WorldWideScience

Sample records for adeno-associated viral variant

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

    Science.gov (United States)

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

    2015-01-01

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

  2. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  3. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  4. 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...

  5. 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…

  6. A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

    NARCIS (Netherlands)

    Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

    Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

  7. 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.

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

    Science.gov (United States)

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

    2017-06-13

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

  9. DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

    Directory of Open Access Journals (Sweden)

    Maria Schnödt

    2016-01-01

    Full Text Available Adeno-associated viral (AAV vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss and self-complementary (sc AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV. Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

  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. 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...... recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2...... 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....

  12. 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.

  13. 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...

  14. Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function

    NARCIS (Netherlands)

    Blits, B; Oudega, M.; Boer, G J; Bartlett Bunge, M; Verhaagen, J

    2003-01-01

    To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding

  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

    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...

  16. Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

    Directory of Open Access Journals (Sweden)

    Nagesh Pulicherla

    Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

  17. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

    Directory of Open Access Journals (Sweden)

    Vreugdenhil Erno

    2010-07-01

    Full Text Available Abstract Background This study compared the transduction efficiencies of an adeno-associated viral (AAV vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP, with a lentiviral (LV vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed, to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 108 or 1 × 109 genomic copies of AAV1-GFP and 1 × 105 transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

  18. 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. © 2014 Wiley Periodicals, Inc.

  19. Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

    Directory of Open Access Journals (Sweden)

    Verhaagen Joost

    2010-02-01

    Full Text Available Abstract Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs and several axon guidance molecules, including all members of the secreted (class 3 Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV mediated expression of short hairpin RNAs (shRNAs to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1 and Neuropilin 2 (Npn-2. Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

  20. Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

    Science.gov (United States)

    Mason, J B; Gurda, B L; Van Wettere, A; Engiles, J B; Wilson, J M; Richardson, D W

    2017-01-01

    Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Randomised in vivo experiment. We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the

  1. Adeno-Associated Viral Vector (Serotype 2)-Nerve Growth Factor for Patients With Alzheimer Disease: A Randomized Clinical Trial.

    Science.gov (United States)

    Rafii, Michael S; Tuszynski, Mark H; Thomas, Ronald G; Barba, David; Brewer, James B; Rissman, Robert A; Siffert, Joao; Aisen, Paul S

    2018-03-26

    Nerve growth factor (NGF) is an endogenous neurotrophic factor that prevents the death and augments the functional state of cholinergic neurons of the basal forebrain, a cell population that undergoes extensive degeneration in Alzheimer disease (AD). To determine whether stereotactically guided intracerebral injections of adeno-associated viral vector (serotype 2)-nerve growth factor (AAV2-NGF) are well tolerated and exhibit preliminary evidence of impact on cognitive decline in mild to moderate AD-associated dementia. In a multicenter phase 2 trial, 49 participants with mild to moderate AD were randomly assigned in a 1:1 ratio to receive stereotactically guided intracerebral injections of AAV2-NGF or sham surgery. Participants were enrolled between November 2009 and December 2012. Analyses began in February 2015. The study was conducted at 10 US academic medical centers. Eligibility required a diagnosis of mild to moderate dementia due to AD and individuals aged 55 to 80 years. A total of 39 participants did not pass screening; the most common reason was Mini-Mental State Examination scores below cutoff. Analyses were intention-to-treat. Stereotactically guided intracerebral injections of AAV2-NGF into the nucleus basalis of Meynert of each hemisphere or sham surgery. Change from baseline on the Alzheimer Disease Assessment Scale-cognitive subscale at month 24. Among 49 participants, 21 (43%) were women, 42 (86%) self-identified as white, and the mean (SD) age was 68 (6.4) years. AAV2-NGF was safe and well-tolerated through 24 months. No significant difference was noted between the treatment group and placebo on the primary outcome measure, the Alzheimer Disease Assessment Scale-cognitive subscale (mean [SD] score, 14.52 [4.66] vs 9.11 [4.65], P = .17). This multicenter randomized clinical trial demonstrated the feasibility of sham-surgery-controlled stereotactic gene delivery studies in patients with AD. AAV2-NGF delivery was well-tolerated but did not

  2. Targeted CNS delivery using human MiniPromoters and demonstrated compatibility with adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Charles N de Leeuw

    2014-01-01

    Full Text Available Critical for human gene therapy is the availability of small promoters tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters (MiniPs using computational biology and phylogenetic conservation. MiniPs were tested in mouse as single-copy knock-ins at the Hprt locus on the X chromosome and evaluated for lacZ reporter expression in central nervous system (CNS and non–CNS tissue. Eighteen novel MiniPs driving expression in mouse brain were identified, 2 MiniPs for driving pan-neuronal expression and 17 MiniPs for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPs exhibit similar cell type specificity when delivered via adeno-associated virus vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy-number effects or genomic location, and results in constructs predisposed to success in adeno-associated virus. These MiniPs are immediately applicable for preclinical studies toward gene therapy in humans and are publicly available to facilitate basic and clinical research, and human gene therapy.

  3. Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation.

    Science.gov (United States)

    Saunders, Arpiar; Sabatini, Bernardo L

    2015-07-01

    Recombinant adeno-associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre-On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre-Off) can be used in conjunction with Cre-On rAAVs or genomic Cre-reporter alleles to study brain circuits. Intracranial injection of Cre-On/Cre-Off rAAVs into spatially intermingled Cre(+) and Cre(-) neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre(+) neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre-Off rAAV systems, including caveats and methods of validation. Copyright © 2015 John Wiley & Sons, Inc.

  4. 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.

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

    Science.gov (United States)

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

    2015-08-01

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

  6. 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.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

    Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

    2013-01-01

    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.

  10. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  11. Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

    Science.gov (United States)

    Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

    2014-04-01

    Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

  12. Adeno-Associated Viral Vector-Mediated mTOR Inhibition by Short Hairpin RNA Suppresses Laser-Induced Choroidal Neovascularization

    Directory of Open Access Journals (Sweden)

    Tae Kwann Park

    2017-09-01

    Full Text Available Choroidal neovascularization (CNV is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF, the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA, which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV. Keywords: retinal neovascularization, choroidal neovascularization, adeno-associated virus, mTOR, RNA interference, mTOR shRNA, autophagy

  13. Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

    Science.gov (United States)

    Drouin, Lauren M; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni; Baker, Timothy S; Agbandje-McKenna, Mavis

    2016-10-01

    The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid

  14. 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...

  15. Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

    Science.gov (United States)

    Fu, Haiyan; DiRosario, Julianne; Kang, Lu; Muenzer, Joseph; McCarty, Douglas M

    2010-07-01

    Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

  16. 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.

  17. 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.

  18. Pharmacology of Recombinant Adeno-associated Virus Production

    Directory of Open Access Journals (Sweden)

    Magalie Penaud-Budloo

    2018-03-01

    Full Text Available Recombinant adeno-associated viral (rAAV vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input used in each platform and which related impurities can be expected in final products (output. The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious, residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses, and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.

  19. Adeno-associated virus rep protein synthesis during productive infection

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  20. Adeno-associated virus-mediated gene transfer

    OpenAIRE

    Srivastava, Arun

    2008-01-01

    Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of whic...

  1. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    International Nuclear Information System (INIS)

    Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2008-01-01

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution. Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 Å resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination

  2. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    Directory of Open Access Journals (Sweden)

    Yarong Liu

    2014-01-01

    Full Text Available Adeno-associated virus type 2 (AAV2 is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs. We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

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

    International Nuclear Information System (INIS)

    Malkinson, Mertyn; Winocour, Ernest

    2005-01-01

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

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

    Science.gov (United States)

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

    2006-07-01

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

  5. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

    Science.gov (United States)

    Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude

    2016-01-01

    Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

  6. Adeno-associated virus-mediated gene transfer.

    Science.gov (United States)

    Srivastava, Arun

    2008-09-01

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

  7. Ultrasound Targeted Microbubble Destruction Stimulates Cellular Endocytosis in Facilitation of Adeno-Associated Virus Delivery

    Directory of Open Access Journals (Sweden)

    Lian-Fang Du

    2013-05-01

    Full Text Available The generally accepted mechanism for ultrasound targeted microbubble destruction (UTMD to enhance drug and gene delivery is through sonoporation. However, passive uptake of adeno-associated virus (AAV into cells following sonoporation does not adequately explain observations of enhanced transduction by UTMD. This study investigated alternative mechanisms of UTMD enhancement in AAV delivery. UTMD significantly enhanced transduction efficiency of AAV in a dose-dependent manner. UTMD stimulated a persistent uptake of AAV into the cytoplasm and nucleus. This phenomenon occurred over several hours, suggesting that some viral particles are endocytosed by cells rather than exclusively passing through pores created by sonoporation. Additionally, UTMD enhanced clathrin expression and accumulation at the plasma membrane suggesting greater clathrin-mediated endocytosis following UTMD. Transmission electron microscopy (TEM revealed that UTMD stimulated formation of clathrin-coated pits (CPs and uncoated pits (nCPs. Furthermore, inhibition of clathrin-mediated endocytosis partially blocked the enhancement of AAV uptake following UTMD. The results of this study implicate endocytosis as a mechanism that contributes to UTMD-enhanced AAV delivery.

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Science.gov (United States)

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

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

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

    Science.gov (United States)

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

    1996-07-15

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

  11. Factors influencing recombinant adeno-associated virus production.

    Science.gov (United States)

    Salvetti, A; Orève, S; Chadeuf, G; Favre, D; Cherel, Y; Champion-Arnaud, P; David-Ameline, J; Moullier, P

    1998-03-20

    Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary

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

    Science.gov (United States)

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

    2011-05-01

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

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

    Science.gov (United States)

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

    2018-02-05

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

  14. Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

    Science.gov (United States)

    Mays, Lauren E.; Wang, Lili; Tenney, Rebeca; Bell, Peter; Nam, Hyun-Joo; Lin, Jianping; Gurda, Brittney; Van Vliet, Kim; Mikals, Kyle; Agbandje-McKenna, Mavis

    2013-01-01

    Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33. PMID:23720715

  15. 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.

  16. Recombinant adeno-associated virus: efficient transduction of the rat VMH and clearance from blood.

    Directory of Open Access Journals (Sweden)

    Margriet A van Gestel

    Full Text Available To promote the efficient and safe application of adeno-associated virus (AAV vectors as a gene transfer tool in the central nervous system (CNS, transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH. No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×109 genomic copies of AAV would be fully cleared from blood after 2 days.

  17. Random Plant Viral Variants Attain Temporal Advantages During Systemic Infections and in Turn Resist other Variants of the Same Virus.

    Science.gov (United States)

    Zhang, Xiao-Feng; Guo, Jiangbo; Zhang, Xiuchun; Meulia, Tea; Paul, Pierce; Madden, Laurence V; Li, Dawei; Qu, Feng

    2015-10-20

    Infection of plants with viruses containing multiple variants frequently leads to dominance by a few random variants in the systemically infected leaves (SLs), for which a plausible explanation is lacking. We show here that SL dominance by a given viral variant is adequately explained by its fortuitous lead in systemic spread, coupled with its resistance to superinfection by other variants. We analyzed the fate of a multi-variant turnip crinkle virus (TCV) population in Arabidopsis and N. benthamiana plants. Both wild-type and RNA silencing-defective plants displayed a similar pattern of random dominance by a few variant genotypes, thus discounting a prominent role for RNA silencing. When introduced to plants sequentially as two subpopulations, a twelve-hour head-start was sufficient for the first set to dominate. Finally, SLs of TCV-infected plants became highly resistant to secondary invasions of another TCV variant. We propose that random distribution of variant foci on inoculated leaves allows different variants to lead systemic movement in different plants. The leading variants then colonize large areas of SLs, and resist the superinfection of lagging variants in the same areas. In conclusion, superinfection resistance is the primary driver of random enrichment of viral variants in systemically infected plants.

  18. Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

    Science.gov (United States)

    Diprimio, Nina; Bowles, Dawn E.; Hirsch, Matthew L.; Monahan, Paul E.; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration. PMID:22593151

  19. Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

    Science.gov (United States)

    Bell, Peter; Vandenberghe, Luk H; Wilson, James M

    2014-06-01

    Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

  20. 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.

  1. Selective expansion of viral variants following experimental transmission of a reconstituted feline immunodeficiency virus quasispecies.

    Directory of Open Access Journals (Sweden)

    Brian J Willett

    Full Text Available Following long-term infection with virus derived from the pathogenic GL8 molecular clone of feline immunodeficiency virus (FIV, a range of viral variants emerged with distinct modes of interaction with the viral receptors CD134 and CXCR4, and sensitivities to neutralizing antibodies. In order to assess whether this viral diversity would be maintained following subsequent transmission, a synthetic quasispecies was reconstituted comprising molecular clones bearing envs from six viral variants and its replicative capacity compared in vivo with a clonal preparation of the parent virus. Infection with either clonal (Group 1 or diverse (Group 2 challenge viruses, resulted in a reduction in CD4+ lymphocytes and an increase in CD8+ lymphocytes. Proviral loads were similar in both study groups, peaking by 10 weeks post-infection, a higher plateau (set-point being achieved and maintained in study Group 1. Marked differences in the ability of individual viral variants to replicate were noted in Group 2; those most similar to GL8 achieved higher viral loads while variants such as the chimaeras bearing the B14 and B28 Envs grew less well. The defective replication of these variants was not due to suppression by the humoral immune response as virus neutralising antibodies were not elicited within the study period. Similarly, although potent cellular immune responses were detected against determinants in Env, no qualitative differences were revealed between animals infected with either the clonal or the diverse inocula. However, in vitro studies indicated that the reduced replicative capacity of variants B14 and B28 in vivo was associated with altered interactions between the viruses and the viral receptor and co-receptor. The data suggest that viral variants with GL8-like characteristics have an early, replicative advantage and should provide the focus for future vaccine development.

  2. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    International Nuclear Information System (INIS)

    Lerch, Thomas F.; Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2009-01-01

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning. Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication

  3. Stable producer cell lines for adeno-associated virus (AAV) assembly.

    Science.gov (United States)

    Chadeuf, Gilliane; Salvetti, Anna

    2010-10-01

    Stable producer cell lines containing both the rep and cap genes and recombinant adeno-associated virus (rAAV) vectors can be infected with a helper virus to provide reliable and efficient production of rAAV stocks. However, the development of these cell lines is time-consuming. The procedure described here is therefore recommended only for studies requiring the production of high amounts of rAAV, such as preclinical studies performed in large animals.

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

    OpenAIRE

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

    1994-01-01

    Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthe...

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

    Science.gov (United States)

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

    2015-10-15

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

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

    Science.gov (United States)

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

    2008-03-01

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

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

    Science.gov (United States)

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

    2011-06-01

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

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

    International Nuclear Information System (INIS)

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna

    2005-01-01

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

  9. Transmission of single and multiple viral variants in primary HIV-1 subtype C infection.

    Directory of Open Access Journals (Sweden)

    Vladimir Novitsky

    2011-02-01

    Full Text Available To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1,2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80% cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64% cases, and transmission of multiple variants was evident in 8 of 25 (32% cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96% cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively.

  10. 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.

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

    International Nuclear Information System (INIS)

    Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

    2006-01-01

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

  12. A random forest classifier for detecting rare variants in NGS data from viral populations

    Directory of Open Access Journals (Sweden)

    Raunaq Malhotra

    Full Text Available We propose a random forest classifier for detecting rare variants from sequencing errors in Next Generation Sequencing (NGS data from viral populations. The method utilizes counts of varying length of k-mers from the reads of a viral population to train a Random forest classifier, called MultiRes, that classifies k-mers as erroneous or rare variants. Our algorithm is rooted in concepts from signal processing and uses a frame-based representation of k-mers. Frames are sets of non-orthogonal basis functions that were traditionally used in signal processing for noise removal. We define discrete spatial signals for genomes and sequenced reads, and show that k-mers of a given size constitute a frame.We evaluate MultiRes on simulated and real viral population datasets, which consist of many low frequency variants, and compare it to the error detection methods used in correction tools known in the literature. MultiRes has 4 to 500 times less false positives k-mer predictions compared to other methods, essential for accurate estimation of viral population diversity and their de-novo assembly. It has high recall of the true k-mers, comparable to other error correction methods. MultiRes also has greater than 95% recall for detecting single nucleotide polymorphisms (SNPs and fewer false positive SNPs, while detecting higher number of rare variants compared to other variant calling methods for viral populations. The software is available freely from the GitHub link https://github.com/raunaq-m/MultiRes. Keywords: Sequencing error detection, Reference free methods, Next-generation sequencing, Viral populations, Multi-resolution frames, Random forest classifier

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

    Science.gov (United States)

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

    1999-07-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

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

    2008-06-01

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

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

    Science.gov (United States)

    Nash, Kevin R; Gordon, Marcia N

    2016-01-01

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

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

    Science.gov (United States)

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

    1998-12-01

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

  18. 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.

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

    Science.gov (United States)

    Wu, Zhijian; Wu, Xiaobing; Cao, Hui; Dong, Xiaoyan; Wang, Hong; Hou, Yunde

    2002-02-01

    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/DeltaUL2, 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/DeltaUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28x10(4) 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.

  20. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B

    DEFF Research Database (Denmark)

    Miesbach, Wolfgang; Meijer, Karina; Coppens, Michiel

    2018-01-01

    Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving expre...

  1. Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice

    Directory of Open Access Journals (Sweden)

    Hung-Bo Hsiao

    2014-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    Saunders, Arpiar; Johnson, Caroline A.; Sabatini, Bernardo L.

    2012-01-01

    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. PMID:22866029

  4. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

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

    Science.gov (United States)

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

    2003-10-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  7. Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

    Science.gov (United States)

    Gray-Edwards, Heather L; Randle, Ashley N; Maitland, Stacy A; Benatti, Hector R; Hubbard, Spencer M; Canning, Peter F; Vogel, Matthew B; Brunson, Brandon L; Hwang, Misako; Ellis, Lauren E; Bradbury, Allison M; Gentry, Atoska S; Taylor, Amanda R; Wooldridge, Anne A; Wilhite, Dewey R; Winter, Randolph L; Whitlock, Brian K; Johnson, Jacob A; Holland, Merilee; Salibi, Nouha; Beyers, Ronald J; Sartin, James L; Denney, Thomas S; Cox, Nancy R; Sena-Esteves, Miguel; Martin, Douglas R

    2018-03-01

    Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and β subunits separately (TSD α + β) injected at high (1.3 × 10 13 vector genomes) or low (4.2 × 10 12 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + β sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + β), and ganglioside clearance was most widespread in the TSD α + β high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

  8. Adeno-associated virus vectors can be efficiently produced without helper virus.

    Science.gov (United States)

    Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

    1998-07-01

    The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

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

    Science.gov (United States)

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

    1994-09-01

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

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

    Science.gov (United States)

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

    2013-04-01

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

  11. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

    Science.gov (United States)

    Flannery, J G; Zolotukhin, S; Vaquero, M I; LaVail, M M; Muzyczka, N; Hauswirth, W W

    1997-06-24

    We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

  12. Computer-aided identification of polymorphism sets diagnostic for groups of bacterial and viral genetic variants

    Directory of Open Access Journals (Sweden)

    Huygens Flavia

    2007-08-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs and genes that exhibit presence/absence variation have provided informative marker sets for bacterial and viral genotyping. Identification of marker sets optimised for these purposes has been based on maximal generalized discriminatory power as measured by Simpson's Index of Diversity, or on the ability to identify specific variants. Here we describe the Not-N algorithm, which is designed to identify small sets of genetic markers diagnostic for user-specified subsets of known genetic variants. The algorithm does not treat the user-specified subset and the remaining genetic variants equally. Rather Not-N analysis is designed to underpin assays that provide 0% false negatives, which is very important for e.g. diagnostic procedures for clinically significant subgroups within microbial species. Results The Not-N algorithm has been incorporated into the "Minimum SNPs" computer program and used to derive genetic markers diagnostic for multilocus sequence typing-defined clonal complexes, hepatitis C virus (HCV subtypes, and phylogenetic clades defined by comparative genome hybridization (CGH data for Campylobacter jejuni, Yersinia enterocolitica and Clostridium difficile. Conclusion Not-N analysis is effective for identifying small sets of genetic markers diagnostic for microbial sub-groups. The best results to date have been obtained with CGH data from several bacterial species, and HCV sequence data.

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

    International Nuclear Information System (INIS)

    Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.; Murrin, L. Charles; Lockridge, Oksana

    2006-01-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

  15. Defective-interfering particles of the human parvovirus adeno-associated virus

    International Nuclear Information System (INIS)

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

    1979-01-01

    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

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

    Science.gov (United States)

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

    2005-01-01

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

  17. Novel strategy for generation and titration of recombinant adeno-associated virus vectors.

    Science.gov (United States)

    Shiau, Ai-Li; Liu, Pu-Ste; Wu, Chao-Liang

    2005-01-01

    Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

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

    Science.gov (United States)

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

    2018-01-01

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

  19. Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy.

