratory studies of a Brazilian strain of Aedes albopictus as a potential vector of Mayaro and Oropouche viruses.

    Science.gov (United States)

    Smith, G C; Francy, D B

    1991-03-01

    The vector efficiency of colonized Aedes albopictus from Brazil was assessed for Mayaro (MAY) and Oropouche (ORO) viruses. Female mosquitoes, 3-4 days old, were fed on a MAY-infected hamster with a viremia level of 5.3 log10 Vero cell plaque-forming units (PFU) of virus/ml or an ORO-infected hamster circulating 7.3 log10 PFU/ml. Mayaro infection rates among fed mosquitoes were 16.9 and 11% at 6, 13 and 20 days postfeeding, respectively, and 1/2 and 2/2 infected mosquitoes transmitted virus on days 13 and 20, respectively. Only 13, 5 and 3% of mosquitoes were infected with ORO virus at 6, 13 and 20 days, respectively, and no transmission occurred. Mosquitoes were also fed on 3 dilutions of MAY virus-blood suspensions in membrane feeders. The infection rate among mosquitoes fed the highest concentration (7.7 log10 PFU/ml) was 11/13 (85%), and 5/11 (46%) infected mosquitoes transmitted virus.

  11. Efficient Transient Expression of Recombinant Proteins in Plants by the Novel pEff Vector Based on the Genome of Potato Virus X

    Science.gov (United States)

    Mardanova, Eugenia S.; Blokhina, Elena A.; Tsybalova, Liudmila M.; Peyret, Hadrien; Lomonossoff, George P.; Ravin, Nikolai V.

    2017-01-01

    Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5–10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants. PMID:28293244

  12. A Hierarchical Approach Embedding Hydrologic and Population Modeling for a West Nile Virus Vector Prediction

    Science.gov (United States)

    Jian, Y.; Silvestri, S.; Marani, M.; Saltarin, A.; Chillemi, G.

    2012-12-01

    We applied a hierarchical state space model to predict the abundance of Cx.pipiens (a West Nile Virus vector) in the Po River Delta Region, Northeastern Italy. The study area has large mosquito abundance, due to a favorable environment and climate as well as dense human population. Mosquito data were collected on a weekly basis at more than 20 sites from May to September in 2010 and 2011. Cx.pipiens was the dominant species in our samples, accounting for about 90% of the more than 300,000 total captures. The hydrological component of the model accounted for evapotranspiration, infiltration and deep percolation to infer, in a 0D context, the local dynamics of soil moisture as a direct exogenous forcing of mosquito dynamics. The population model had a Gompertz structure, which included exogenous meteorological forcings and delayed internal dynamics. The models were coupled within a hierarchical statistical structure to overcome the relatively short length of the samples by exploiting the large number of concurrent observations available. The results indicated that Cx.pipiens abundance had significant density dependence at 1 week lag, which approximately matched its development time from larvae to adult. Among the exogenous controls, temperature, daylight hours, and soil moisture explained most of the dynamics. Longer daylight hours and lower soil moisture values resulted in higher abundance. The negative correlation of soil moisture and mosquito population can be explained with the abundance of water in the region (e.g. due to irrigation) and the preference for eutrophic habitats by Cx.pipien. Variations among sites were explained by land use factors as represented by distance to the nearest rice field and NDVI values: the carrying capacity decreased with increased distance to the nearest rice filed, while the maximum growth rate was positively related with NDVI. The model shows a satisfactory performance in predicting (potentially one week in advance) mosquito

  13. Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

    Science.gov (United States)

    Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis

    2012-01-01

    Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures

  14. The Tomato spotted wilt virus genome is processed differentially in its plant host Arachis hypogaea and its thrips vector Frankliniella fusca

    OpenAIRE

    Stephen John Fletcher; Anita Shrestha; Jonathan Peters; Carroll, Bernard J.; Rajagopalbabu Srinivasan; Pappu, Hanu R.; Neena Mitter

    2016-01-01

    Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus is the type member of the Tospovirus genus with a broad host range of more than 900 plant species. Interactions between these viruses and their plant hosts and insect vectors via RNA interference pathways are likely a key determinant of pathogenicity. The current investigation, for the first time, compares biogenesis of small RNAs be...

  15. Inhibition of avian tumor viruses by vector-based RNA interference

    Science.gov (United States)

    RNA interference (RNAi) has been shown to reduce the replication of certain animal viruses both in cell culture and in live animals. We developed RNAi-based anti-viral strategies against two important chicken pathogens: avian leukosis virus (ALV) and Marek’s Disease virus MDV). Entry plasmids conta...