    Science.gov (United States)

    Negrete, Alejandro; Esteban, Geoffrey; Kotin, Robert M

    2007-09-01

    A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 x 10(15) particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 x 10(16) particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

  20. Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo.

    Directory of Open Access Journals (Sweden)

    Rajiv R Mohan

    Full Text Available Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5 impedes corneal neovascularization (CNV in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10(12 vg/ml application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05, 66% (p<0.001, and 63% (p<0.01 reduction at early (day 5, mid (day 10, and late (day 14 stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57-65, p<0.5, and CD31 immunoblotting (62-67%, p<0.05 supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic and up-regulated PEDF (anti-angiogenic genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5-mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.

  1. Adeno-associated virus (AAV-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice

    Directory of Open Access Journals (Sweden)

    Bading Hilmar

    2007-12-01

    Full Text Available Abstract Background Calcium/calmodulin-dependent protein kinase IV (CaMKIV controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB. This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice. Results We used recombinant adeno-associated virus (rAAV-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test. Conclusion Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.

  2. Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity

    Directory of Open Access Journals (Sweden)

    Justin Judd

    2012-01-01

    Full Text Available Adeno-associated virus (AAV-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase. The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.

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

    International Nuclear Information System (INIS)

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2012-01-01

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

  4. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.

    Science.gov (United States)

    Whiteway, Alistair; Deru, Wale; Prentice, H Grant; Anderson, Robert

    2003-12-01

    Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.

  5. Use of self-complementary adeno-associated virus serotype 2 as a tracer for labeling axons: implications for axon regeneration.

    Directory of Open Access Journals (Sweden)

    Yingpeng Liu

    Full Text Available Various types of tracers are available for use in axon regeneration, but they require an extra operational tracer injection, time-consuming immunohistochemical analysis and cause non-specific labeling. Considerable efforts over the past years have explored other methodologies, especially the use of viral vectors, to investigate axon regeneration after injury. Recent studies have demonstrated that self-complementary Adeno-Associated Virus (scAAV induced a high transduction efficiency and faster expression of transgenes. Here, we describe for the first time the use of scAAV2-GFP to label long-projection axons in the corticospinal tract (CST, rubrospinal tract (RST and the central axons of dorsal root ganglion (DRG in the normal and lesioned animal models. We found that scAAV2-GFP could efficiently transduce neurons in the sensorimotor cortex, red nucleus and DRG. Strong GFP expression could be transported anterogradely along the axon to label the numerous axon fibers from CST, RST and central axons of DRG separately. Comparison of the scAAV2 vector with single-stranded (ss AAV2 vector in co-labeled sections showed that the scAAV2 vector induced a faster and stronger transgene expression than the ssAAV2 vector in DRG neurons and their axons. In both spinal cord lesion and dorsal root crush injury models, scAAV-GFP could efficiently label the lesioned and regenerated axons around the lesion cavity and the dorsal root entry zone (DREZ respectively. Further, scAAV2-GFP vector could be combined with traditional tracer to specifically label sensory and motor axons after spinal cord lesion. Thus, we show that using scAAV2-GFP as a tracer is a more effective and efficient way to study axon regeneration following injury.

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

    Science.gov (United States)

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

    2017-10-15

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

  7. Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.

    Science.gov (United States)

    Lamartina, S; Roscilli, G; Rinaudo, D; Delmastro, P; Toniatti, C

    1998-09-01

    Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

  8. 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

    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...

  9. Peripheral immunophenotype and viral promoter variants during the asymptomatic phase of feline immunodeficiency virus infection.

    Science.gov (United States)

    Murphy, B; Hillman, C; McDonnel, S

    2014-01-22

    Feline immunodeficiency virus (FIV)-infected cats enter a clinically asymptomatic phase during chronic infection. Despite the lack of overt clinical disease, the asymptomatic phase is characterized by persistent immunologic impairment. In the peripheral blood obtained from cats experimentally infected with FIV-C for approximately 5 years, we identified a persistent inversion of the CD4/CD8 ratio. We cloned and sequenced the FIV-C long terminal repeat containing the viral promoter from cells infected with the inoculating virus and from in vivo-derived peripheral blood mononuclear cells and CD4 T cells isolated at multiple time points throughout the asymptomatic phase. Relative to the inoculating virus, viral sequences amplified from cells isolated from all of the infected animals demonstrated multiple single nucleotide mutations and a short deletion within the viral U3, R and U5 regions. A transcriptionally inactivating proviral mutation in the U3 promoter AP-1 site was identified at multiple time points from all of the infected animals but not within cell-associated viral RNA. In contrast, no mutations were identified within the sequence of the viral dUTPase gene amplified from PBMC isolated at approximately 5 years post-infection relative to the inoculating sequence. The possible implications of these mutations to viral pathogenesis are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Sendai virosomal infusion of an adeno-associated virus-derived construct containing neuropeptide Y into primary rat brain cultures.

    Science.gov (United States)

    Wu, P; de Fiebre, C M; Millard, W J; Elmstrom, K; Gao, Y; Meyer, E M

    1995-05-05

    A novel neuronal gene-delivery system was investigated in primary neuron-enriched cultures with respect to driving the expression of neuropeptide Y (NPY). This delivery system consists of an adeno-associated virus-derived (AAV) plasmid, pJDT95npy, encapsulated in reconstituted Sendai virosomes. pJDT95npy contains full length rat NPY cDNA inserted downstream from the P40 promoter in a cap-gene deleted AAV-derived construct. The rep-sequences under control of the P5 and P19 promoters are intact. Virosomally encapsulated pJDT95npy drove the expression of NPY mRNAs, predominantly by P40. Total cellular NPY immunoreactivity and release in the presence of depolarization increased following pJDT95npy-transfection. Neither empty virosomes nor virosomes containing pJDT95 affected NPY mRNA expression or immunoreactivity. This study demonstrates that an AAV-derived plasmid can drive exogenous gene expression in intact neurons after infusion by Sendai virosomes.

  11. 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.

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

    Science.gov (United States)

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

    2009-11-01

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

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

    Science.gov (United States)

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

    2018-03-05

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

  14. 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.

  15. Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

    OpenAIRE

    Zhong, Li; Li, Baozheng; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Cooper, Mario; Herzog, Roland W.; Zolotukhin, Irene; Warrington, Kenneth H.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

    2008-01-01

    Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively...

  16. Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

    Science.gov (United States)

    Hartman, Emily C; Jakobson, Christopher M; Favor, Andrew H; Lobba, Marco J; Álvarez-Benedicto, Ester; Francis, Matthew B; Tullman-Ercek, Danielle

    2018-04-11

    Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

  17. 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.

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

    Science.gov (United States)

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

    2010-01-01

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

  19. 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.

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

    International Nuclear Information System (INIS)

    Zheng, Liu; Weilun, Zhang; Minghong, Jiang; Yaxi, Zhang; Shilian, Liu; Yanxin, Liu; Dexian, Zheng

    2012-01-01

    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. 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. 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. 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

  1. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    International Nuclear Information System (INIS)

    Prasad, C. Krishna; Meyers, Craig; Zhan Dejin; You Hong; Chiriva-Internati, Maurizio; Mehta, Jawahar L.; Liu Yong; Hermonat, Paul L.

    2003-01-01

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

  2. 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.

  3. 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.

  4. Definition of herpes simplex virus type 1 helper activities for adeno-associated virus early replication events.

    Directory of Open Access Journals (Sweden)

    Nathalie Alazard-Dany

    2009-03-01

    Full Text Available The human parvovirus Adeno-Associated Virus (AAV type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1; whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP complex (UL5/8/52 and the single-stranded DNA-Binding Protein (ICP8 were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42 was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.

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

    Science.gov (United States)

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

    2008-10-01

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

  6. Isolation of uv-sensitive variants of human FL cells by a viral suicide method

    International Nuclear Information System (INIS)

    Shiomi, T.; Sato, K.

    1979-01-01

    A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m 2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability

  7. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

    Science.gov (United States)

    Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

    2015-01-01

    Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

  8. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

    Science.gov (United States)

    Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J

    2009-06-29

    It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with

  9. Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

    Directory of Open Access Journals (Sweden)

    Thuy Le

    Full Text Available BACKGROUND: It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL were obtained from a specimen bank (from 2004-2007. The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36% detected by deep sequencing; the majority of these (95% were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53. The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%. When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016. CONCLUSIONS/SIGNIFICANCE: Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional

  10. Systemic Errors in Quantitative Polymerase Chain Reaction Titration of Self-Complementary Adeno-Associated Viral Vectors and Improved Alternative Methods

    Science.gov (United States)

    Fagone, Paolo; Wright, J. Fraser; Nathwani, Amit C.; Nienhuis, Arthur W.; Davidoff, Andrew M.

    2012-01-01

    Abstract Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities. PMID:22428975

  11. Mosquito bottlenecks alter viral mutant swarm in a tissue and time-dependent manner with contraction and expansion of variant positions and diversity.

    Science.gov (United States)

    Patterson, Edward I; Khanipov, Kamil; Rojas, Mark M; Kautz, Tiffany F; Rockx-Brouwer, Dedeke; Golovko, Georgiy; Albayrak, Levent; Fofanov, Yuriy; Forrester, Naomi L

    2018-01-01

    Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.

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

    Science.gov (United States)

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

    1997-01-01

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

  13. Suppression of cancer growth in mice by adeno-associated virus vector-mediated IFN-beta expression driven by hTERT promoter.

    Science.gov (United States)

    He, Ling Feng; Wang, Yi Gang; Xiao, Tian; Zhang, Kang Jiang; Li, Gong Chu; Gu, Jin Fa; Chu, Liang; Tang, Wen Hao; Tan, Wen-Song; Liu, Xin Yuan

    2009-12-28

    Adeno-associated virus (AAV) has rapidly become a promising gene delivery vehicle for its excellent advantages of non-immunogenic, low pathogenicity and long-term gene expression in vivo. However, a major obstacle in development of effective AAV vector is the lack of tissue specificity, which caused low efficiency of AAV transfer to target cells. The application of human telomerase reverse transcriptase (hTERT) promoter is a prior targeting strategy for AAV in cancer gene therapy as hTERT activity is transcriptionally upregulated in most cancer cells. In the present work, we investigated whether AAV-mediated human interferon beta (IFN-beta) gene driven by hTERT promoter could specifically express in tumor cells and suppress tumor cell growth. Our data demonstrated that hTERT promoter-driven IFN-beta expression was the tumor-specific, decreased the cell viability of tumor cells but not normal cells, and induced tumor cell apoptosis via activation of caspase pathway and release of cytochrome c. AAV-mediated IFN-beta expression driven by hTERT promoter significantly suppressed the growth of colorectal cancer and lung cancer xenograft in mice and resulted in tumor cells death in vivo. These data suggested that AAVs in combination with hTERT-mediated IFN-beta expression could exert potential antitumor activity and provide a novel targeting approach to clinical gene therapy of varieties of cancers.

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

    International Nuclear Information System (INIS)

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig; Jameson, J. Larry

    2008-01-01

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

  15. Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

    Directory of Open Access Journals (Sweden)

    Akikazu Ishihara

    2012-01-01

    Full Text Available Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad, serotype 2 adeno-associated virus vectors (rAAV2, or self-complementary (sc AAV2 vectors carrying green fluorescent protein (GFP. Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

  16. Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

    Science.gov (United States)

    Bull, Marta; Learn, Gerald; Genowati, Indira; McKernan, Jennifer; Hitti, Jane; Lockhart, David; Tapia, Kenneth; Holte, Sarah; Dragavon, Joan; Coombs, Robert; Mullins, James; Frenkel, Lisa

    2009-09-22

    Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence

  17. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    OpenAIRE

    Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV cap...

  18. A CASE OF EPSTEIN-BARR VIRAL INFECTION PROCEEDED UNDER THE MASK OF THE SYSTEMIC VARIANT OF THE JUVENILE RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    T.M. Bzarova

    2007-01-01

    Full Text Available The article describes the treatment of Epstein–Barr viral infection under the mask of the systemic variant of the juvenile rheumatoid arthritis. The clinical presentations of the disease included fever, rash, lymphadenopathy, hepatomegaly, polyarticular syndrome and high lab activity indices. the serologic research uncovered the antibodies to the Epstein–Barr virus in diagnostic titers, which allowed the researchers to verify the diagnosis. A child underwent the treatment with the immunoglobulin of a man with the high concentration of antibodies to cytomegalovirus, which induced the remission of the systemic representations, articular syndrome accompanied B normalization of the lab activity indices and reduction of the antibody titers towards the Epstein–Barr virus.Key words: children, treatment, immune globulin intravenous, septic syndrome, epstein–barr virus.

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

    Science.gov (United States)

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

    2010-09-01

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

  20. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

    Science.gov (United States)

    Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1999-06-01

    Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

  1. Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

    Science.gov (United States)

    Zhong, Li; Li, Baozheng; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Cooper, Mario; Herzog, Roland W.; Zolotukhin, Irene; Warrington, Kenneth H.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

    2008-01-01

    Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells ≈10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an ≈10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy. PMID:18511559

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

    Science.gov (United States)

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

    2016-05-01

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

  3. 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.

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Chang Jin

    2009-11-01

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

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

    Science.gov (United States)

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

    2002-02-05

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

  7. 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.

  8. 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.

  9. Viral RNA levels and env variants in semen and tissues of mature male rhesus macaques infected with SIV by penile inoculation.

    Directory of Open Access Journals (Sweden)

    Francis Fieni

    Full Text Available HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1-9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA in the axillary lymph node (6.48 ± 0.50 were significantly higher than in the genital tract tissues: testis (3.67 ± 2.16; p<0.05, epididymis (3.08 ± 1.19; p<0.0001, prostate (3.36 ± 1.30; p<0.01, and seminal vesicle (2.67 ± 1.50; p<0.0001. Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.

  10. Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

    Science.gov (United States)

    Gorgoglione, Bartolomeo; Carpio, Yamila; Secombes, Christopher J; Taylor, Nick G H; Lugo, Juana María; Estrada, Mario Pablo

    2015-12-01

    Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens. Copyright © 2015

  11. POLYMORPHIC VARIANTS OF THE GENE OF INTERFERON LAMBDA 3 AND FEATURES OF IMMUNE RESPONSE IN CHILDREN WITH CHRONIC VIRAL HEPATITIS C

    Directory of Open Access Journals (Sweden)

    T. B. Sentsova

    2017-01-01

    Full Text Available To study the immune manifestations of the interferon-lambda 3 genepolymorphism in chronic viral hepatitis C, 110 Russian children (54 girls and 56 boys with chronic HCV infection aged from 3 to 17 years were examined. All children were on combined therapy (pegylated interferon + ribavirin. It was found that among the studied polymorphic variants of the IFN-λ 3 gene in children with chronic HCV infection, T allele of the marker rs12979860 is associated with infection and chronization of HCV. The T/T rs12979860 genotype of the IFN-λ3 gene is unfavorable for the course of chronic HCV infection due to low levels of activated T-lymphocytes, intactness of the proinflammatory cytokines TNF-α, IL-6, IL-1α, and interferon-γ inducible protein IP-10. The revealed relation of the polymorphic variants of C/C + C/T locus rs12979860 of INF-λ3 gene with the expression of activated T-lymphocytes discloses the protective nature of these genotypes to the development of chronic HCV infection in children. 

  12. Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

    Science.gov (United States)

    Ryschkewitsch, C F; Friedlaender, J S; Mgone, C S; Jobes, D V; Agostini, H T; Chima, S C; Alpers, M P; Koki, G; Yanagihara, R; Stoner, G L

    2000-07-01

    The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

  13. Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants.

    Science.gov (United States)

    Matthew, Ashley N; Zephyr, Jacqueto; Hill, Caitlin J; Jahangir, Muhammad; Newton, Alicia; Petropoulos, Christos J; Huang, Wei; Kurt-Yilmaz, Nese; Schiffer, Celia A; Ali, Akbar

    2017-07-13

    A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156, and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC 50 values ≤5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.

  14. [Human Papilloma virus in Quechua women from Jujuy with high frequency of cervical cancer: viral types and HPV-16 variants].

    Science.gov (United States)

    Picconi, Maria Alejandra; Gronda, Jorge; Alonio, Lidia V; Villa, Luisa L; Sichero, Laura; Miranda, Sergio; Barcena, Martin; Teyssie, Angelica

    2002-01-01

    Human Papillomaviruses (HPVs) are etiologically associated to cervical carcinoma. In order to evaluate HPV infection and its relationship with the high frequency of this neoplasia in Quechua women from Jujuy (Argentina), 271 cervical samples from preneoplastic and neoplastic lesions (biopsies) and normal controls (cytologies) were studied. Detection and typing were performed using PCR-RFLP or PCR-hybridization and the HPV-16 variability in L1 and E6 genes (by PCR-hybridization) was analysed. HPV was detected in 52% of controls, 91% of low-grade lesions, 97% of high-grade lesions and 100% of invasive carcinomas, corresponding 55% to HPV-16. HPV-16 European variants were predominant, most of them being non-prototypic strains. The high frequency of high risk infection types and the raised proportion of HPV-16 non-prototypic variants related to a greater oncogenic potential could explain, in part, the high cervical cancer frequency of this native population. These data may contribute to disease control and vaccinal formulation.

  15. The prevalence of the pre-existing hepatitis C viral variants and the evolution of drug resistance in patients treated with the NS3-4a serine protease inhibitor telaprevir

    Energy Technology Data Exchange (ETDEWEB)

    Rong, Libin [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory

    2008-01-01

    Telaprevir (VX-950), a novel hepatitis C virus (HCV) NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients infected with HCV genotype 1. Some patients experience viral breakthrough, which has been shown to be associated with emergence of telaprevir-resistant HCV variants during treatment. The exact mechanisms underlying the rapid selection of drug resistant viral variants during dosing are not fully understood. In this paper, we develop a two-strain model to study the pre-treatment prevalence of the mutant virus and derive an analytical solution of the mutant frequency after administration of the protease inhibitor. Our analysis suggests that the rapid increase of the mutant frequency during therapy is not due to mutant growth but rather due to the rapid and profound loss of wild-type virus, which uncovers the pre-existing mutant variants. We examine the effects of backward mutation and hepatocyte proliferation on the pre-existence of the mutant virus and the competition between wild-type and drug resistant virus during therapy. We then extend the simple model to a general model with multiple viral strains. Mutations during therapy do not play a significant role in the dynamics of various viral strains, although they are capable of generating low levels of HCV variants that would otherwise be completely suppressed because of fitness disadvantages. Hepatocyte proliferation may not affect the pretreatment frequency of mutant variants, but is able to influence the quasispecies dynamics during therapy. It is the relative fitness of each mutant strain compared with wild-type that determines which strain(s) will dominate the virus population. The study provides a theoretical framework for exploring the prevalence of pre-existing mutant variants and the evolution of drug resistance during treatment with other protease inhibitors or HCV polymerase inhibitors.

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

    Science.gov (United States)

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

    2012-01-01

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

  17. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    Science.gov (United States)

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-11-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  18. 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

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

    Science.gov (United States)

    Shi, Xin; Wang, Jianzhong; Qin, Yan

    2014-12-01

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

  20. Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.

    Directory of Open Access Journals (Sweden)

    Barbara Bogner

    Full Text Available Adeno associated virus (AAV is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc genomes in the anterior segment of the eye.AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE, iris and chamber angle including trabecular meshwork, with scAAV2(Y444F and scAAV2(triple being the most efficient.This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene

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

    Science.gov (United States)

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

    1997-03-01

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

  2. Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

    Science.gov (United States)

    Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

    2017-01-01

    Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a

  3. Adeno-Associated Virus Serotype 9–Driven Expression of BAG3 Improves Left Ventricular Function in Murine Hearts With Left Ventricular Dysfunction Secondary to a Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Tijana Knezevic, PhD

    2016-12-01

    Full Text Available Mutations in Bcl-2–associated athanogene 3 (BAG3 were associated with skeletal muscle dysfunction and dilated cardiomyopathy. Retro-orbital injection of an adeno-associated virus serotype 9 expressing BAG3 (rAAV9-BAG3 significantly (p < 0.0001 improved left ventricular ejection fraction, fractional shortening, and stroke volume 9 days post-injection in mice with cardiac dysfunction secondary to a myocardial infarction. Furthermore, myocytes isolated from mice 3 weeks after injection showed improved cell shortening, enhanced systolic [Ca2+]i and increased [Ca2+]i transient amplitudes, and increased maximal L-type Ca2+ current amplitude. These results suggest that BAG3 gene therapy may provide a novel therapeutic option for the treatment of heart failure.

  4. PNPLA 3 I148M genetic variant associates with insulin resistance and baseline viral load in HCV genotype 2 but not in genotype 3 infection

    DEFF Research Database (Denmark)

    Rembeck, Karolina; Maglio, Cristina; Lagging, Martin

    2012-01-01

    ABSTRACT: BACKGROUND: Hepatic steatosis in HCV patients has been postulated as a risk factor associated with a higher frequency of fibrosis and cirrhosis. A single genetic variant, PNPLA3 I148M, has been widely associated with increased hepatic steatosis. Previous studies of the PNPLA3 I148M...... sequence variant in HCV infected individuals have reported an association between this variant and prevalence of steatosis, fibrosis, and cirrhosis. To evaluate the impact of PNPLA3 I148M variant on metabolic traits and treatment response in HCV genotype 2 and 3 infected patients. METHODS: Three hundred...

  5. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    International Nuclear Information System (INIS)

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  6. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  7. Human interleukin-10 delivered intrathecally by self-complementary adeno-associated virus 8 induces xenogeneic transgene immunity without clinical neurotoxicity in swine.

    Science.gov (United States)

    Unger, Mark D; Pleticha, Josef; Heilmann, Lukas F; Newman, Laura K; Maus, Timothy P; Beutler, Andreas S

    2018-05-25

    Intrathecal interleukin-10 delivered by plasmid or viral gene vectors has been proposed for clinical testing because it is effective for chronic pain in rodents, a potential therapeutic for various human diseases, and was found to be non-toxic in dogs, when the human interleukin-10 ortholog was tested. However, recent studies in swine testing porcine interleukin-10 demonstrated fatal neurotoxicity. To deliver vector-encoded human interleukin-10 in swine, measure expression of the transgene in cerebrospinal fluid, and monitor animals for signs of neurotoxicity. Human interleukin-10 levels peaked 2 weeks after vector administration followed by a rapid decline that occurred concomitant with the emergence of anti-human interleukin-10 antibodies in the cerebrospinal fluid and serum. Animals remained neurologically healthy throughout the study period. This study suggests that swine are not idiosyncratically sensitive to intrathecal interleukin-10 because, recapitulating previous reports in dogs, they suffered no clinical neurotoxicity from the human ortholog. These results strongly infer that toxicity of intrathecal interleukin-10 in large animal models was previously overlooked because of a species mismatch between transgene and host. The present study further suggests that swine were protected from interleukin-10 by a humoral immune response against the xenogeneic cytokine. Future safety studies of interleukin-10 or related therapeutics may require syngeneic large animal models. This article is protected by copyright. All rights reserved.

  8. Transmission of different variants of PCV2 and viral dynamics in a research facility with pigs mingled from PMWS-affected herds and non-affected herds

    DEFF Research Database (Denmark)

    Dupont, Kitt; Hjulsager, Charlotte Kristiane; Kristensen, C.S.

    2009-01-01

    Post-weaning Multisystemic Wasting Syndrome (PMWS) has been identified in most swine-producing countries worldwide. The disease has resulted in significant health challenges and economic damage to the swine industry. The aim of this study was to determine horizontal transmission of porcine...... the aisle and pens in other compartments). By DNA sequence analysis, eight variants of genotype PCV-2b were identified in the research facility. From the spread of these PCV2-variants it was concluded that PCV2 primarily infects through close contact and nose-to-nose contact. PCV2 genome sequences were...... obtained from selected pigs at arrival to the research facility and again when the same pigs developed PMWS. This analysis showed that pigs from PMWS-affected herds developed PMWS caused by the same variant of PCV2 as they carried when entering the research facility. In contrast, pigs from non...

  9. Viral Hepatitis

    Science.gov (United States)

    ... Home A-Z Health Topics Viral hepatitis Viral hepatitis > A-Z Health Topics Viral hepatitis (PDF, 90 ... liver. Source: National Cancer Institute Learn more about hepatitis Watch a video. Learn who is at risk ...

  10. Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle.

    Science.gov (United States)

    Serre, Stéphanie B N; Jensen, Sanne B; Ghanem, Lubna; Humes, Daryl G; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M

    2016-06-01

    Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research

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

    Science.gov (United States)

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

    2016-01-01

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

  12. 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.

  13. The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

    Directory of Open Access Journals (Sweden)

    Anna C. Maurer

    2018-05-01

    Full Text Available Summary: The adeno-associated virus (AAV vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

  14. Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology.

    Science.gov (United States)

    Sizemore, Rachel J; Seeger-Armbruster, Sonja; Hughes, Stephanie M; Parr-Brownlie, Louise C

    2016-04-01

    Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients. Copyright © 2016 the American Physiological Society.

  15. Comparison between Roche and Xpert in HIV-1 RNA quantitation: A high concordance between the two techniques except for a CRF02_AG subtype variant with high viral load titters detected by Roche but undetected by Xpert.

    Science.gov (United States)

    Avidor, Boaz; Matus, Natalia; Girshengorn, Shirley; Achsanov, Svetlana; Gielman, Simona; Zeldis, Irene; Schweitzer, Inbal; Adler, Amos; Turner, Dan

    2017-08-01

    HIV-1 viral load (VL) testing is important to predict viral progression and to monitor the response to antiretroviral therapy. New HIV-1 VL tests are continuously introduced to the market. Their performance is usually compared to Abbott and/or Roche HIV-1 VL assays, as reference. The Xpert HIV-1 VL test was recently introduced, but its performance compared to Roche has not been sufficiently studied. To compare the Xpert assay with Roche and to assess its use in the HIV clinical laboratory. A total of 383 plasma samples of HIV-1 infected patients previously tested by Roche, were retrospectively tested by Xpert to determine concordance between the two assays. Samples included a diversity of HIV-1 subtypes and a wide range of VLs. There was a high concordance between the two assays, except for a CRF02_AG subtype variant with high VL titters, that was detected by Roche but undetected by Xpert. The 5' long terminal repeat gene region of this virus, targeted by the Xpert assay, was amplified and sequenced. A 25 nucleotide insert was identified, but was unmatched to any known sequences of HIV-1. This particular insert, however could not explain the false-negativity by the Xpert assay. This study underlines the challenge to routine VL testing due to the high genetic diversity of HIV-1. Clinicians should, therefore be advised that a negative VL in cases where the clinical picture does not match the laboratory report, might in fact be, a false-negative result of the VL assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Viral Meningitis

    Science.gov (United States)

    ... better from treatment such as an antiviral medicine. Antibiotics do not help viral infections, so they are not useful in the treatment of viral meningitis. However, antibiotics do fight bacteria, so they are very important ...

  17. Pharyngitis - viral

    Science.gov (United States)

    ... throat is due to a viral infection. The antibiotics will not help. Using them to treat viral infections helps bacteria become resistant to antibiotics. With some sore throats (such as those caused ...

  18. Viral Marketing

    OpenAIRE

    Sorina Raula Gîrboveanu; Silvia Puiu

    2008-01-01

    With consumers showing increasing resistance to traditional forms of advertising such as TV or newspaper ads, marketers have turned to alternate strategies, including viral marketing. Viral marketing exploits existing social networks by encouraging customers to share product information with their friends.In our study we are able to directly observe the effectiveness of person to person word of mouth advertising for hundreds of thousands of products for the first time

  19. Valuable Virality

    NARCIS (Netherlands)

    Akpinar, E.; Berger, Jonah

    2017-01-01

    Given recent interest in social media, many brands now create content that they hope consumers will view and share with peers. While some campaigns indeed go “viral,” their value to the brand is limited if they do not boost brand evaluation or increase purchase. Consequently, a key question is how

  20. Viral Cre-LoxP tools aid genome engineering in mammalian cells.

    Science.gov (United States)

    Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

    2017-01-01

    Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

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

    Science.gov (United States)

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

    2017-07-18

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

  2. Cellulase variants

    Science.gov (United States)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  3. Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: Involvement of viral RNA sequences and the reverse transcriptase enzyme

    NARCIS (Netherlands)

    Abbink, Truus E. M.; Beerens, Nancy; Berkhout, Ben

    2004-01-01

    Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation

  4. Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    Science.gov (United States)

    Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ~68% and ~74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which leads to ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy. PMID:18834608

  5. Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

    Science.gov (United States)

    Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

    2009-03-09

    The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

  6. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  7. Holoprosencephaly Variant

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2003-01-01

    Full Text Available The clinical manifestations in 15 patients (6 boys and 9 girls with middle interhemispheric variant (MIH of holoprosencephaly (HPE were compared with classic subtypes (alobar, semilobar, and lobar of HPE in a multicenter study at Stanford University School of Medicine and Lucile Packard Children’s Hospital; Children’s Hospital of Philadelphia; University of California at San Francisco; Texas Scottish Rite Hospital, Dallas; and Kennedy Krieger Institute, Baltimore, MD.

  8. 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

  9. ACVP-02: Plasma SIV/SHIV RNA Viral Load Measurements through the AIDS and Cancer Virus Program Quantitative Molecular Diagnostics Core | Frederick National Laboratory for Cancer Research

    Science.gov (United States)

    The SIV plasma viral load assay performed by the Quantitative Molecular Diagnostics Core (QMDC) utilizes reagents specifically designed to detect and accurately quantify the full range of SIV/SHIV viral variants and clones in common usage in the rese

  10. ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

    Science.gov (United States)

    Pellet, J; Tafforeau, L; Lucas-Hourani, M; Navratil, V; Meyniel, L; Achaz, G; Guironnet-Paquet, A; Aublin-Gex, A; Caignard, G; Cassonnet, P; Chaboud, A; Chantier, T; Deloire, A; Demeret, C; Le Breton, M; Neveu, G; Jacotot, L; Vaglio, P; Delmotte, S; Gautier, C; Combet, C; Deleage, G; Favre, M; Tangy, F; Jacob, Y; Andre, P; Lotteau, V; Rabourdin-Combe, C; Vidalain, P O

    2010-01-01

    Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

  11. Switches for multiple behavioral states and a viral-based approach to non-invasive whole-brain cargo delivery (Conference Presentation)

    Science.gov (United States)

    Gradinaru, Viviana

    2017-05-01

    Over the past years we have worked on: (1) Viral-based approaches to non-invasive whole-brain cargo delivery: Genetically-encoded tools that can be used to visualize, monitor, and modulate mammalian neurons are revolutionizing neuroscience. These tools are particularly powerful in rodents and invertebrate models where intersectional transgenic strategies are available to restrict their expression to defined cell populations. However, use of genetic tools in non-transgenic animals is often hindered by the lack of vectors capable of safe, efficient, and specific delivery to the desired cellular targets. To begin to address these challenges, we have developed an in vivo Cre-based selection platform (CREATE) for identifying adeno-associated viruses (AAVs) that more efficiently transduce genetically defined cell populations. Our platform's novelty and power arises from the additional selective pressure imparted by a recovery step that amplifies only those capsid variants that have functionally transduced a genetically-defined, Cre-expressing target cell population. The Cre-dependent capsid recovery works within heterogeneous tissue samples without the need for additional steps such as selective capsid recovery approaches that require cell sorting or secondary adenovirus infection. As a first test of the CREATE platform, we selected for viruses that transduced the brain after intravascular delivery and found a novel vector, AAV-PHP.B, that is 40- to 90-fold more efficient at transducing the brain than the current standard, AAV9. AAV-PHP.B transduces most neuronal types and glia across the brain. We also demonstrate here how whole-body tissue clearing can facilitate transduction maps of systemically delivered genes. Since CNS disorders are notoriously challenging due to the restrictive nature of the blood brain barrier our discovery that recombinant vectors can be engineered to overcome this barrier is enabling for the whole field. With the exciting advances in gene

  12. De novo assembly of highly diverse viral populations

    Directory of Open Access Journals (Sweden)

    Yang Xiao

    2012-09-01

    Full Text Available Abstract Background Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. Results We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/ viral-genomics-analysis-software. Conclusions We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.

  13. Mobil Viral Pazarlama

    OpenAIRE

    Barutçu, Süleyman

    2011-01-01

    OBJECTIVE: Mobile Viral Marketing, with using mobile phones, is one of the most importantinnovations after Word of Mouth Marketing performed by face to face amongpeople and Viral Marketing performed in the İnternet. The main objective of thisstudy is to call marketing communicators’ and academicians’ attentions whowant to increase the recognition of companies’ products, services and brands tobecome a current issue in the marketplace using Mobile Viral Marketingapplications by reason of techno...

  14. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  15. High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

    Directory of Open Access Journals (Sweden)

    Sandra J Kuhlman

    2008-04-01

    Full Text Available We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs that express GFP, dsRedExpress, or channelrhodopsin (ChR2 upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

  16. [Emergent viral infections

    NARCIS (Netherlands)

    Galama, J.M.D.

    2001-01-01

    The emergence and re-emergence of viral infections is an ongoing process. Large-scale vaccination programmes led to the eradication or control of some viral infections in the last century, but new viruses are always emerging. Increased travel is leading to a rise in the importation of exotic

  17. Viral Haemorrhagic Septicaemia Virus

    DEFF Research Database (Denmark)

    Olesen, Niels Jørgen; Skall, Helle Frank

    2013-01-01

    This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus.......This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus....

  18. Viral Disease Networks?

    Science.gov (United States)

    Gulbahce, Natali; Yan, Han; Vidal, Marc; Barabasi, Albert-Laszlo

    2010-03-01

    Viral infections induce multiple perturbations that spread along the links of the biological networks of the host cells. Understanding the impact of these cascading perturbations requires an exhaustive knowledge of the cellular machinery as well as a systems biology approach that reveals how individual components of the cellular system function together. Here we describe an integrative method that provides a new approach to studying virus-human interactions and its correlations with diseases. Our method involves the combined utilization of protein - protein interactions, protein -- DNA interactions, metabolomics and gene - disease associations to build a ``viraldiseasome''. By solely using high-throughput data, we map well-known viral associated diseases and predict new candidate viral diseases. We use microarray data of virus-infected tissues and patient medical history data to further test the implications of the viral diseasome. We apply this method to Epstein-Barr virus and Human Papillomavirus and shed light into molecular development of viral diseases and disease pathways.

  19. Understanding Image Virality

    Science.gov (United States)

    2015-06-07

    Example non-viral images. Figure 1: Top: Images with high viral scores in our dataset depict internet “celebrity” memes ex. “Grumpy Cat”; Bottom: Images...of images that is most similar to ours is the concurrently introduced viral meme generator of Wang et al., that combines NLP and Computer Vision (low...doing any of our tasks. The test included questions about widely spread Reddit memes and jargon so that anyone familiar with Reddit can easily get a high

  20. Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains.

    Directory of Open Access Journals (Sweden)

    Yoichiro Shinohara

    Full Text Available Adeno-associated virus (AAV vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength and 0.2-kb (70% astrocyte specificity promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.

  1. Viral hemorrhagic septicemia

    Science.gov (United States)

    Batts, William N.; Winton, James R.

    2012-01-01

    Viral hemorrhagic septicemia (VHS) is one of the most important viral diseases of finfish worldwide. In the past, VHS was thought to affect mainly rainbow trout Oncorhynchus mykiss reared at freshwater facilities in Western Europe where it was known by various names including Egtved disease and infectious kidney swelling and liver degeneration (Wolf 1988). Today, VHS is known as an important source of mortality for cultured and wild fish in freshwater and marine environments in several regions of the northern hemisphere (Dixon 1999; Gagné et al. 2007; Kim and Faisal 2011; Lumsden et al. 2007; Marty et al. 1998, 2003; Meyers and Winton 1995; Skall et al. 2005b; Smail 1999; Takano et al. 2001). Viral hemorrhagic septicemia is caused by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus of the family Rhabdoviridae

  2. Hepatitis viral aguda

    Directory of Open Access Journals (Sweden)

    Héctor Rubén Hernández Garcés

    1998-10-01

    Full Text Available Se realizó una revisión bibliográfica de las hepatitis virales agudas sobre aspectos vinculados a su etiología. Se tuvieron en cuenta además algunos datos epidemiológicos, las formas clínicas más importantes, los exámenes complementarios con especial énfasis en los marcadores virales y el diagnóstico positivoA bibliographical review of acute viral hepatitis was made taking into account those aspects connected with its etiology. Some epidemiological markers, the most important clinical forms, and the complementary examinations with special emphasis on the viral markers and the positive diagnosis were also considered

  3. Wastewater viral community

    Data.gov (United States)

    U.S. Environmental Protection Agency — The dataset contains the information used to generate the figures in the manuscript. The data describes the viral loss measured at all steps of sample processing,...

  4. Viral pathogenesis in diagrams

    National Research Council Canada - National Science Library

    Tremblay, Michel; Berthiaume, Laurent; Ackermann, Hans-Wolfgang

    2001-01-01

    .... The 268 diagrams in Viral Pathogenesis in Diagrams were selected from over 800 diagrams of English and French virological literature, including one derived from a famous drawing by Leonardo da Vinci...

  5. Zoonotic Viral Deseases and Virus Discovery

    DEFF Research Database (Denmark)

    Nielsen, Sandra Cathrine Abel

    Viruses are the most abundant organisms on earth and are ubiquitous in all environments where life is present. They are capable of infecting all cellular forms of life, sometimes causing disease in the infected host. This thesis is broadly divided into two main sections with three projects...... program of wildlife, and with the purpose of preventing the next disease emerging from these animals. Numerous viruses were detected of which many were novel variants, thus reaffirming the notion that attention should be focused at these animals. Near-complete viral genome sequencing was performed...

  6. Metabolism goes viral.

    Science.gov (United States)

    Miyake-Stoner, Shigeki J; O'Shea, Clodagh C

    2014-04-01

    Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2003-01-01

    The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness.......The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness....

  8. Novel Parvovirus and Related Variant in Human Plasma

    Science.gov (United States)

    Fryer, Jacqueline F.; Kapoor, Amit; Minor, Philip D.; Delwart, Eric

    2006-01-01

    We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from 106 copies/mL plasma. PMID:16494735

  9. Effect of oligonucleotide primers in determining viral variability within hosts

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2004-12-01

    Full Text Available Abstract Background Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. Results To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient. Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Conclusions Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.

  10. Effect of oligonucleotide primers in determining viral variability within hosts.

    Science.gov (United States)

    Bracho, Maria Alma; García-Robles, Inmaculada; Jiménez, Nuria; Torres-Puente, Manuela; Moya, Andrés; González-Candelas, Fernando

    2004-12-09

    Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV) populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient). Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.

  11. Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H

    2012-06-06

    RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

  12. Effects of anti-cocaine vaccine and viral gene transfer of cocaine hydrolase in mice on cocaine toxicity including motor strength and liver damage.

    Science.gov (United States)

    Gao, Yang; Geng, Liyi; Orson, Frank; Kinsey, Berma; Kosten, Thomas R; Shen, Xiaoyun; Brimijoin, Stephen

    2013-03-25

    In developing an vivo drug-interception therapy to treat cocaine abuse and hinder relapse into drug seeking provoked by re-encounter with cocaine, two promising agents are: (1) a cocaine hydrolase enzyme (CocH) derived from human butyrylcholinesterase and delivered by gene transfer; (2) an anti-cocaine antibody elicited by vaccination. Recent behavioral experiments showed that antibody and enzyme work in a complementary fashion to reduce cocaine-stimulated locomotor activity in rats and mice. Our present goal was to test protection against liver damage and muscle weakness in mice challenged with massive doses of cocaine at or near the LD50 level (100-120 mg/kg, i.p.). We found that, when the interceptor proteins were combined at doses that were only modestly protective in isolation (enzyme, 1mg/kg; antibody, 8 mg/kg), they provided complete protection of liver tissue and motor function. When the enzyme levels were ~400-fold higher, after in vivo transduction by adeno-associated viral vector, similar protection was observed from CocH alone. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  13. Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.

    Science.gov (United States)

    Chang, Qing; Wang, Jianjun; Li, Qi; Kim, Yeunjung; Zhou, Binfei; Wang, Yunfeng; Li, Huawei; Lin, Xi

    2015-08-01

    Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1(-/-) mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0-P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner's membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1(-/-) mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  14. [Viral hepatitis in travellers].