  16. Host feeding patterns of established and potential mosquito vectors of West Nile virus in the eastern United States.

    Science.gov (United States)

    Apperson, Charles S; Hassan, Hassan K; Harrison, Bruce A; Savage, Harry M; Aspen, Stephen E; Farajollahi, Ary; Crans, Wayne; Daniels, Thomas J; Falco, Richard C; Benedict, Mark; Anderson, Michael; McMillen, Larry; Unnasch, Thomas R

    2004-01-01

    An important variable in determining the vectorial capacity of mosquito species for arthropod-borne infections is the degree of contact of the vector and the vertebrate reservoir. This parameter can be estimated by examining the host-feeding habits of vectors. Serological and polymerase chain reaction based methods have been used to study the host-feedings patterns of 21 mosquito species from New York, New Jersey, and Tennessee, 19 of which previously have been found infected with West Nile virus. Mammalophilic mosquito species in New Jersey and New York fed primarily upon white-tailed deer, while those from Memphis, Tennessee, fed mainly upon domestic dogs. A total of 24 different avian host species were detected among the avian-derived blood meals. American Robin, Northern Cardinal, Northern Mockingbird, Tufted Titmouse, and Brown-headed Cowbird were common avian hosts, while blood meals derived from the American Crow were relatively rare. Although the majority of common host species were potentially among the most abundant birds at each location, the proportion of blood meals from the most commonly fed upon avian species was greater than was predicted based upon the likely abundance of these species alone. These findings suggest that vector species for West Nile virus may preferentially feed upon certain avian hosts.

  17. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available BACKGROUND: Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS. METHODS AND FINDINGS: This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP. CONCLUSION: This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  18. Development of conditionally replicating integrase defective lentiviral vectors for Epstein-Barr virus gene therapy

    OpenAIRE

    Blasi, Maria

    2012-01-01

    Integrase defective lentiviral vectors show promise for achieving gene expression without integration, preserving some benefits of LVs, whereas reducing the potentially detrimental risk of insertional mutagenesis. Numerous reports supported the ability of these vectors to confer long-term gene expression in slowly dividing cell types for potentially corrective gene expression. These reports also highlighted additional applications of these vectors as delivery platforms for alternate integrati...

  19. Ecological Fitness of Non-vector Planthopper Sogatella furcifera on Rice Plants Infected with Rice Black Streaked Dwarf Virus

    Institute of Scientific and Technical Information of China (English)

    HE Xiao-chan; XU Hong-xing; ZHENG Xu-song; YANG Ya-jun; GAO Guang-chun; PAN Jian-hong; LU Zhong-xian

    2012-01-01

    We evaluated the effects of rice black streak dwarf virus (RBSDV)-infested rice plants on the ecological parameters and its relevant defensive and detoxification enzymes of white-backed planthopper (WBPH) in laboratory for exploring the relationship between RBSDV and the non-vector planthopper.The results showed that nymph survival rate,female adult weight and fecundity,and egg hatchability of WBPH fed on RBSDV-infested rice plants did not markedly differ from those on healthy plants,whereas the female adult longevity and egg duration significantly shortened on diseased plants.Furthermore,significantly higher activities of defensive enzymes (dismutase,catalase and peroxidase) and detoxification enzymes (acetylcholinesterase,carboxylesterase and glutathione S-transferase) were found in WBPH adults fed on infected plants.Results implied that infestation by RBSDV increased the ecological fitness of non-vector planlhopper population.

  20. Synchrony of sylvatic dengue isolations: a multi-host, multi-vector SIR model of dengue virus transmission in Senegal.

    Directory of Open Access Journals (Sweden)

    Benjamin M Althouse

    Full Text Available Isolations of sylvatic dengue-2 virus from mosquitoes, humans and non-human primates in Senegal show synchronized multi-annual dynamics over the past 50 years. Host demography has been shown to directly affect the period between epidemics in other pathogen systems, therefore, one might expect unsynchronized multi-annual cycles occurring in hosts with dramatically different birth rates and life spans. However, in Senegal, we observe a single synchronized eight-year cycle across all vector species, suggesting synchronized dynamics in all vertebrate hosts. In the current study, we aim to explore two specific hypotheses: 1 primates with different demographics will experience outbreaks of dengue at different periodicities when observed as isolated systems, and that coupling of these subsystems through mosquito biting will act to synchronize incidence; and 2 the eight-year periodicity of isolations observed across multiple primate species is the result of long-term cycling in population immunity in the host populations. To test these hypotheses, we develop a multi-host, multi-vector Susceptible, Infected, Removed (SIR model to explore the effects of coupling multiple host-vector systems of dengue virus transmission through cross-species biting rates. We find that under small amounts of coupling, incidence in the host species synchronize. Long-period multi-annual dynamics are observed only when prevalence in troughs reaches vanishingly small levels (< 10(-10, suggesting that these dynamics are inconsistent with sustained transmission in this setting, but are consistent with local dengue virus extinctions followed by reintroductions. Inclusion of a constant introduction of infectious individuals into the system causes the multi-annual periods to shrink, while the effects of coupling remain the same. Inclusion of a stochastic rate of introduction allows for multi-annual periods at a cost of reduced synchrony. Thus, we conclude that the eight-year period