    Science.gov (United States)

    Abreu, Cândida

    2007-01-01

    Considering the geographical asymmetric distribution of viral hepatitis A, B and E, having a much higher prevalence in the less developed world, travellers from developed countries are exposed to a considerable and often underestimated risk of hepatitis infection. In fact a significant percentage of viral hepatitis occurring in developed countries is travel related. This results from globalization and increased mobility from tourism, international work, humanitarian and religious missions or other travel related activities. Several studies published in Europe and North America shown that more than 50% of reported cases of hepatitis A are travel related. On the other hand frequent outbreaks of hepatitis A and E in specific geographic areas raise the risk of infection in these restricted zones and that should be clearly identified. Selected aspects related with the distribution of hepatitis A, B and E are reviewed, particularly the situation in Portugal according to the published studies, as well as relevant clinical manifestations and differential diagnosis of viral hepatitis. Basic prevention rules considering enteric transmitted hepatitis (hepatitis A and hepatitis E) and parenteral transmitted (hepatitis B) are reviewed as well as hepatitis A and B immunoprophylaxis. Common clinical situations and daily practice "pre travel" advice issues are discussed according to WHO/CDC recommendations and the Portuguese National Vaccination Program. Implications from near future availability of a hepatitis E vaccine, a currently in phase 2 trial, are highlighted. Potential indications for travellers to endemic countries like India, Nepal and some regions of China, where up to 30% of sporadic cases of acute viral hepatitis are caused by hepatitis E virus, are considered. Continued epidemiological surveillance for viral hepatitis is essential to recognize and control possible outbreaks, but also to identify new viral hepatitis agents that may emerge as important global health

  15. CDKL5 variants

    Science.gov (United States)

    Kalscheuer, Vera M.; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A.; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E.S.; Cobb, Stuart R.

    2017-01-01

    Objective: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. Methods: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. Results: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. Conclusions: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain. PMID:29264392

  16. Treating viral hemorrhagic fever.

    NARCIS (Netherlands)

    Mairuhu, A.T.; Brandjes, D.P.; Gorp, E. van

    2003-01-01

    Viral hemorrhagic fevers are illnesses associated with a number of geographically restricted, mostly tropical areas. Over recent decades a number of new hemorrhagic fever viruses have emerged. Advances in our understanding of the pathophysiology of these diseases have improved our initial supportive

  17. HIV Viral Load

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  18. HIV and Viral Hepatitis

    Science.gov (United States)

    ... common causes of viral hepatitis are hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV). HBV and HCV are common ... gov/ mmwr/ preview/ mmwrhtml/ rr5516a1. htm? s_ cid= rr5516a1_ e. The Numbers • • Of people with HIV in the ...

  19. Immigration and viral hepatitis

    NARCIS (Netherlands)

    S. Sharma (Suraj); M. Carballo (Manuel); J.J. Feld (Jordan J.); H.L.A. Janssen (Harry)

    2015-01-01

    textabstractWHO estimates reveal that the global prevalence of viral hepatitis may be as high as 500 million, with an annual mortality rate of up to 1.3 million individuals. The majority of this global burden of disease is borne by nations of the developing world with high rates of vertical and

  20. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2007-01-01

    Trials have assessed bile acids for patients with viral hepatitis, but no consensus has been reached regarding their usefulness.......Trials have assessed bile acids for patients with viral hepatitis, but no consensus has been reached regarding their usefulness....

  1. Hepatitis A through E (Viral Hepatitis)

    Science.gov (United States)

    ... Treatment Eating, Diet, & Nutrition Clinical Trials Wilson Disease Hepatitis (Viral) View or Print All Sections What is Viral Hepatitis? Viral hepatitis is an infection that causes liver inflammation ...

  2. Value of Sharing: Viral Advertisement

    OpenAIRE

    Duygu Aydın; Aşina Gülerarslan; Süleyman Karaçor; Tarık Doğan

    2013-01-01

    Sharing motivations of viral advertisements by consumers and the impacts of these advertisements on the perceptions for brand will be questioned in this study. Three fundamental questions are answered in the study. These are advertisement watching and sharing motivations of individuals, criteria of liking viral advertisement and the impact of individual attitudes for viral advertisement on brand perception respectively. This study will be carried out via a viral advertise...

  3. Viral Marketing and Academic Institution

    OpenAIRE

    Koktová, Silvie

    2010-01-01

    This bachelor thesis examines modern and constantly developing kind of internet marketing -- the so called viral marketing. It deals with its origin, principle, process, advantages and disadvantages, types of viral marketing and presumptions of creating successful viral campaign. The aim of the theoretical part is especially the understanding of viral marketing as one of the effective instruments of contemporary marketing. In this theoretical part the thesis also elaborates a marketing school...

  4. Dengue viral infections

    OpenAIRE

    Malavige, G; Fernando, S; Fernando, D; Seneviratne, S

    2004-01-01

    Dengue viral infections are one of the most important mosquito borne diseases in the world. They may be asymptomatic or may give rise to undifferentiated fever, dengue fever, dengue haemorrhagic fever (DHF), or dengue shock syndrome. Annually, 100 million cases of dengue fever and half a million cases of DHF occur worldwide. Ninety percent of DHF subjects are children less than 15 years of age. At present, dengue is endemic in 112 countries in the world. No vaccine is available for preventing...

  5. Viral membrane fusion

    International Nuclear Information System (INIS)

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

  6. [History of viral hepatitis].

    Science.gov (United States)

    Fonseca, José Carlos Ferraz da

    2010-01-01

    The history of viral hepatitis goes back thousands of years and is a fascinating one. When humans were first infected by such agents, a natural repetitive cycle began, with the capacity to infect billions of humans, thus decimating the population and causing sequelae in thousands of lives. This article reviews the available scientific information on the history of viral hepatitis. All the information was obtained through extensive bibliographic review, including original and review articles and consultations on the internet. There are reports on outbreaks of jaundice epidemics in China 5,000 years ago and in Babylon more than 2,500 years ago. The catastrophic history of great jaundice epidemics and pandemics is well known and generally associated with major wars. In the American Civil War, 40,000 cases occurred among Union troops. In 1885, an outbreak of catarrhal jaundice affected 191 workers at the Bremen shipyard (Germany) after vaccination against smallpox. In 1942, 28,585 soldiers became infected with hepatitis after inoculation with the yellow fever vaccine. The number of cases of hepatitis during the Second World War was estimated to be 16 million. Only in the twentieth century were the main agents causing viral hepatitis identified. The hepatitis B virus was the first to be discovered. In this paper, through reviewing the history of major epidemics caused by hepatitis viruses and the history of discovery of these agents, singular peculiarities were revealed. Examples of this include the accidental or chance discovery of the hepatitis B and D viruses.

  7. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  8. Variants of cellobiohydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Bott, Richard R.; Foukaraki, Maria; Hommes, Ronaldus Wilhelmus; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Nikolaev, Igor; Sandgren, Mats; Van Lieshout, Johannes Franciscus Thomas; Van Stigt Thans, Sander

    2018-04-10

    Disclosed are a number of homologs and variants of Hypocrea jecorina Ce17A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  9. Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

    OpenAIRE

    Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

    2003-01-01

    For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

  10. Viral hepatitis in women of reproductive age

    Directory of Open Access Journals (Sweden)

    I.A. Zaytsev

    2017-04-01

    Full Text Available Annually in Ukraine, about 17 thousands of newborns are at risk of vertical infection with hepatitis B and C. Identification of infected women at the stage of family planning is the best way to prevent infection in newborns, and therefore it must be performed strictly in accordance with established norms. In case of detection of hepatitis, further tactics depend on the variant of the virus: in case of hepatitis C, pre-pregnancy treatment is preferable. In case of hepatitis B — pregnancy with subsequent simultaneous vaccination of the newborn. Antiviral therapy is possible in women with high viral load to prevent intrauterine infection. Similar tactics should be followed in case of in vitro fertilisation too. The text of the lecture is illustrated by clinical examples. The lecture is intended for infectious disease physicians and obstetrician-gynecologists.

  11. Dengue viral infections

    OpenAIRE

    Gurugama Padmalal; Garg Pankaj; Perera Jennifer; Wijewickrama Ananda; Seneviratne Suranjith

    2010-01-01

    Dengue viral infections are one of the most important mosquito-borne diseases in the world. Presently dengue is endemic in 112 countries in the world. It has been estimated that almost 100 million cases of dengue fever and half a million cases of dengue hemorrhagic fever (DHF) occur worldwide. An increasing proportion of DHF is in children less than 15 years of age, especially in South East and South Asia. The unique structure of the dengue virus and the pathophysiologic responses of the host...

  12. Migraine Variants in Children

    Science.gov (United States)

    ... Headaches in Children FAQ Migraine Variants In Children Children Get Migraines Too! Learn More Migraine Information Find Help Doctors & Resources Get Connected Join the Conversation Follow Us on Social Media Company About News Resources Privacy Policy Contact Phone: ...

  13. Viral marketing as epidemiological model

    OpenAIRE

    Rodrigues, Helena Sofia; Fonseca, Manuel

    2015-01-01

    In epidemiology, an epidemic is defined as the spread of an infectious disease to a large number of people in a given population within a short period of time. In the marketing context, a message is viral when it is broadly sent and received by the target market through person-to-person transmission. This specific marketing communication strategy is commonly referred as viral marketing. Due to this similarity between an epidemic and the viral marketing process and because the understanding of...

  14. Viral quasispecies profiles as the result of the interplay of competition and cooperation.

    Science.gov (United States)

    Arbiza, Juan; Mirazo, Santiago; Fort, Hugo

    2010-05-10

    Viral quasispecies can be regarded as a swarm of genetically related mutants. A common approach employed to describe viral quasispecies is by means of the quasispecies equation (QE). However, a main criticism of QE is its lack of frequency-dependent selection. This can be overcome by an alternative formulation for the evolutionary dynamics: the replicator-mutator equation (RME). In turn, a problem with the RME is how to quantify the interaction coefficients between viral variants. Here, this is addressed by adopting an ecological perspective and resorting to the niche theory of competing communities, which assumes that the utilization of resources primarily determines ecological segregation between competing individuals (the different viral variants that constitute the quasispecies). This provides a theoretical framework to estimate quantitatively the fitness landscape. Using this novel combination of RME plus the ecological concept of niche overlapping for describing a quasispecies we explore the population distributions of viral variants that emerge, as well as the corresponding dynamics. We observe that the population distribution requires very long transients both to A) reach equilibrium and B) to show a clear dominating master sequence. Based on different independent and recent experimental evidence, we find that when some cooperation or facilitation between variants is included in appropriate doses we can solve both A) and B). We show that a useful quantity to calibrate the degree of cooperation is the Shannon entropy. In order to get a typical quasispecies profile, at least within the considered mathematical approach, it seems that pure competition is not enough. Some dose of cooperation among viral variants is needed. This has several biological implications that might contribute to shed light on the mechanisms operating in quasispecies dynamics and to understand the quasispecies as a whole entity.

  15. Equine viral arteritis

    Directory of Open Access Journals (Sweden)

    Kosec Marjan

    2016-01-01

    Full Text Available Equine viral arteritis (EVA is a contagious disease of equids caused by equine artheritis virus (EAV, widespread in most countries in the world, where patients are diagnosed. The infection usually starts asymptomatic. Clinical signs indicate respiratory infection of different intensity and also abortions are present at different stages of gestation. Large prevalence of this disease in the world has become a growing economic problem. The disease is specific to a particular kind of animals, and it affects only equids (horses, donkeys, mules, mule and zebras. In countries where the infection has been confirmed, the percentage of positive animals differ. Likewise, there is difference in percentage among certain animal kinds. The highest percentage of positive animals has been found in totters and the lowest in cold-blooded.

  16. Viral pathogen discovery

    Science.gov (United States)

    Chiu, Charles Y

    2015-01-01

    Viral pathogen discovery is of critical importance to clinical microbiology, infectious diseases, and public health. Genomic approaches for pathogen discovery, including consensus polymerase chain reaction (PCR), microarrays, and unbiased next-generation sequencing (NGS), have the capacity to comprehensively identify novel microbes present in clinical samples. Although numerous challenges remain to be addressed, including the bioinformatics analysis and interpretation of large datasets, these technologies have been successful in rapidly identifying emerging outbreak threats, screening vaccines and other biological products for microbial contamination, and discovering novel viruses associated with both acute and chronic illnesses. Downstream studies such as genome assembly, epidemiologic screening, and a culture system or animal model of infection are necessary to establish an association of a candidate pathogen with disease. PMID:23725672

  17. Human papillomavirus type-16 variants in Quechua aboriginals from Argentina.

    Science.gov (United States)

    Picconi, María Alejandra; Alonio, Lidia Virginia; Sichero, Laura; Mbayed, Viviana; Villa, Luisa Lina; Gronda, Jorge; Campos, Rodolfo; Teyssié, Angélica

    2003-04-01

    Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions. Copyright 2003 Wiley-Liss, Inc.

  18. Influence of population diversity on neurovirulence potential of plaque purified L-Zagreb variants.

    Science.gov (United States)

    Ivancic-Jelecki, Jelena; Forcic, Dubravko; Jagusic, Maja; Kosutic-Gulija, Tanja; Mazuran, Renata; Balija, Maja Lang; Isakov, Ofer; Shomron, Noam

    2016-04-29

    Despite continuing research efforts, determinants of mumps virus virulence are still largely unknown. One of consequences of this is difficulty in striking a balance between efficacy and safety of live attenuated mumps vaccines. Among mumps vaccine strains associated with occurrence of postvaccinal aseptic meningitis is L-Zagreb, developed by further attenuation of vaccine strain L-3. Starting from an archived L-Zagreb sample with suboptimal neuroattenuation score, we isolated different viral variants and compared their genetic and phenotypic properties, in investigation of neurovirulence markers. Six different L-Zagreb variants were isolated by plaque purification. Their neurovirulent status was determined by rat-based neurovirulence test; population structure was determined by deep sequencing. We isolated one well neuroattenuated viral variant, two marginally neuroattenuated, and three insufficiently neuroattenuated. No genetic markers of neurovirulence could be identified. None of variants had detectable amounts of defective interfering particles. Two characteristics set insufficiently neuroattenuated variants apart from less-neurovirulent ones: elevated variability level in regions 1293-3314, 5363-7773 and 9382-11657, and/or elevated number of mutations present in frequencies ≥ 1%. The most neurovirulent variants possessed both of these features. Distinctive heterogeneity profiles were obtained for insufficiently neuroattenuated L-Zagreb variants. No markers that would discriminate between marginally and well neuroattenuated variants were identified. The findings of this study may serve as a guideline during development of an improved L3/L-Zagreb vaccine strain. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Viral myositis in children.

    Science.gov (United States)

    Magee, Haley; Goldman, Ran D

    2017-05-01

    Question I recently evaluated a child in my clinic after an emergency department visit where she presented having woken up that morning refusing to walk and was crawling around the house. The parents reported she was getting over a cold, and I recall similar cases of myositis during the H1N1 influenza epidemic a few years ago. What are the key features of myositis that I should recognize? Which investigations are needed to confirm the diagnosis and how should affected patients be managed? Answer Benign acute childhood myositis is a mild and self-limited sudden onset of lower extremity pain during or following recovery from a viral illness. Presentation can include tiptoe gait or refusal to walk, secondary to symmetric bilateral lower extremity pain that resolves quickly, usually within 3 days. In general, no investigation is needed except in severe cases for which screening bloodwork and a urine myoglobin test can confirm the diagnosis and rule out complications. Myoglobinuria and highly elevated creatine phosphokinase levels are rare but should be a consideration for admission to hospital. Prognosis is excellent and management might include rest and analgesia. Copyright© the College of Family Physicians of Canada.

  20. Dengue viral infections

    Directory of Open Access Journals (Sweden)

    Gurugama Padmalal

    2010-01-01

    Full Text Available Dengue viral infections are one of the most important mosquito-borne diseases in the world. Presently dengue is endemic in 112 countries in the world. It has been estimated that almost 100 million cases of dengue fever and half a million cases of dengue hemorrhagic fever (DHF occur worldwide. An increasing proportion of DHF is in children less than 15 years of age, especially in South East and South Asia. The unique structure of the dengue virus and the pathophysiologic responses of the host, different serotypes, and favorable conditions for vector breeding have led to the virulence and spread of the infections. The manifestations of dengue infections are protean from being asymptomatic to undifferentiated fever, severe dengue infections, and unusual complications. Early recognition and prompt initiation of appropriate supportive treatment are often delayed resulting in unnecessarily high morbidity and mortality. Attempts are underway for the development of a vaccine for preventing the burden of this neglected disease. This review outlines the epidemiology, clinical features, pathophysiologic mechanisms, management, and control of dengue infections.

  1. Emerging zoonotic viral diseases.

    Science.gov (United States)

    Wang, L-F; Crameri, G

    2014-08-01

    Zoonotic diseases are infectious diseases that are naturally transmitted from vertebrate animals to humans and vice versa. They are caused by all types of pathogenic agents, including bacteria, parasites, fungi, viruses and prions. Although they have been recognised for many centuries, their impact on public health has increased in the last few decades due to a combination of the success in reducing the spread of human infectious diseases through vaccination and effective therapies and the emergence of novel zoonotic diseases. It is being increasingly recognised that a One Health approach at the human-animal-ecosystem interface is needed for effective investigation, prevention and control of any emerging zoonotic disease. Here, the authors will review the drivers for emergence, highlight some of the high-impact emerging zoonotic diseases of the last two decades and provide examples of novel One Health approaches for disease investigation, prevention and control. Although this review focuses on emerging zoonotic viral diseases, the authors consider that the discussions presented in this paper will be equally applicable to emerging zoonotic diseases of other pathogen types.

  2. Viral Hepatitis: A through E and Beyond

    Science.gov (United States)

    ... National Digestive Diseases Information Clearinghouse What is viral hepatitis? Viral hepatitis is inflammation of the liver caused by ... and serious. Drugs are available to treat chronic hepatitis. 4 Viral Hepatitis: A through E and Beyond What else ...

  3. Neuroanatomy goes viral!

    Directory of Open Access Journals (Sweden)

    Jonathan eNassi

    2015-07-01

    Full Text Available The nervous system is complex not simply because of the enormous number of neurons it contains but by virtue of the specificity with which they are connected. Unraveling this specificity is the task of neuroanatomy. In this endeavor, neuroanatomists have traditionally exploited an impressive array of tools ranging from the Golgi method to electron microscopy. An ideal method for studying anatomy would label neurons that are interconnected, and, in addition, allow expression of foreign genes in these neurons. Fortuitously, nature has already partially developed such a method in the form of neurotropic viruses, which have evolved to deliver their genetic material between synaptically connected neurons while largely eluding glia and the immune system. While these characteristics make some of these viruses a threat to human health, simple modifications allow them to be used in controlled experimental settings, thus enabling neuroanatomists to trace multi-synaptic connections within and across brain regions. Wild-type neurotropic viruses, such as rabies and alpha-herpes virus, have already contributed greatly to our understanding of brain connectivity, and modern molecular techniques have enabled the construction of recombinant forms of these and other viruses. These newly engineered reagents are particularly useful, as they can target genetically defined populations of neurons, spread only one synapse to either inputs or outputs, and carry instructions by which the targeted neurons can be made to express exogenous proteins, such as calcium sensors or light-sensitive ion channels, that can be used to study neuronal function. In this review, we address these uniquely powerful features of the viruses already in the neuroanatomist's toolbox, as well as the aspects of their biology that currently limit their utility. Based on the latter, we consider strategies for improving viral tracing methods by reducing toxicity, improving control of transsynaptic

  4. Histone variants and lipid metabolism

    NARCIS (Netherlands)

    Borghesan, Michela; Mazzoccoli, Gianluigi; Sheedfar, Fareeba; Oben, Jude; Pazienza, Valerio; Vinciguerra, Manlio

    2014-01-01

    Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis

  5. Viral-mediated Zif268 expression in the prefrontal cortex protects against gonadectomy-induced working memory, long-term memory, and social interaction deficits in male rats.

    Science.gov (United States)

    Dossat, Amanda M; Jourdi, Hussam; Wright, Katherine N; Strong, Caroline E; Sarkar, Ambalika; Kabbaj, Mohamed

    2017-01-06

    In humans, some males experience reductions in testosterone levels, as a natural consequence of aging or in the clinical condition termed hypogonadism, which are associated with impaired cognitive performance and mood disorder(s). Some of these behavioral deficits can be reversed by testosterone treatment. Our previous work in rats reported that sex differences in the expression of the transcription factor Zif268, a downstream target of testosterone, within the medial prefrontal cortex (mPFC) mediates sex differences in social interaction. In the present study, we aimed to examine the effects of gonadectomy (GNX) in male rats on mPFC Zif268 expression, mood and cognitive behaviors. We also examined whether reinstitution of Zif268 in GNX rats will correct some of the behavioral deficits observed following GNX. Our results show that GNX induced a downregulation of Zif268 protein in the mPFC, which was concomitant with impaired memory in the y-maze and spontaneous object recognition test, reduced social interaction time, and depression-like behaviors in the forced swim test. Reinstitution of mPFC Zif268, using a novel adeno-associated-viral (AAV) construct, abrogated GNX-induced working memory and long-term memory impairments, and reductions in social interaction time, but not GNX-induced depression-like behaviors. These findings suggest that mPFC Zif268 exerts beneficial effects on memory and social interaction, and could be a potential target for novel treatments for behavioral impairments observed in hypogonadal and aged men with declining levels of gonadal hormones. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Viral Evolution Core | FNLCR Staging

    Science.gov (United States)

    Brandon F. Keele, Ph.D. PI/Senior Principal Investigator, Retroviral Evolution Section Head, Viral Evolution Core Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research Frederick, MD 21702-1201 Tel: 301-846-173

  7. FastStats: Viral Hepatitis

    Science.gov (United States)

    ... Submit What's this? Submit Button NCHS Home Viral Hepatitis Recommend on Facebook Tweet Share Compartir Data are for the U.S. Morbidity Number of new hepatitis A cases: 1,239 (2014) Number of new ...

  8. Microbiological diagnostics of viral hepatitis

    OpenAIRE

    HASDEMİR, Ufuk

    2016-01-01

    Viral hepatitis is an infection that primarily affects the liverbut may also have systemic clinical manifestations. The vastmajority of viral hepatitis are caused by one of five hepatotropicviruses: hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D (delta) virus (HDV), andhepatitis E virus (HEV) (Table I) [1]. HBV, HCV, and HDValso cause chronic hepatitis, whereas HAV does not. HEVcauses acute hepatitis in normal hosts but can cause protractedand chronic he...

  9. Viral infections in transplant recipients.

    Science.gov (United States)

    Razonable, R R; Eid, A J

    2009-12-01

    Solid organ and hematopoietic stem cell transplant recipients are uniquely predisposed to develop clinical illness, often with increased severity, due to a variety of common and opportunistic viruses. Patients may acquire viral infections from the donor (donor-derived infections), from reactivation of endogenous latent virus, or from the community. Herpes viruses, most notably cytomegalovirus and Epstein Barr virus, are the most common among opportunistic viral pathogens that cause infection after solid organ and hematopoietic stem cell transplantation. The polyoma BK virus causes opportunistic clinical syndromes predominantly in kidney and allogeneic hematopoietic stem cell transplant recipients. The agents of viral hepatitis B and C present unique challenges particularly among liver transplant recipients. Respiratory viral illnesses due to influenza, respiratory syncytial virus, and parainfluenza virus may affect all types of transplant recipients, although severe clinical disease is observed more commonly among lung and allogeneic hematopoietic stem cell transplant recipients. Less common viral infections affecting transplant recipients include those caused by adenoviruses, parvovirus B19, and West Nile virus. Treatment for viruses with proven effective antiviral drug therapies should be complemented by reduction in the degree of immunosuppression. For others with no proven antiviral drugs for therapy, reduction in the degree of immunosuppression remains as the sole effective strategy for management. Prevention of viral infections is therefore of utmost importance, and this may be accomplished through vaccination, antiviral strategies, and aggressive infection control measures.

  10. Variants of glycoside hydrolases

    Science.gov (United States)

    Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  11. Accurate genotyping across variant classes and lengths using variant graphs

    DEFF Research Database (Denmark)

    Sibbesen, Jonas Andreas; Maretty, Lasse; Jensen, Jacob Malte

    2018-01-01

    of read k-mers to a graph representation of the reference and variants to efficiently perform unbiased, probabilistic genotyping across the variation spectrum. We demonstrate that BayesTyper generally provides superior variant sensitivity and genotyping accuracy relative to existing methods when used...... collecting a set of candidate variants across discovery methods, individuals and databases, and then realigning the reads to the variants and reference simultaneously. However, this realignment problem has proved computationally difficult. Here, we present a new method (BayesTyper) that uses exact alignment...... to integrate variants across discovery approaches and individuals. Finally, we demonstrate that including a ‘variation-prior’ database containing already known variants significantly improves sensitivity....

  12. Beyond viral suppression of HIV

    DEFF Research Database (Denmark)

    Lazarus, Jeffrey V.; Safreed-Harmon, Kelly; Barton, Simon E

    2016-01-01

    BACKGROUND: In 2016, the World Health Organization (WHO) adopted a new Global Health Sector Strategy on HIV for 2016-2021. It establishes 15 ambitious targets, including the '90-90-90' target calling on health systems to reduce under-diagnosis of HIV, treat a greater number of those diagnosed......, and ensure that those being treated achieve viral suppression. DISCUSSION: The WHO strategy calls for person-centered chronic care for people living with HIV (PLHIV), implicitly acknowledging that viral suppression is not the ultimate goal of treatment. However, it stops short of providing an explicit target...... for health-related quality of life. It thus fails to take into account the needs of PLHIV who have achieved viral suppression but still must contend with other intense challenges such as serious non-communicable diseases, depression, anxiety, financial stress, and experiences of or apprehension about HIV...

  13. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals.

    Science.gov (United States)

    Ahmed, Asma; Felmlee, Daniel J

    2015-12-18

    There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR) rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

  14. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals

    Directory of Open Access Journals (Sweden)

    Asma Ahmed

    2015-12-01

    Full Text Available There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

  15. Variants of Moreau's sweeping process

    International Nuclear Information System (INIS)

    Siddiqi, A.H.; Manchanda, P.

    2001-07-01

    In this paper we prove the existence and uniqueness of two variants of Moreau's sweeping process -u'(t) is an element of Nc (t) (u(t)), where in one variant we replace u(t) by u'(t) in the right-hand side of the inclusion and in the second variant u'(t) and u(t) are respectively replaced by u''(t) and u'(t). (author)

  16. Immune responses to rAAV6: The influence of canine parvovirus vaccination and neonatal administration of viral vector

    Directory of Open Access Journals (Sweden)

    Andrea L H Arnett

    2011-11-01

    Full Text Available Recombinant adeno-associated viral (rAAV vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV. rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, one month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

  17. Hairy cell leukemia-variant

    International Nuclear Information System (INIS)

    Quadri, Mohammad I.; Al-Sheikh, Iman H.

    2001-01-01

    Hairy cell leukaemia variant is a very rare chronic lymphoproliferative disorder and is closely related to hairy cell leukemia. We hereby describe a case of hairy cell leukaemia variant for the first time in Saudi Arabia. An elderly Saudi man presented with pallor, massive splenomegaly, and moderate hepatomegaly. Hemoglobin was 7.7 g/dl, Platelets were 134 x109/l and white blood count was 140x10 9/l with 97% being abnormal lymphoid cells with cytoplasmic projections. The morphology, cytochemistry, and immunophenotype of the lymphoid cells were classical of hairy cell leukaemia variant. The bone marrow was easily aspirated and findings were consistent with hairy cell leukaemia variant. (author)

  18. Recycling Endosomes and Viral Infection.

    Science.gov (United States)

    Vale-Costa, Sílvia; Amorim, Maria João

    2016-03-08

    Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral-host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

  19. Ventilator and viral induced inflammation

    NARCIS (Netherlands)

    Hennus, M.P.

    2013-01-01

    This thesis expands current knowledge on ventilator induced lung injury and provides insights on the immunological effects of mechanical ventilation during viral respiratory infections. The experimental studies in the first part of this thesis improve our understanding of how mechanical ventilation

  20. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  1. Viral hepatitis and hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Juei-Low, Sung [ed.; National Taiwan University College of Medicine, Taipei (Republic of China Taiwan). Department of Internal Medicine; Ding-Shinn, Chen [ed.; National Taiwan University College of Medicine, Taipei (Republic of China Taiwan). Hepatitis Research Center National Taiwan University College of Medicine, Taipei (Republic of China Taiwan). Graduate Institute of Clinical Medicine

    1990-01-01

    Two papers in this volume are in INIS scope, respectively dealing with MRI in the study of viral hepatitis and hepatocellular carcinoma, and The use of {sup 131}I-labeled Lipidol in the diagnosis of hepato-cellular carcinoma. (H.W.). refs.; figs.; tabs.

  2. Viral hepatitis and hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Sung Juei-Low; Chen Ding-Shinn

    1990-01-01

    Two papers in this volume are in INIS scope, respectively dealing with MRI in the study of viral hepatitis and hepatocellular carcinoma, and The use of 131 I-labeled Lipidol in the diagnosis of hepato-cellular carcinoma. (H.W.). refs.; figs.; tabs

  3. Mast cells in viral infections

    Directory of Open Access Journals (Sweden)

    Piotr Witczak

    2012-04-01

    Full Text Available  There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9, but also RIG-I-like and NOD-like molecules. Furthermore, mast cells generate various mediators, cytokines and chemokines which modulate the intensity of inflammation and regulate the course of innate and adaptive anti-viral immunity. Indirect evidence for the role of mast cells in viral infections is also provided by clinical observations and results of animal studies. Currently, more and more data indicate that mast cells can be infected by some viruses (dengue virus, adenoviruses, hantaviruses, cytomegaloviruses, reoviruses, HIV-1 virus. It is also demonstrated that mast cells can release pre formed mediators as well as synthesize de novo eicosanoids in response to stimulation by viruses. Several data indicate that virus-stimulated mast cells secrete cytokines and chemokines, including interferons as well as chemokines with a key role in NK and Tc lymphocyte influx. Moreover, some information indicates that mast cell stimulation via TLR3, TLR7/8 and TLR9 can affect their adhesion to extracellular matrix proteins and chemotaxis, and influence expression of some membrane molecules. Critical analysis of current data leads to the conclusion that it is not yet possible to make definitive statements about the role of mast cells in innate and acquired defense mechanisms developing in the course of viral infection and/or pathomechanisms of viral diseases.

  4. Product Variant Master as a Means to Handle Variant Design

    DEFF Research Database (Denmark)

    Hildre, Hans Petter; Mortensen, Niels Henrik; Andreasen, Mogens Myrup

    1996-01-01

    be implemented in the CAD system I-DEAS. A precondition for high degree of computer support is identification of a product variant master from which new variants can be derived. This class platform defines how a product build up fit certain production methods and rules governing determination of modules...

  5. Adeno-associated virus LPL(S447X) gene therapy in LDL receptor knockout mice

    NARCIS (Netherlands)

    Rip, Jaap; Sierts, Jeroen A.; Vaessen, Stefan F. C.; Kastelein, John J. P.; Twisk, Jaap; Kuivenhoven, Jan Albert

    2007-01-01

    BACKGROUND: Overexpression of lipoprotein lipase (LPL) protects against atherosclerosis in genetically engineered mice. We tested whether a gene therapy vector that delivers human (h) LPL(S447X) cDNA to skeletal muscle could induce similar effects. METHODS: LDL receptor knockout (LDLr-/-) mice were

  6. Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

    Science.gov (United States)

    Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

    2007-02-28

    The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

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

    Czech Academy of Sciences Publication Activity Database

    Tadokoro, T.; Miyanohara, A.; Navarro, M.; Kamizato, K.; Juhás, Štefan; Juhásová, Jana; Maršala, S.; Platoshyn, O.; Curtis, E.; Gabel, B.; Ciacci, J. D.; Lukáčová, N.; Bimbová, K.; Maršala, M.

    2017-01-01

    Roč. 125, č. 13 (2017), č. článku e55770. ISSN 1940-087X R&D Projects: GA MŠk(CZ) LO1609 Institutional support: RVO:67985904 Keywords : AAV9 * adult mouse Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Technologies involving the manipulation of cells, tissues, organs or the whole organism (assisted reproduction) Impact factor: 1.232, year: 2016

  8. Canine parvovirus (CPV-2) variants circulating in Nigerian dogs

    Science.gov (United States)

    Apaa, T. T.; Daly, J. M.; Tarlinton, R. E.

    2016-01-01

    Canine parvovirus type 2 (CPV-2) is a highly contagious viral disease with three variants (CPV-2a, CPV-2b and CPV-2c) currently circulating in dogs worldwide. The main aim of this study was to determine the prevalent CPV-2 variant in faecal samples from 53 dogs presenting with acute gastroenteritis suspected to be and consistent with CPV-2 to Nigerian Veterinary Clinics in 2013–2014. Seventy-five per cent of these dogs tested positive for CPV-2 in a commercial antigen test and/or by PCR. Partial sequencing of the VP2 gene of six of these demonstrated them to be CPV-2a. Most of the dogs (60 per cent) were vaccinated, with 74 per cent of them puppies less than six months old. PMID:27933190

  9. Surveillance for Viral Hepatitis - United States, 2014

    Science.gov (United States)

    ... Resource Center Anonymous Feedback Viral Hepatitis Surveillance for Viral Hepatitis – United States, 2014 Recommend on Facebook Tweet Share ... Cases Hepatitis A Hepatitis B Hepatitis C Discussion Hepatitis A virus Index PAGE DESCRIPTION Table 2.1 Reported ...

  10. Faktor Risiko Non Viral Pada Karsinoma Nasofaring

    Directory of Open Access Journals (Sweden)

    Sukri Rahman

    2015-09-01

    Full Text Available Abstrak           Latar belakang: Karsinoma nasofaring adalah tumor ganas epitel nasofaring yang sampai saat ini penyebabnya belum diketahui, infeksi virus Epstein Barr dilaporkan sebagai faktor dominan terjadinya karsinoma nasofaring tetapi faktor non viral juga berperan untuk timbulnya keganasan nasofaring. Tujuan: Untuk mengetahui faktor non viral  yang dapat meningkatkan kejadian karsinoma nasofaring sehingga dapat mencegah dan menghindari faktor-faktor non viral tersebut. Tinjauan Pustaka: Karsinoma nasofaring merupakan tumor ganas epitel nasofaring yang penyebabnya berhubungan dengan faktor viral dan non viral diantaranya asap rokok, ikan asin, formaldehid, genetik, asap kayu bakar , debu kayu, infeksi kronik telinga hidung tenggorok, alkohol dan obat tradisional. Kesimpulan: Pembuktian secara klinis dan ilmiah terhadap faktor non viral sebagai penyebab timbulnya karsinoma nasofaring masih belum dapat dijelaskan secara pasti. Faktor non viral merupakan salah satu faktor risiko yang dapat meningkatkan angka kejadian timbulnya keganasan nasofaring Kata kunci: karsinoma nasofaring, faktor risiko, non viral AbstractBackground: Nasopharyngeal carcinoma is a malignant epithelial nasopharyngeal tumor that until now the cause still unknown, Epstein barr virus infection had reported as predominant occurance of nasopharyngeal carcinoma but non viral factors may also contribute to the onset of the incidence of nasopharyngeal malignancy. Purpose: To find non viral factors that may increase the incidence of nasopharyngel carcinoma in order to prevent and avoid non-viral factors Literature: Nasopharyngeal carcinoma is a malignant tumor that causes nasopharyngeal epithelium associated with viral and non-viral factors such as cigarette smoke, salt fish, formaldehyde, genetic, wood smoke ,wood dust, ear nose throat chronic infections, alcohol, and traditional medicine. Conclusion: Clinically and scientifically proving the non-viral factors as

  11. Assembly of viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2014-01-01

    textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow

  12. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

    Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

    2016-01-01

    Viral envelope glycoproteins are major targets for antibodies that bind to and inactivate viral particles. The capacity of a viral vaccine to induce virus-neutralizing antibodies is often used as a marker for vaccine efficacy. Yet the number of known neutralization target epitopes is restricted o...

  13. Viral commercials: the consumer as marketeer

    NARCIS (Netherlands)

    Ketelaar, P.E.; Lucassen, P.; Kregting, G.H.J.

    2010-01-01

    Research into the reasons why consumers pass along viral commercials: their motives, the content characteristics of viral commercials and the medium context in which viral commercials appear. Based on the uses and gratifications perspective this study has determined which motives of consumers,

  14. Viral diseases and human evolution

    Directory of Open Access Journals (Sweden)

    Leal Élcio de Souza

    2000-01-01

    Full Text Available The interaction of man with viral agents was possibly a key factor shaping human evolution, culture and civilization from its outset. Evidence of the effect of disease, since the early stages of human speciation, through pre-historical times to the present suggest that the types of viruses associated with man changed in time. As human populations progressed technologically, they grew in numbers and density. As a consequence different viruses found suitable conditions to thrive and establish long-lasting associations with man. Although not all viral agents cause disease and some may in fact be considered beneficial, the present situation of overpopulation, poverty and ecological inbalance may have devastating effets on human progress. Recently emerged diseases causing massive pandemics (eg., HIV-1 and HCV, dengue, etc. are becoming formidable challenges, which may have a direct impact on the fate of our species.

  15. APLASTIC ANEMIA AND VIRAL HEPATITIS

    Directory of Open Access Journals (Sweden)

    Laura Cudillo

    2009-11-01

    Liver histology is characterized by T cell infiltrating the parenchyma as reported in acute hepatitis. Recently in HAA it has been demonstrated intrahepatic  and blood lymphocytes with  T cell repertoire similar to that of confirmed viral acute hepatitis. The expanded T cell clones return to a normal distribution after response to immunosuppressive treatment, suggesting the antigen or T cell clearance. Therapeutic options are the same as acquired aplastic anemia.

  16. [Immunotherapy for refractory viral infections].

    Science.gov (United States)

    Morio, Tomohiro; Fujita, Yuriko; Takahashi, Satoshi

    Various antiviral agents have been developed, which are sometimes associated with toxicity, development of virus-resistant strain, and high cost. Virus-specific T-cell (VST) therapy provides an alternative curative therapy that can be effective for a prolonged time without eliciting drug resistance. VSTs can be directly separated using several types of capture devices and can be obtained by stimulating peripheral blood mononuclear cells with viral antigens (virus, protein, or peptide) loaded on antigen-presenting cells (APC). APC can be transduced with virus-antigen coding plasmid or pulsed with overlapping peptides. VST therapy has been studied in drug non-responsive viral infections after hematopoietic cell transplantation (HCT). Several previous studies have demonstrated the efficacy of VST therapy without significant severe GVHD. In addition, VSTs from a third-party donor have been prepared and administered for post-HCT viral infection. Although target viruses of VSTs include herpes virus species and polyomavirus species, a wide variety of pathogens, such as papillomavirus, intracellular bacteria, and fungi, can be treated by pathogen-specific T-cells. Perhaps, these specific T-cells could be used for opportunistic infections in other immunocompromised hosts in the near future.

  17. Evolution of simeprevir-resistant variants over time by ultra-deep sequencing in HCV genotype 1b.

    Science.gov (United States)

    Akuta, Norio; Suzuki, Fumitaka; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Ikeda, Kenji; Kumada, Hiromitsu

    2014-08-01

    Using ultra-deep sequencing technology, the present study was designed to investigate the evolution of simeprevir-resistant variants (amino acid substitutions of aa80, aa155, aa156, and aa168 positions in HCV NS3 region) over time. In Toranomon Hospital, 18 Japanese patients infected with HCV genotype 1b, received triple therapy of simeprevir/PEG-IFN/ribavirin (DRAGON or CONCERT study). Sustained virological response rate was 67%, and that was significantly higher in patients with IL28B rs8099917 TT than in those with non-TT. Six patients, who did not achieve sustained virological response, were tested for resistant variants by ultra-deep sequencing, at the baseline, at the time of re-elevation of viral loads, and at 96 weeks after the completion of treatment. Twelve of 18 resistant variants, detected at re-elevation of viral load, were de novo resistant variants. Ten of 12 de novo resistant variants become undetectable over time, and that five of seven resistant variants, detected at baseline, persisted over time. In one patient, variants of Q80R at baseline (0.3%) increased at 96-week after the cessation of treatment (10.2%), and de novo resistant variants of D168E (0.3%) also increased at 96-week after the cessation of treatment (9.7%). In conclusion, the present study indicates that the emergence of simeprevir-resistant variants after the start of treatment could not be predicted at baseline, and the majority of de novo resistant variants become undetectable over time. Further large-scale prospective studies should be performed to investigate the clinical utility in detecting simeprevir-resistant variants. © 2014 Wiley Periodicals, Inc.

  18. Laboratory procedures to generate viral metagenomes.

    Science.gov (United States)

    Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

    2009-01-01

    This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

  19. Viral suppression of multiple escape mutants by de novo CD8+ T cell responses in a human immunodeficiency virus-1 Infected elite suppressor

    Directory of Open Access Journals (Sweden)

    Siliciano Robert F

    2011-08-01

    Full Text Available Abstract Elite suppressors or controllers (ES are HIV-1 infected patients who maintain undetectable viral loads without treatment. While HLA-B*57-positive ES are usually infected with virus that is unmutated at CTL epitopes, a single, dominant variant containing CTL escape mutations is typically seen in plasma during chronic infection. We describe an ES who developed seven distinct and rare escape variants at an HLA-B*57-restricted Gag epitope over a five year period. Interestingly, he developed proliferative, de novo CTL responses that suppressed replication of each of these variants. These responses, in combination with low viral fitness of each variant, may contribute to sustained elite control in this ES.

  20. Evaluation of Viral Meningoencephalitis Cases

    Directory of Open Access Journals (Sweden)

    Handan Ilhan

    2012-08-01

    Full Text Available AIM: To evaluate retrospectively adult cases of viral encephalitis. METHOD: Fifteen patients described viral encephalitis hospitalized between the years 2006-2011 follow-up and treatment at the infectious diseases clinic were analyzed retrospectively. RESULTS: Most of the patients (%60 had applied in the spring. Fever (87%, confusion (73%, neck stiffness (73%, headache (73%, nausea-vomiting (33%, loss of consciousness (33%, amnesia (33%, agitation (20%, convulsion (%20, focal neurological signs (13%, Brudzinski-sign (13% were most frequently encountered findings. Electroencephalography test was applied to 13 of 14 patients, and pathological findings compatible with encephalitis have been found. Radiological imaging methods such as CT and MRI were performed in 9 of the 14 patients, and findings consistent with encephalitis were reported. All of initial cerebrospinal fluid (CSF samples were abnormal. The domination of the first examples was lymphocytes in 14 patients; only one patient had an increase in neutrophilic cells have been found. CSF protein level was high in nine patients, and low glucose level was detected in two patients. Herpes simplex virus polymerized chain reaction (PCR analyze was performed to fourteen patients CSF. Only two of them (14% were found positive. One of the patients sample selectively examined was found to be Parvovirus B19 (+, the other patient urine sample Jacobs-creutzfeld virus PCR was found to be positively. Empiric acyclovir therapy was given to all patients. Neuropsychiatric squeal developed at the one patient. CONCLUSION: The cases in the forefront of change in mental status viral meningoencephalitis should be considered and empirical treatment with acyclovir should be started. [TAF Prev Med Bull 2012; 11(4.000: 447-452

  1. Encefalitis virales en la infancia

    Directory of Open Access Journals (Sweden)

    Monserrat Téllez de Meneses

    2013-09-01

    Full Text Available La encefalitis viral es una enfermedad grave que implica el compromiso inflamatorio del parénquima cerebral. Las infecciones virales del SNC ocurren con frecuencia como complicación de infecciones virales sistémicas. Más de 100 virus están implicados como agentes causales, entre los cuales el virus Herpes simplex tipo I, es el agente causal más frecuente de encefalitis no epidémica en todos los grupos poblacionales del mundo; es el responsable de los casos más graves en todas las edades. Muchos de los virus para los cuales existe vacunas también pueden causar encefalitis como: sarampión, paperas, polio, rabia, rubéola, varicela. El virus produce una inflamación del tejido cerebral, la cual puede evolucionar a una destrucción de neuronas, provocar hemorragia y daño cerebral, dando lugar a encefalitis graves, como la encefalitis necrotizante o hemorrágica, con mucho peor pronóstico, produciendo secuelas graves, incluso la muerte. El cuadro clínico, incluye la presencia de cefalea, fiebre y alteración de la conciencia, de rápida progresión. El pronóstico de las encefalitis víricas es variable, algunos casos son leves, con recuperación completa, sin embargo existen casos graves que pueden ocasionar secuelas importantes a nivel cerebral. Es fundamental realizar un diagnóstico lo antes posible, a través de pruebas de laboratorio (bioquímica, PCR, cultivos y de neuroimagen (TAC, RM y ante todo, la instauración de un tratamiento precoz para evitar la evolución del proceso y sus posibles complicaciones. El pronóstico empeora si se retrasa la instauración del tratamiento.

  2. Viral diseases of marine invertebrates

    Science.gov (United States)

    Johnson, P. T.

    1984-03-01

    Approximately 40 viruses are known from marine sponges; turbellarian and monogenetic flatworms; cephalopod, bivalve, and gastropod mollusks; nereid polychaetes; and isopod and decapod crustaceans. Most of the viruses can be tentatively assigned to the Herpesviridae, Baculoviridae, Iridoviridae, Adenoviridae, Papovaviridae, Reoviridae, “Birnaviridae”, Bunyaviridae, Rhabdoviridae, and Picornaviridae. Viruslike particles found in oysters might be representatives of the Togaviridae and Retroviridae. Enveloped single-stranded RNA viruses from crustaceans have developmental and morphological characteristics intermediate between families, and some show evidence of relationships to the Paramyxoviridae as well as the Bunyaviridae or Rhabdoviridae. Certain small viruses of shrimp cannot be assigned, even tentatively, to a particular family. Some viruses cause disease in wild and captive hosts, others are associated with disease states but may not be primary instigators, and many occur in apparently normal animals. The frequency of viral disease in natural populations of marine invertebrates is unknown. Several viruses that cause disease in captive animals, with or without experimental intervention, have also been found in diseased wild hosts, including herpeslike viruses of crabs and oysters, iridovirus of octopus, and reolike and bunyalike viruses of crabs. Iridolike viruses have been implicated in massive mortalities of cultured oysters. Baculoviruses, and IHHN virus, which is of uncertain affinities, cause economically damaging diseases in cultured penaeid shrimp. Double or multiple viral infection is common in crabs. For example, a reolike virus and associated rhabdolike virus act synergistically to cause paralytic and fatal disease in Callinectes sapidus. Information on host range, most susceptible stage, and viral latency is available only for viruses of shrimp. One baculovirus attacks five species of New World penaeid shrimp. IHHN virus infects three species of

  3. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Waldhoer, M; Lüttichau, H R

    2001-01-01

    expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.......A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...

  4. A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

    Science.gov (United States)

    Wang, Xiaoyu; Zhou, Defang; Wang, Guihua; Huang, Libo; Zheng, Qiankun; Li, Chengui; Cheng, Ziqiang

    2017-12-04

    The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Viral Organization of Human Proteins

    Science.gov (United States)

    Wuchty, Stefan; Siwo, Geoffrey; Ferdig, Michael T.

    2010-01-01

    Although maps of intracellular interactions are increasingly well characterized, little is known about large-scale maps of host-pathogen protein interactions. The investigation of host-pathogen interactions can reveal features of pathogenesis and provide a foundation for the development of drugs and disease prevention strategies. A compilation of experimentally verified interactions between HIV-1 and human proteins and a set of HIV-dependency factors (HDF) allowed insights into the topology and intricate interplay between viral and host proteins on a large scale. We found that targeted and HDF proteins appear predominantly in rich-clubs, groups of human proteins that are strongly intertwined among each other. These assemblies of proteins may serve as an infection gateway, allowing the virus to take control of the human host by reaching protein pathways and diversified cellular functions in a pronounced and focused way. Particular transcription factors and protein kinases facilitate indirect interactions between HDFs and viral proteins. Discerning the entanglement of directly targeted and indirectly interacting proteins may uncover molecular and functional sites that can provide novel perspectives on the progression of HIV infection and highlight new avenues to fight this virus. PMID:20827298

  6. Viral Infection in Renal Transplant Recipients

    Directory of Open Access Journals (Sweden)

    Jovana Cukuranovic

    2012-01-01

    Full Text Available Viruses are among the most common causes of opportunistic infection after transplantation. The risk for viral infection is a function of the specific virus encountered, the intensity of immune suppression used to prevent graft rejection, and other host factors governing susceptibility. Although cytomegalovirus is the most common opportunistic pathogen seen in transplant recipients, numerous other viruses have also affected outcomes. In some cases, preventive measures such as pretransplant screening, prophylactic antiviral therapy, or posttransplant viral monitoring may limit the impact of these infections. Recent advances in laboratory monitoring and antiviral therapy have improved outcomes. Studies of viral latency, reactivation, and the cellular effects of viral infection will provide clues for future strategies in prevention and treatment of viral infections. This paper will summarize the major viral infections seen following transplant and discuss strategies for prevention and management of these potential pathogens.

  7. Viral Advertising: Branding Effects from Consumers’ Perspectives

    OpenAIRE

    Jiang, Yueqing

    2012-01-01

    Viral advertising is popular for its high viral transmission results online. Its increased impacts on the social media users have been noticed by the author. At the same time, viewers’ negative attitudes toward traditional advertisements become obvious which can be regarded as the phenomenon of advertisement avoidance. It arouses author’s interests to know how the viral advertising reduces the viewers’ negative emotions and its performances in branding online. This paper is going to look into...

  8. Viral Advertising on Facebook in Vietnam

    OpenAIRE

    Tran, Phuong

    2014-01-01

    The purpose of this thesis is to explore which factors affect the effectiveness of viral advertising on Facebook in Vietnam. The quantitative research method is applied in this research and the sample is Vietnamese Facebook users. After the data analysis stage using SPSS, it became clear that weak ties, perceptual affinity and emotions have an impact on the effectiveness of viral advertising. The results provide a pratical implication of how to make an Ad which can go viral on Facebook. Moreo...

  9. Viral diseases of northern ungulates

    Directory of Open Access Journals (Sweden)

    K. Frölich

    2000-03-01

    Full Text Available This paper describes viral diseases reported in northern ungulates and those that are a potential threat to these species. The following diseases are discussed: bovine viral diarrhoea/mucosal disease (BVD/MD, alphaherpesvirus infections, malignant catarrhal fever (MCF, poxvirus infections, parainfluenza type 3 virus infection, Alvsborg disease, foot-and-mouth disease, epizootic haemorrhage disease of deer and bluetongue disease, rabies, respiratory syncytial virus infection, adenovirus infection, hog-cholera, Aujeszky's disease and equine herpesvirus infections. There are no significant differences in antibody prevalence to BVDV among deer in habitats with high, intermediate and low density of cattle. In addition, sequence analysis from the BVDV isolated from roe deer (Capreolus capreolus showed that this strain was unique within BVDV group I. Distinct BVDV strains might circulate in free-ranging roe deer populations in Germany and virus transmission may be independent of domestic livestock. Similar results have been obtained in a serological survey of alpha-herpesviruses in deer in Germany. Malignant catarrhal fever was studied in fallow deer (Cervus dama in Germany: the seroprevalence and positive PCR results detected in sheep originating from the same area as the antibody-positive deer might indicate that sheep are the main reservoir animals. Contagious ecthyma (CE is a common disease in domestic sheep and goats caused by the orf virus. CE has been diagnosed in Rocky Mountain bighorn sheep (Ovis canadensis, mountain goats (Oreamnos americanus, Dall sheep (Ovis dalli, chamois (Rupkapra rupi-capra, muskox {Ovibos moschatus and reindeer (Rangifer tarandus. Most parainfluenza type 3 virus infections are mild or clinically undetectable. Serological surveys in wildlife have been successfully conducted in many species. In 1985, a new disease was identified in Swedish moose (Alces alces, designated as Alvsborg disease. This wasting syndrome probably

  10. Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2006-11-01

    Full Text Available Abstract Background We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1 variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY, creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism. Results Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R. This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity. Conclusion The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY can be corrected by a second site mutation in Env (GIA-SKY-G431R that affects the interaction with the CD4 receptor.

  11. Viral hepatitis in the elderly.

    Science.gov (United States)

    Carrion, Andres F; Martin, Paul

    2012-05-01

    As life expectancy continues to rise, elderly adults represent a rapidly growing proportion of the population. The likelihood of complications of acute and chronic liver disease and overall mortality are higher in elderly populations. Several physiological changes associated with aging, greater prevalence of co-morbid conditions, and cumulative exposure to hepatotropic viruses and environmental hepatotoxins may contribute to worse outcomes of viral hepatitis in the elderly. Although pharmacotherapy for hepatitis B and C continues to evolve, the efficacy, tolerability, and side effects of these agents have not been studied extensively in elderly adults. Immunization against hepatitis A and B in naïve elderly adults is an important public health intervention that needs to be revised and broadened.

  12. Viral hepatitis vaccination during pregnancy.

    Science.gov (United States)

    Zhao, Yueyuan; Jin, Hui; Zhang, Xuefeng; Wang, Bei; Liu, Pei

    2016-04-02

    Viral hepatitis is a serious global public health problem. It is also a common cause of jaundice and gestational complications in pregnant women. Moreover, infected mothers can transmit the virus to their fetus or neonate, which may increase disease burden and decrease quality of life. To date, commercial vaccines have been developed for hepatitis A, B, and E and are available to the general population. The Advisory Committee on Immunization Practices currently accepts emergency vaccination against hepatitis A and B during pregnancy due to benefits that overweight the potential risks. While there are limited data from trials with limited numbers of samples that suggest the efficacy or safety of hepatitis B and E vaccines in pregnant women, additional data are necessary to provide evidence of vaccination during pregnancy.

  13. Viral ancestors of antiviral systems.

    Science.gov (United States)

    Villarreal, Luis P

    2011-10-01

    All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the 'Big Bang' theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  14. Viral Ancestors of Antiviral Systems

    Directory of Open Access Journals (Sweden)

    Luis P. Villarreal

    2011-10-01

    Full Text Available All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the ‘Big Bang’ theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  15. Molecular Epidemiology of Viral Gastroenteritis in Hajj pilgrimage

    KAUST Repository

    Padron Regalado, Eriko

    2014-05-01

    Hajj is the annual gathering of Islam practitioners in Mecca, Saudi Arabia. During the event, gastrointestinal infections are usually experienced and outbreaks have always been a concern; nevertheless, a deep and integrative study of the etiological agents has never been carried out. Here, I describe for the first time the epidemiology of pathogenic enteric viruses during Hajj 2011, 2012 and 2013. The focus of this study was the common enteric viruses Astrovirus, Norovirus, Rotavirus and Adenovirus. An enzyme Immunoassay established their presence in 14.9%, 15.0% and 6.6% of the reported cases of acute diarrhea for 2011, 2012 and 2013, respectively. For the three years of study, Astrovirus accounted for the majority of the viral infections. To our knowledge, this is the first time an epidemiological study depicts Astrovirus as the main viral agent of gastroenteritis in a mass gathering event. Hajj is rich in strains of Astrovirus, Norovirus and Rotavirus. A first screening by RT-PCR resulted in ten different genotypes. Strains HAstV 2, HAstV 1 and HAstV 5 were identified for Astrovirus. GI.6, GII.3, GII.4 and GII.1 were described for Norovirus and G1P[8], G4P[8] and G3P[8] were found for Rotavirus. The majority of the Astrovirus isolates could not be genotyped suggesting the presence of a new variant(s). Cases like this encourage the use of metagenomics (and nextgeneration sequencing) as a state-of-the-art technology in clinical diagnosis. A sample containing Adenovirus particles is being used to standardize a process for detection directly from stool samples and results will be obtained in the near future. The overall findings of the present study support the concept of Hajj as a unique mass gathering event that potentiates the transmission of infectious diseases. The finding of Norovirus GII.4 Sydney, a variant originated from Australia, suggests that Hajj is a receptor of infectious diseases worldwide. This work is part of the Hajj project, a collaborative

  16. Data-variant kernel analysis

    CERN Document Server

    Motai, Yuichi

    2015-01-01

    Describes and discusses the variants of kernel analysis methods for data types that have been intensely studied in recent years This book covers kernel analysis topics ranging from the fundamental theory of kernel functions to its applications. The book surveys the current status, popular trends, and developments in kernel analysis studies. The author discusses multiple kernel learning algorithms and how to choose the appropriate kernels during the learning phase. Data-Variant Kernel Analysis is a new pattern analysis framework for different types of data configurations. The chapters include

  17. Acute Pancreatitis in acute viral hepatitis

    Directory of Open Access Journals (Sweden)

    S K.C.

    2011-03-01

    Full Text Available Introduction: The association of acute viral hepatitis and acute pancreatitis is well described. This study was conducted to find out the frequency of pancreatic involvement in acute viral hepatitis in the Nepalese population. Methods: Consecutive patients of acute viral hepatitis presenting with severe abdominal pain between January 2005 and April 2010 were studied. Patients with history of significant alcohol consumption and gall stones were excluded. Acute viral hepatitis was diagnosed by clinical examination, liver function test, ultrasound examination and confirmed by viral serology. Pancreatitis was diagnosed by clinical presentation, biochemistry, ultrasound examination and CT scan. Results: Severe abdominal pain was present in 38 of 382 serologically-confirmed acute viral hepatitis patients. Twenty five patients were diagnosed to have acute pancreatitis. The pancreatitis was mild in 14 and severe in 11 patients. The etiology of pancreatitis was hepatitis E virus in 18 and hepatitis A virus in 7 patients. Two patients died of complications secondary to shock. The remaining patients recovered from both pancreatitis and hepatitis on conservative treatment. Conclusions: Acute pancreatitis occurred in 6.5 % of patients with acute viral hepatitis. Cholelithiasis and gastric ulcers are the other causes of severe abdominal pain. The majority of the patients recover with conservative management. Keywords: acute viral hepatitis, acute pancreatitis, pain abdomen, hepatitis E, hepatitis A, endemic zone

  18. Viral marketing: the use of surprise

    NARCIS (Netherlands)

    Lindgreen, A.; Vanhamme, J.; Clarke, I.; Flaherty, T.B.

    2005-01-01

    Viral marketing involves consumers passing along a company's marketing message to their friends, family, and colleagues. This chapter reviews viral marketing campaigns and argues that the emotion of surprise often is at work and that this mechanism resembles that of word-of-mouth marketing.

  19. Emerging Viral Diseases of Tomato Crops

    NARCIS (Netherlands)

    Hanssen, I.M.; Lapidot, M.; Thomma, B.P.H.J.

    2010-01-01

    Viral diseases are an important limiting factor in many crop production systems. Because antiviral products are not available, control strategies rely on genetic resistance or hygienic measures to prevent viral diseases, or on eradication of diseased crops to control such diseases. Increasing

  20. Origins and challenges of viral dark matter.

    Science.gov (United States)

    Krishnamurthy, Siddharth R; Wang, David

    2017-07-15

    The accurate classification of viral dark matter - metagenomic sequences that originate from viruses but do not align to any reference virus sequences - is one of the major obstacles in comprehensively defining the virome. Depending on the sample, viral dark matter can make up from anywhere between 40 and 90% of sequences. This review focuses on the specific nature of dark matter as it relates to viral sequences. We identify three factors that contribute to the existence of viral dark matter: the divergence and length of virus sequences, the limitations of alignment based classification, and limited representation of viruses in reference sequence databases. We then discuss current methods that have been developed to at least partially circumvent these limitations and thereby reduce the extent of viral dark matter. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Ethical Considerations in Research Participation Virality.

    Science.gov (United States)

    Ellis-Barton, Carol

    2016-07-01

    This article seeks to commence and encourage discussion around the upcoming ethical challenges of virality in network structures. When the call for participation in a research project on lupus in Ireland went from an advertisement in a newsletter to a meme (unit of transmissible information) on a closed Facebook page, the ethical considerations of virality were raised. The article analyzes the Association of Internet Researchers guidelines, Facebook policies, and the context of privacy in relation to virality. Virality creates the leverage for methodological pluralism. The nature of the inquiry can determine the method rather than the other way around. Viral ethical considerations are evolving due to the cyber world becoming the primary meme of communication, with flexibility in the researcher's protocol providing opportunities for efficient, cost-effective, and diverse recruitment. © The Author(s) 2016.

  2. GCPII Variants, Paralogs and Orthologs

    Czech Academy of Sciences Publication Activity Database

    Hlouchová, Klára; Navrátil, Václav; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

    2012-01-01

    Roč. 19, č. 9 (2012), s. 1316-1322 ISSN 0929-8673 R&D Projects: GA ČR GAP304/12/0847 Institutional research plan: CEZ:AV0Z40550506 Keywords : PSMA * GCPIII * NAALADase L * splice variants * homologs * PSMAL Subject RIV: CE - Biochemistry Impact factor: 4.070, year: 2012

  3. Odontogenic keratocyst: a peripheral variant.

    Science.gov (United States)

    Vij, H; Vij, R; Gupta, V; Sengupta, S

    2011-01-01

    Odontogenic keratocyst, which is developmental in nature, is an intraosseous lesion though on rare occasions it may occur in an extraosseous location. The extraosseous variant is referred to as peripheral odontogenic keratocyst. Though, clinically, peripheral odontogenic keratocyst resembles the gingival cyst of adults, it has histologic features that are pathognomonic of odontogenic keratocyst. This article presents a case of this uncommon entity.

  4. The influence of TAP1 and TAP2 gene polymorphisms on TAP function and its inhibition by viral immune evasion proteins.

    Science.gov (United States)

    Praest, P; Luteijn, R D; Brak-Boer, I G J; Lanfermeijer, J; Hoelen, H; Ijgosse, L; Costa, A I; Gorham, R D; Lebbink, R J; Wiertz, E J H J

    2018-06-04

    Herpesviruses encode numerous immune evasion molecules that interfere with the immune system, particularly with certain stages in the MHC class I antigen presentation pathway. In this pathway, the transporter associated with antigen processing (TAP) is a frequent target of viral immune evasion strategies. This ER-resident transporter is composed of the proteins TAP1 and TAP2, and plays a crucial role in the loading of viral peptides onto MHC class I molecules. Several variants of TAP1 and TAP2 occur in the human population, some of which are linked to autoimmune disorders and susceptibility to infections. Here, we assessed the influence of naturally occurring TAP variants on peptide transport and MHC class I expression. In addition, we tested the inhibitory capacity of three viral immune evasion proteins, the TAP inhibitors US6 from human cytomegalovirus, ICP47 from herpes simplex virus type 1 and BNLF2a from Epstein-Barr virus, for a series of TAP1 and TAP2 variants. Our results suggest that these TAP polymorphisms have no or limited effect on peptide transport or MHC class I expression. Furthermore, our study indicates that the herpesvirus-encoded TAP inhibitors target a broad spectrum of TAP variants; inhibition of TAP is not affected by the naturally occurring polymorphisms of TAP tested in this study. Our findings suggest that the long-term coevolution of herpesviruses and their host did not result in selection of inhibitor-resistant TAP variants in the human population. Copyright © 2018. Published by Elsevier Ltd.

  5. HIV Viral Load: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... this page: https://medlineplus.gov/labtests/hivviralload.html HIV Viral Load To use the sharing features on this page, please enable JavaScript. What is an HIV Viral Load? An HIV viral load is a ...

  6. Assembly of viral genomes from metagenomes

    Directory of Open Access Journals (Sweden)

    Saskia L Smits

    2014-12-01

    Full Text Available Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

  7. Swine Influenza/Variant Influenza Viruses

    Science.gov (United States)

    ... Address What's this? Submit What's this? Submit Button Influenza Types Seasonal Avian Swine Variant Pandemic Other Information on Swine Influenza/Variant Influenza Virus Language: English (US) Español Recommend ...

  8. Análisis de variantes de HPV-16 como marcador molecular antropólogico

    Directory of Open Access Journals (Sweden)

    Badano, Ines

    2007-01-01

    Full Text Available El Virus Papiloma Humano tipo 16 (HPV16 es el principal responsable del desarrollo del cáncer de cuello uterino. Además de su significado clínico, el estudio de la variación genética en las regiones E6, L1 y LCR de este virus ha permitido identificar variantes específicas de diferentes áreas geográficas. Este descubrimiento sugiere una antigua propagación del HPV16 y su coevolución con el género humano. En este contexto, las variantes podrían servir como marcador molecular antropológico, aportando nuevos datos al análisis de patrones migratorios humanos. El objetivo del trabajo fue determinar las variantes de HPV16 en las regiones genéticas E6 y L1 que infectan mujeres guaraníes de Misiones. Para ello se analizaron 39 muestras de cepillados cervicales de mujeres guaraníes infectadas con HPV16. Las variantes en E6 y L1 se identificaron por PCR e hibridación en dot blot. Los resultados obtenidos fueron: el 77% de las variantes Europeas, 20% Africanas y 3% Asiático Americanas. La baja prevalencia de variantes Asiático Americanas coincide con lo reportado por Picconi y col, 2002 para mujeres quechuas, y haría suponer una limitada diseminación del HPV16 durante la época prehispánica. El predominio de las variantes Europeas podría ser resultado de la colonización española y la inmigración europea, mientras que el tráfico de esclavos negros explicaría la presencia de variantes Africanas. Por otra parte, la hipótesis de competencia viral tampoco puede ser descartada.

  9. Viral Evasion of Natural Killer Cell Activation.

    Science.gov (United States)

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-04-12

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

  10. Viral Evasion of Natural Killer Cell Activation

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-04-01

    Full Text Available Natural killer (NK cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

  11. EXPERIMENTAL LIPOSOMAL VIRAL VACCINE SAFETY

    Directory of Open Access Journals (Sweden)

    Romanova OA

    2016-12-01

    Full Text Available Introduction. With the transport links development there is rather important issue respiratory viral infections spread, especially influenza. The only method controlling influenza is vaccination. Search and development effective and safe vaccines is important. Material and methods. In base SO "Mechnikov Institute Microbiology and Immunology National Ukrainian Academy Medical Sciences" in the scientific theme "Developing new approaches to creating viral vaccines and study specific activity depending of type and degree component`s modification" was created several experimental influenza vaccine with subsequent component`s modification for selecting the most optimal pattern of safety and immunogenicity. In assessing the influenza vaccine safety is using a few criteria, including, reactivity, as measured by the frequency of local and systemic adverse (negative effects, which due to its introduction, and for lipid content drugs, ability to influence oxidation processes. At present study phase was determined: a systemic reaction and local reaction of delayed-type hypersensitivity (foot pad swelling assay;b lipids and proteins peroxidation processes after administration officinal and experimental vaccines (content protein’s carbonyl groups, lipid’s hydroperoxides, activity of glutathione-peroxidase.Study objects were trivalent seasonal influenza vaccine, "Vaxigrip" (Sanofi Pasteur, S.A., France, "Inflexal V" (Biotech Ltd. Berne, Switzerland and experimental vaccine samples. Highest immunogenicity vaccines had undergone improvements and modifications using adjuvant systems and acylation influenza proteins. Liposomes 2 – the experimental influenza vaccine with a liposome negative charge and antigenic composition like split vaccines "Vaksihryp". Liposomes 2.1 - the adjuvantexperimental influenza vaccine with modifications liposomal components (etoniy and chlorophyllipt molecules embedded in liposomal membrane. Liposomes 2.2 - the adjuvant

  12. Coronary artery anatomy and variants

    Energy Technology Data Exchange (ETDEWEB)

    Malago, Roberto; Pezzato, Andrea; Barbiani, Camilla; Alfonsi, Ugolino; Nicoli, Lisa; Caliari, Giuliana; Pozzi Mucelli, Roberto [Policlinico G.B. Rossi, University of Verona, Department of Radiology, Verona (Italy)

    2011-12-15

    Variants and congenital anomalies of the coronary arteries are usually asymptomatic, but may present with severe chest pain or cardiac arrest. The introduction of multidetector CT coronary angiography (MDCT-CA) allows the detection of significant coronary artery stenosis. Improved performance with isotropic spatial resolution and higher temporal resolution provides a valid alternative to conventional coronary angiography (CCA) in many patients. MDCT-CA is now considered the ideal tool for three-dimensional visualization of the complex and tortuous anatomy of the coronary arteries. With multiplanar and volume-rendered reconstructions, MDCT-CA may even outperform CCA in determining the relative position of vessels, thus providing a better view of the coronary vascular anatomy. The purpose of this review is to describe the normal anatomy of the coronary arteries and their main variants based on MDCT-CA with appropriate reconstructions. (orig.)

  13. Modern marketing approach: Concept of viral marketing

    Directory of Open Access Journals (Sweden)

    Mihailović Lidija B.

    2017-01-01

    Full Text Available Today, viral marketing is one of the most effective forms of marketing and it can be used to raise awareness about the product/service of a specific company. This type of marketing thrives in a modern environment, where final users (buyers/clients dominate by spreading messages within themselves. Boosted by the advantages that modern technology brings, viral marketing is booming in the online world. For the first time, small brands have a chance to make their appearance in the global market ad challenge the dominating position of the historically top brands. All they need is content that draws attention and modern, digital culture will help spread their message. Viral marketing is the future. And with the growth of social media and other channels, marketing managers need to be careful, because it is a thin line between a good and a bad (backfired viral campaign.

  14. The Etiology and Pathogenesis of Viral Gastroenteritis.

    Science.gov (United States)

    1984-07-31

    with subsequent seroconversion or susceptibility to illness in a naturally occurring outbreak of Norwalk virus gastroenteritis among American teen ... anorexia , myalgia and malaise. It can be severe, indeed fatal, in the elderly, infant, debilitated or malnourished pa- tient. Viral gastroenteritis

  15. NNDSS - Table II. Hepatitis (viral, acute) C

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Hepatitis (viral, acute) C - 2017. In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the preceding...

  16. NNDSS - Table II. Hepatitis (viral, acute)

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Hepatitis (viral, acute) - 2015.In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the preceding year),...

  17. NNDSS - Table II. Hepatitis (viral, acute)

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Hepatitis (viral, acute) - 2016. In this Table, provisional* cases of selected† notifiable diseases (≥1,000 cases reported during the preceding...

  18. NNDSS - Table II. Hepatitis (viral, acute)

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Hepatitis (viral, acute) - 2014.In this Table, all conditions with a 5-year average annual national total of more than or equals 1,000 cases but...

  19. Microcystic Variant of Urothelial Carcinoma

    Directory of Open Access Journals (Sweden)

    Anthony Kodzo-Grey Venyo

    2013-01-01

    Full Text Available Background. Microcystic variant of urothelial carcinoma is one of the new variants of urothelial carcinoma that was added to the WHO classification in 2004. Aims. To review the literature on microcystic variant of urothelial carcinoma. Methods. Various internet search engines were used to identify reported cases of the tumour. Results. Microscopic features of the tumour include: (i Conspicuous intracellular and intercellular lumina/microcysts encompassed by malignant urothelial or squamous cells. (ii The lumina are usually empty; may contain granular eosinophilic debris, mucin, or necrotic cells. (iii The cysts may be variable in size; round, or oval, up to 2 mm; lined by urothelium which are either flattened cells or low columnar cells however, they do not contain colonic epithelium or goblet cells; are infiltrative; invade the muscularis propria; mimic cystitis cystica and cystitis glandularis; occasionally exhibit neuroendocrine differentiation. (iv Elongated and irregular branching spaces are usually seen. About 17 cases of the tumour have been reported with only 2 patients who have survived. The tumour tends to be of high-grade and high-stage. There is no consensus opinion on the best option of treatment of the tumour. Conclusions. It would prove difficult at the moment to be dogmatic regarding its prognosis but it is a highly aggressive tumour. New cases of the tumour should be reported in order to document its biological behaviour.

  20. Immunity: Insect Immune Memory Goes Viral.

    Science.gov (United States)

    Ligoxygakis, Petros

    2017-11-20

    Adaptive memory in insect immunity has been controversial. In this issue, Andino and co-workers propose that acquisition of viral sequences in the host genome gives rise to anti-sense, anti-viral piRNAs. Such sequences can be regarded as both a genomic archive of past infections and as an armour of potential heritable memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Institute of Medicine's Report on Viral Hepatitis

    Centers for Disease Control (CDC) Podcasts

    2010-05-18

    In this podcast, Dr. John Ward, Director of CDC’s Division of Viral Hepatitis, discusses the 2010 report, Hepatitis and Liver Cancer: A National Strategy for Prevention and Control of Hepatitis B and C, from the Institute of Medicine.  Created: 5/18/2010 by National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP).   Date Released: 5/18/2010.

  2. Viral Immunotherapy to Eradicate Subclinical Brain Metastases

    Science.gov (United States)

    2014-05-01

    flash frozen brain tissue. Hoechst and CFSE labeled cells are readily visualized in fresh CSF. The brightest staining is achieved with Hoechst and is...viral infection in the meninges is in part due to reduction of the effects of suppressor macrophages. The work is complicated by the fact that...therapeutic effect of viral infection in the meninges is in part due to reduction of the effects of suppressor macrophages. • We found that mice cured

  3. Rapid and highly fieldable viral diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    McKnight, Timothy E.

    2016-12-20

    The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.

  4. Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

    International Nuclear Information System (INIS)

    Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer; Hirsch, Hans H.; Rinaldo, Christine Hanssen

    2010-01-01

    High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

  5. Oxygen tension level and human viral infections

    Energy Technology Data Exchange (ETDEWEB)

    Morinet, Frédéric, E-mail: frederic.morinet@sls.aphp.fr [Centre des Innovations Thérapeutiques en Oncologie et Hématologie (CITOH), CHU Saint-Louis, Paris (France); Université Denis Diderot, Sorbonne Paris Cité Paris, Paris (France); Casetti, Luana [Institut Cochin INSERM U1016, Paris (France); François, Jean-Hugues; Capron, Claude [Institut Cochin INSERM U1016, Paris (France); Laboratoire d' Hématologie, Hôpital Ambroise Paré, Boulogne (France); Université de Versailles Saint-Quentin en Yvelynes, Versailles (France); Pillet, Sylvie [Laboratoire de Bactériologie-Virologie-Hygiène, CHU de Saint-Etienne, Saint-Etienne (France); Université de Lyon et Université de Saint-Etienne, Jean Monnet, GIMAP EA3064, F-42023 Saint-Etienne, Lyon (France)

    2013-09-15

    The role of oxygen tension level is a well-known phenomenon that has been studied in oncology and radiotherapy since about 60 years. Oxygen tension may inhibit or stimulate propagation of viruses in vitro as well as in vivo. In turn modulating oxygen metabolism may constitute a novel approach to treat viral infections as an adjuvant therapy. The major transcription factor which regulates oxygen tension level is hypoxia-inducible factor-1 alpha (HIF-1α). Down-regulating the expression of HIF-1α is a possible method in the treatment of chronic viral infection such as human immunodeficiency virus infection, chronic hepatitis B and C viral infections and Kaposi sarcoma in addition to classic chemotherapy. The aim of this review is to supply an updating concerning the influence of oxygen tension level in human viral infections and to evoke possible new therapeutic strategies regarding this environmental condition. - Highlights: • Oxygen tension level regulates viral replication in vitro and possibly in vivo. • Hypoxia-inducible factor 1 (HIF-1α) is the principal factor involved in Oxygen tension level. • HIF-1α upregulates gene expression for example of HIV, JC and Kaposi sarcoma viruses. • In addition to classical chemotherapy inhibition of HIF-1α may constitute a new track to treat human viral infections.

  6. Viral Metagenomics: MetaView Software

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, C; Smith, J

    2007-10-22

    The purpose of this report is to design and develop a tool for analysis of raw sequence read data from viral metagenomics experiments. The tool should compare read sequences of known viral nucleic acid sequence data and enable a user to attempt to determine, with some degree of confidence, what virus groups may be present in the sample. This project was conducted in two phases. In phase 1 we surveyed the literature and examined existing metagenomics tools to educate ourselves and to more precisely define the problem of analyzing raw read data from viral metagenomic experiments. In phase 2 we devised an approach and built a prototype code and database. This code takes viral metagenomic read data in fasta format as input and accesses all complete viral genomes from Kpath for sequence comparison. The system executes at the UNIX command line, producing output that is stored in an Oracle relational database. We provide here a description of the approach we came up with for handling un-assembled, short read data sets from viral metagenomics experiments. We include a discussion of the current MetaView code capabilities and additional functionality that we believe should be added, should additional funding be acquired to continue the work.

  7. Adsorption of viral particles from the blood plasma of patients with viral hepatitis on nanodiamonds.

    Science.gov (United States)

    Baron, A V; Osipov, N V; Yashchenko, S V; Kokotukha, Yu A; Baron, I J; Puzyr, A P; Olkhovskiy, I A; Bondar, V S

    2016-07-01

    Adsorption of viral particles from the blood plasma of patients with viral hepatitis B and C on modified nanodiamonds (MNDs) was shown in the in vitro experiments. PCR method showed the treatment of plasma with MNDs leads to a decrease in the viral load by 2-3 orders of magnitude or more in both cases studied. These results make it possible to predict the applicability of MNDs for the development of new technologies of hemodialysis and plasmapheresis for binding and removal of viral particles from the blood of infected patients.

  8. Characterization of form variants of Xenorhabdus luminescens.

    Science.gov (United States)

    Gerritsen, L J; de Raay, G; Smits, P H

    1992-01-01

    From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed. Images PMID:1622273

  9. An Odyssey to Viral Pathogenesis.

    Science.gov (United States)

    Oldstone, Michael B A

    2016-05-23

    polishing by Karl Habel (a superb senior virologist who left the National Institutes of Health and came to Scripps), and the gifted postdoctoral fellows who joined my laboratory over four decades form the log of my scientific voyage. The strong friendships and collaborations developed with other young but growing experimentalists like Bernie Fields and Abner Notkins are the fabric of the tale I will weave and were pivotal in the establishment of viral pathogenesis as a discipline.

  10. The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is associated with susceptibility to Legionnaires’ disease in humans

    NARCIS (Netherlands)

    Ruiz-Moreno, J.S. (Juan S.); Hamann, L. (Lutz); Shah, J.A. (Javeed A.); A. Verbon (Annelies); Mockenhaupt, F.P. (Frank P.); Puzianowska-Kuznicka, M. (Monika); Naujoks, J. (Jan); Sander, L.E. (Leif E.); Witzenrath, M. (Martin); Cambier, J.C. (John C.); Suttorp, N. (Norbert); Schumann, R.R. (Ralf R.); Jin, L. (Lei); T.R. Hawn; Opitz, B. (Bastian)

    2018-01-01

    textabstractThe cyclic GMP-AMP synthase (cGAS)-STING pathway is central for innate immune sensing of various bacterial, viral and protozoal infections. Recent studies identified the common HAQ and R232H alleles of TMEM173/STING, but the functional consequences of these variants for primary

  11. Novel Genetic Variants of Hepatitis B Virus in Fulminant Hepatitis

    Directory of Open Access Journals (Sweden)

    Jack Bee Chook

    2017-01-01

    Full Text Available Fulminant hepatitis (FH is a life-threatening liver disease characterised by intense immune attack and massive liver cell death. The common precore stop codon mutation of hepatitis B virus (HBV, A1896, is frequently associated with FH, but lacks specificity. This study attempts to uncover all possible viral nucleotides that are specifically associated with FH through a compiled sequence analysis of FH and non-FH cases from acute infection. We retrieved 67 FH and 280 acute non-FH cases of hepatitis B from GenBank and applied support vector machine (SVM model to seek candidate nucleotides highly predictive of FH. Six best candidates with top predictive accuracy, 92.5%, were used to build a SVM model; they are C2129 (85.3%, T720 (83.0%, Y2131 (82.4%, T2013 (82.1%, K2048 (82.1%, and A2512 (82.1%. This model gave a high specificity (99.3%, positive predictive value (95.6%, and negative predictive value (92.1%, but only moderate sensitivity (64.2%. We successfully built a SVM model comprising six variants that are highly predictive and specific for FH: four in the core region and one each in the polymerase and the surface regions. These variants indicate that intracellular virion/core retention could play an important role in the progression to FH.

  12. Clinical Relevance of HLA Gene Variants in HBV Infection

    Directory of Open Access Journals (Sweden)

    Li Wang

    2016-01-01

    Full Text Available Host gene variants may influence the natural history of hepatitis B virus (HBV infection. The human leukocyte antigen (HLA system, the major histocompatibility complex (MHC in humans, is one of the most important host factors that are correlated with the clinical course of HBV infection. Genome-wide association studies (GWASs have shown that single nucleotide polymorphisms (SNPs near certain HLA gene loci are strongly associated with not only persistent HBV infection but also spontaneous HBV clearance and seroconversion, disease progression, and the development of liver cirrhosis and HBV-related hepatocellular carcinoma (HCC in chronic hepatitis B (CHB. These variations also influence the efficacy of interferon (IFN and nucleot(side analogue (NA treatment and response to HBV vaccines. Meanwhile, discrepant conclusions were reached with different patient cohorts. It is therefore essential to identify the associations of specific HLA allele variants with disease progression and viral clearance in chronic HBV infection among different ethnic populations. A better understanding of HLA polymorphism relevance in HBV infection outcome would enable us to elucidate the roles of HLA SNPs in the pathogenesis and clearance of HBV in different areas and ethnic groups, to improve strategies for the prevention and treatment of chronic HBV infection.

  13. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

    Energy Technology Data Exchange (ETDEWEB)

    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T., E-mail: a.t.das@amc.uva.nl

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

  14. Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based or remarkably insensitive (antibody-based. Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A

  15. Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

    Science.gov (United States)

    Metzgar, David; Myers, Christopher A; Russell, Kevin L; Faix, Dennis; Blair, Patrick J; Brown, Jason; Vo, Scott; Swayne, David E; Thomas, Colleen; Stenger, David A; Lin, Baochuan; Malanoski, Anthony P; Wang, Zheng; Blaney, Kate M; Long, Nina C; Schnur, Joel M; Saad, Magdi D; Borsuk, Lisa A; Lichanska, Agnieszka M; Lorence, Matthew C; Weslowski, Brian; Schafer, Klaus O; Tibbetts, Clark

    2010-02-03

    For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence

  16. Expansion of the CRF19_cpx Variant in Spain.

    Science.gov (United States)

    Patiño Galindo, Juan Angel; Torres-Puente, Manoli; Gimeno, Concepción; Ortega, Enrique; Navarro, David; Galindo, María José; Navarro, Laura; Navarro, Vicente; Juan, Amparo; Belda, Josefina; Bracho, María Alma; González-Candelas, Fernando

    2015-08-01

    HIV-1 CRF19_cpx, is a recombinant variant found almost exclusively in Cuba and recently associated to a faster AIDS onset. Infection with this variant leads to higher viral loads and levels of RANTES and CXCR4 co-receptor use. The goal of this study was to assess the presence of CRF19_cpx in the Spanish province of Valencia, given its high pathogenicity. 1294 HIV-1 protease-reverse transcriptase (PR/RT) sequences were obtained in Valencia (Spain), between 2005 and 2014. After subtyping, the detected CRF19_cpx sequences were aligned with 201 CRF19_cpx and 66 subtype D sequences retrieved from LANL, and subjected to maximum-likelihood phylogenetic analyses and Bayesian coalescent reconstructions. The presence of resistance mutations in the PR/RT region of these sequences was also analyzed. Among the 9 CRF19_cpx sequences from different patients found (prevalence <0.1%), 7 grouped in two well-supported clades (groups A, n=4, and B, n=3), suggesting the existence of at least two independent introductions which subsequently started to expand in the studied Spanish region. Unprotected sex between men was the only known transmission route. Coalescent analyses suggested that the introductions in Valencia occurred between 2008 and 2010. Resistance mutations in the RT region were found in all sequences from group A (V139D) and in two sequences from group B (E138A). This study reports for the first time the recent expansion of CRF19_cpx outside Cuba. Our results suggest that CRF19_cpx might become an emerging HIV variant in Spain, affecting Spanish native MSM and not only Cuban migrants. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine.

    Science.gov (United States)

    Dunham, J P; Simmons, H E; Holmes, E C; Stephenson, A G

    2014-10-13

    Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Viral Hepatitis: Information for Gay and Bisexual Men

    Science.gov (United States)

    VIRAL HEPATITIS Information for Gay and Bisexual Men What is viral hepatitis? Viral hepatitis is an infection of the liver caused by one of several ... each virus is spread in different ways. Are gay and bisexual men at risk for viral hepatitis? ...

  19. Raw Sewage Harbors Diverse Viral Populations

    Science.gov (United States)

    Cantalupo, Paul G.; Calgua, Byron; Zhao, Guoyan; Hundesa, Ayalkibet; Wier, Adam D.; Katz, Josh P.; Grabe, Michael; Hendrix, Roger W.; Girones, Rosina; Wang, David; Pipas, James M.

    2011-01-01

    ABSTRACT At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. Importance At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that

  20. APLASTIC ANEMIA AND VIRAL HEPATITIS

    Directory of Open Access Journals (Sweden)

    Laura Cudillo

    2009-11-01

    target organ of the immune  response is the liver as suggested by the time interval between hepatitis and the onset of bone marrow failure.

    Liver histology is characterized by T cell infiltrating the parenchyma as reported in acute hepatitis.

    Recently in HAA it has been demonstrated intrahepatic  and blood lymphocytes with  T cell repertoire similar to that of confirmed viral acute hepatitis. The expanded T cell clones return to a normal distribution after response to immunosuppressive treatment, suggesting the antigen or T cell clearance. Therapeutic options are the same as acquired aplastic anemia.

  1. Shift-Variant Multidimensional Systems.

    Science.gov (United States)

    1985-05-29

    x,y;u,v) is the system response at (x,y) to an unit impulse applied at (u,v). The presence of additive noise in the preceding input-output model of a...space model developed works very effi- ciently to deblur images affected by 2-D linear shift- varying blurs, its use, in presence of noise needs to be...causal linear shift-variant (LSV) system, whose impulse res- ponse is a K-th order degenerate sequence, a K-th order state-space model was obtained

  2. View and review on viral oncology research

    Directory of Open Access Journals (Sweden)

    Parolin Cristina

    2010-05-01

    Full Text Available Abstract To date, almost one and a half million cases of cancer are diagnosed every year in the US and nearly 560,000 Americans are expected to die of cancer in the current year, more than 1,500 people a day (data from the American Cancer Society at http://www.cancer.org/. According to the World Health Organization (WHO, roughly 20% of all cancers worldwide results from chronic infections; in particular, up to 15% of human cancers is characterized by a viral aetiology with higher incidence in Developing Countries. The link between viruses and cancer was one of the pivotal discoveries in cancer research during the past Century. Indeed, the infectious nature of specific tumors has important implications in terms of their prevention, diagnosis, and therapy. In the 21st Century, the research on viral oncology field continues to be vigorous, with new significant and original studies on viral oncogenesis and translational research from basic virology to treatment of cancer. This review will cover different viral oncology aspects, starting from the history of viral oncology and moving to the peculiar features of oncogenic RNA and DNA viruses, with a special focus on human pathogens.

  3. Molecular imaging of oncolytic viral therapy

    Directory of Open Access Journals (Sweden)

    Dana Haddad

    2014-01-01

    Full Text Available Oncolytic viruses have made their mark on the cancer world as a potential therapeutic option, with the possible advantages of reduced side effects and strengthened treatment efficacy due to higher tumor selectivity. Results have been so promising, that oncolytic viral treatments have now been approved for clinical trials in several countries. However, clinical studies may benefit from the ability to noninvasively and serially identify sites of viral targeting via molecular imaging in order to provide safety, efficacy, and toxicity information. Furthermore, molecular imaging of oncolytic viral therapy may provide a more sensitive and specific diagnostic technique to detect tumor origin and, more importantly, presence of metastases. Several strategies have been investigated for molecular imaging of viral replication broadly categorized into optical and deep tissue imaging, utilizing several reporter genes encoding for fluorescence proteins, conditional enzymes, and membrane protein and transporters. Various imaging methods facilitate molecular imaging, including computer tomography, magnetic resonance imaging, positron emission tomography, single photon emission CT, gamma-scintigraphy, and photoacoustic imaging. In addition, several molecular probes are used for medical imaging, which act as targeting moieties or signaling agents. This review will explore the preclinical and clinical use of in vivo molecular imaging of replication-competent oncolytic viral therapy.

  4. Pediatric Viral Exanthema: A Review Article

    Directory of Open Access Journals (Sweden)

    Mohammed Jafar Saffar

    2017-04-01

    Full Text Available Context Many diseases caused by viral agents are associated with fever and cutaneous manifestations. Viral exanthema is a widespread nonspecific skin rash, commonly characterized by generalized eruption of erythematous macules and papular lesions. Although these rashes are mostly benign and self-limited, some may be serious and life-threatening. Differentiation between severe and benign types is clinically important and life-saving. Evidence Acquisition In this narrative review, electronic databases, including Google Scholar, Science Direct, PubMed (including Medline, Web of Science, Scientific Information Database, and Scopus, were searched. We conducted a narrative review of papers published on pediatric viral exanthema during 2000 - 2016. The used keywords included “viral exanthema”, “fever”, and “skin rash”. Articles on skin rash, caused by drug reactions or nonviral exanthema, were excluded. Results Different viral agents can cause different types of skin reactions. Cutaneous manifestations and skin rashes can be categorized, based on the form of the rash (macular, papular, vesicular, blistery, petechial, and purpuric or the general term, which denotes illnesses such as measles-like morbilliform rash, rubella or rubelliform rash, and scarlatiniform rash, a scarlet-fever like infection. Conclusions Based on the findings, a systematic approach relying on accurate history-taking and analysis of epidemiological cues and rash characteristics is of great significance.

  5. Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells.

    Directory of Open Access Journals (Sweden)

    Claudia V Filomatori

    2017-03-01

    Full Text Available The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs, relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of

  6. Viral vector-based influenza vaccines

    Science.gov (United States)

    de Vries, Rory D.; Rimmelzwaan, Guus F.

    2016-01-01

    ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

  7. CT images of infantile viral encephalitis

    International Nuclear Information System (INIS)

    Sugimoto, Tateo; Okazaki, Hitoshi; Woo, Man

    1985-01-01

    Cranial CT scanning was undertaken in 40 patients with infantile viral encephalitis seen from 1977 to 1983. According to the pathogenic viruses, abnormal CT findings were detected most frequently in cases of herpes simplex encephalitis (HSE), followed by non-eruptive viral encephalitis, measles encephalitis, and rubella encephalitis in that order, which coincided well with neurological prognosis. Although CT findings lay within a normal range in cases of measles encephalitis, except a case in which cerebral ventricle was slightly dilated, the degree of consciousness disturbance was unfavorable and it persisted long. This revealed that there is no distinct correlation between the degree of consciousness disturbance and CT findings. Normal CT findings were detected in 13% of patients aged less than 5 years and 76.5% of patients aged 5 years or more. In many patients who had an attack of viral encephalitis at the age of 5 years or more, epileptic seizures occurred frequently, even though CT findings were normal. (Namekawa, K.)

  8. V-GAP: Viral genome assembly pipeline

    KAUST Repository

    Nakamura, Yoji

    2015-10-22

    Next-generation sequencing technologies have allowed the rapid determination of the complete genomes of many organisms. Although shotgun sequences from large genome organisms are still difficult to reconstruct perfect contigs each of which represents a full chromosome, those from small genomes have been assembled successfully into a very small number of contigs. In this study, we show that shotgun reads from phage genomes can be reconstructed into a single contig by controlling the number of read sequences used in de novo assembly. We have developed a pipeline to assemble small viral genomes with good reliability using a resampling method from shotgun data. This pipeline, named V-GAP (Viral Genome Assembly Pipeline), will contribute to the rapid genome typing of viruses, which are highly divergent, and thus will meet the increasing need for viral genome comparisons in metagenomic studies.

  9. Hepatitis C Virus: Viral Quasispecies and Genotypes

    Directory of Open Access Journals (Sweden)

    Kyoko Tsukiyama-Kohara

    2017-12-01

    Full Text Available Hepatitis C virus (HCV mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs, which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  10. Hepatitis C Virus: Viral Quasispecies and Genotypes.

    Science.gov (United States)

    Tsukiyama-Kohara, Kyoko; Kohara, Michinori

    2017-12-22

    Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  11. Viral haemorrhagic fever and vascular alterations.

    Science.gov (United States)

    Aleksandrowicz, P; Wolf, K; Falzarano, D; Feldmann, H; Seebach, J; Schnittler, H

    2008-02-01

    Pathogenesis of viral haemorrhagic fever (VHF) is closely associated with alterations of the vascular system. Among the virus families causing VHF, filoviruses (Marburg and Ebola) are the most fatal, and will be focused on here. After entering the body, Ebola primarily targets monocytes/macrophages and dendritic cells. Infected dendritic cells are largely impaired in their activation potency, likely contributing to the immune suppression that occurs during filovirus infection. Monocytes/macrophages, however, immediately activate after viral contact and release reasonable amounts of cytokines that target the vascular system, particularly the endothelial cells. Some underlying molecular mechanisms such as alteration of the vascular endothelial cadherin/catenin complex, tyrosine phosphorylation, expression of cell adhesion molecules, tissue factor and the effect of soluble viral proteins released from infected cells to the blood stream will be discussed.

  12. V-GAP: Viral genome assembly pipeline

    KAUST Repository

    Nakamura, Yoji; Yasuike, Motoshige; Nishiki, Issei; Iwasaki, Yuki; Fujiwara, Atushi; Kawato, Yasuhiko; Nakai, Toshihiro; Nagai, Satoshi; Kobayashi, Takanori; Gojobori, Takashi; Ototake, Mitsuru

    2015-01-01

    Next-generation sequencing technologies have allowed the rapid determination of the complete genomes of many organisms. Although shotgun sequences from large genome organisms are still difficult to reconstruct perfect contigs each of which represents a full chromosome, those from small genomes have been assembled successfully into a very small number of contigs. In this study, we show that shotgun reads from phage genomes can be reconstructed into a single contig by controlling the number of read sequences used in de novo assembly. We have developed a pipeline to assemble small viral genomes with good reliability using a resampling method from shotgun data. This pipeline, named V-GAP (Viral Genome Assembly Pipeline), will contribute to the rapid genome typing of viruses, which are highly divergent, and thus will meet the increasing need for viral genome comparisons in metagenomic studies.

  13. Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy

    Directory of Open Access Journals (Sweden)

    Marcin P Komorowski

    2016-01-01

    Full Text Available Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

  14. Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants.

    Science.gov (United States)

    Paradowska, Edyta; Jabłońska, Agnieszka; Studzińska, Mirosława; Suski, Patrycja; Kasztelewicz, Beata; Zawilińska, Barbara; Wiśniewska-Ligier, Małgorzata; Dzierżanowska-Fangrat, Katarzyna; Woźniakowska-Gęsicka, Teresa; Czech-Kowalska, Justyna; Lipka, Bożena; Kornacka, Maria; Pawlik, Dorota; Tomasik, Tomasz; Kosz-Vnenchak, Magdalena; Leśnikowski, Zbigniew J

    2013-09-01

    Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Viral diseases in honey bee queens

    DEFF Research Database (Denmark)

    Francis, Roy Mathew

    Honey bees are important insects for human welfare, due to pollination as well as honey production. Viral diseases strongly impact honey bee health, especially since the spread of varroa mites. This dissertation deals with the interactions between honey bees, viruses and varroa mites. A new tool...... was developed to diagnose three viruses in honey bees. Quantitative PCR was used to investigate the distribution of two popular viruses in five different tissues of 86 honey bee queens. Seasonal variation of viral infection in honey bee workers and varroa mites were determined by sampling 23 colonies under...

  16. Importance of viral diseases in irradiated persons

    International Nuclear Information System (INIS)

    Blaha, M.; Jebavy, L.; Merka, V.; Horacek, J.

    1988-01-01

    A preliminary study was performed aimed at establishing the incidence of some viral diseases in radiation syndrome patients and the significance of the diseases for prognosis. In the study, 77 patients with syndromologically identical acute hematological forms of radiation sickness, mainly leukemic patients suffering from severe blood formation suppression and/or hematoblastosis were examined for concurrent herpes simplex virus and cytomegalovirus infections. Active viruses were isolated in almost 30% of the patients; nearly 90% of the patients were serologically positive, shedding antibodies. The findings thus confirmed the view that viral disease, especially in immunocompromised patients, has a critical effect on the survival of radiation sickness sufferers. (L.O.). 12 refs

  17. Viral pneumonias: Typical and atypical findings

    International Nuclear Information System (INIS)

    Westhoff-Bleck, M.; Bleck, J.S.; Schirg, E.

    1987-01-01

    The clinical and radiological features of viral pneumonias are summarized and discussed. Although viral infections of the lung belong to atypical pneumonias they demonstrate not always the radiographic pattern of an interstitial pneumonia. Characteristic radiographic findings are quite rare. In most cases the microbial etiology cannot be predicted from chest radiographs. The appearance varies depending on the virulence of the organism and the resistence of the host. In this regard knowledge of epidemiological data as well as patients condition and underlying disease is of utmost importance. Differentiation between community- and hospital-acquired infection may be very helpful. (orig.) [de

  18. Viral gastroenteritis in daily pediatric practice

    International Nuclear Information System (INIS)

    Kracmarova, R.; Plisek, S.

    2011-01-01

    Diarrhoeal disease is one of the most common causes of an acute examination and hospitalisation of a child. Portion of a viral etiology of intestinal diseases is increasing in connection with an improvement of social and economical conditions. The most common viral agents are rotaviruses, caliciviruses, adenoviruses and astroviruses, but also other viruses cause an intestinal disease. The most severe clinical course is expected from the rotaviral and noroviral infection. The dehydratation, which could be less or more severe, often complicated the infection. The treatment is symptomatic. The most important role for the prevention of rotavirus disease is played by the vaccination. (author)

  19. UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication.

    Directory of Open Access Journals (Sweden)

    Peng-Nien Huang

    2017-05-01

    Full Text Available Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR in host cells. This study found that enterovirus A71 (EVA71 utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1, a key endoplasmic reticulum protein (ER involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice. These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity.

  20. Identification of viral infections in the prostate and evaluation of their association with cancer

    Directory of Open Access Journals (Sweden)

    Calderon-Cardenas German

    2010-06-01

    Full Text Available Abstract Background Several viruses with known oncogenic potential infect prostate tissue, among these are the polyomaviruses BKV, JCV, and SV40; human papillomaviruses (HPVs, and human cytomegalovirus (HCMV infections. Recently, the Xenotropic Murine Leukemia Virus-related gammaretrovirus (XMRV was identified in prostate tissue with a high prevalence observed in prostate cancer (PC patients homozygous for the glutamine variant of the RNASEL protein (462Q/Q. Association studies with the R462Q allele and non-XMRV viruses have not been reported. We assessed associations between prostate cancer, prostate viral infections, and the RNASEL 462Q allele in Mexican cancer patients and controls. Methods 130 subjects (55 prostate cancer cases and 75 controls were enrolled in the study. DNA and RNA isolated from prostate tissues were screened for the presence of viral genomes. Genotyping of the RNASEL R462Q variant was performed by Taqman method. Results R/R, R/Q, and Q/Q frequencies for R462Q were 0.62, 0.38, and 0.0 for PC cases and 0.69, 0.24, and 0.07 for controls, respectively. HPV sequences were detected in 11 (20.0% cases and 4 (5.3% controls. XMRV and HCMV infections were detected in one and six control samples, respectively. The risk of PC was significantly increased (Odds Ratio = 3.98; 95% CI: 1.17-13.56, p = 0.027 by infection of the prostatic tissue with HPV. BKV, JCV, and SV40 sequences were not detected in any of the tissue samples examined. Conclusions We report a positive association between PC and HPV infection. The 462Q/Q RNASEL genotype was not represented in our PC cases; thus, its interaction with prostate viral infections and cancer could not be evaluated.

  1. Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Niko eBeerenwinkel

    2012-09-01

    Full Text Available Many viruses, including the clinically relevant RNA viruses HIV and HCV, exist in large populations and display high genetic heterogeneity within and between infected hosts. Assessing intra-patient viral genetic diversity is essential for understanding the evolutionary dynamics of viruses, for designing effective vaccines, and for the success of antiviral therapy. Next-generation sequencing technologies allow the rapid and cost-effective acquisition of thousands to millions of short DNA sequences from a single sample. However, this approach entails several challenges in experimental design and computational data analysis. Here, we review the entire process of inferring viral diversity from sample collection to computing measures of genetic diversity. We discuss sample preparation, including reverse transcription and amplification, and the effect of experimental conditions on diversity estimates due to in vitro base substitutions, insertions, deletions, and recombination. The use of different next-generation sequencing platforms and their sequencing error profiles are compared in the context of various applications of diversity estimation, ranging from the detection of single nucleotide variants to the reconstruction of whole-genome haplotypes. We describe the statistical and computational challenges arising from these technical artifacts, and we review existing approaches, including available software, for their solution. Finally, we discuss open problems, and highlight successful biomedical applications and potential future clinical use of next-generation sequencing to estimate viral diversity.

  2. Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus.

    Science.gov (United States)

    Kong, Byung-Whi; Foster, Linda K; Foster, Douglas N

    2008-03-01

    The avian metapneumovirus (AMPV) genome encodes the fusion (F), small hydrophobic (SH), and attachment glycoprotein (G) as envelope glycoproteins. The F and G proteins mainly function to allow viral entry into host cells during the early steps of the virus life cycle. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes. Sequence analysis was used to determine if any differences between avian or mammalian cell propagated subtype C AMPV could be detected for the 1.8kb G gene. As a result, the complete 1.8kb G gene was found to be present when AMPV was propagated in our immortal turkey turbinate (TT-1) cell line regardless of passage number. Surprisingly, AMPV propagated for 15 or more passages in mammalian Vero cells revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. Although the Vero cell propagated AMPV genome contained a small 122 nucleotide fragment of the G gene, no other mRNA variants were detected from either mammalian or avian propagated AMPV. The G gene truncation might be caused by cellular molecular mechanisms that are species-specific. The lack of viral gene deletions suggests that avian cell propagated AMPV will provide a better alternative host for live recombinant vaccine development based on a reverse genetics system.

  3. Good Friends, Bad News - Affect and Virality in Twitter

    DEFF Research Database (Denmark)

    Hansen, Lars Kai; Arvidsson, Adam; Nielsen, Finn Årup

    2011-01-01

    The link between affect, defined as the capacity for sentimental arousal on the part of a message, and virality, defined as the probability that it be sent along, is of significant theoretical and practical importance, e.g. for viral marketing. The basic measure of virality in Twitter is the prob......The link between affect, defined as the capacity for sentimental arousal on the part of a message, and virality, defined as the probability that it be sent along, is of significant theoretical and practical importance, e.g. for viral marketing. The basic measure of virality in Twitter...

  4. Recombinant viruses as vaccines against viral diseases

    Directory of Open Access Journals (Sweden)

    A.P.D. Souza

    2005-04-01

    Full Text Available Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

  5. Viral skin diseases of the rabbit.

    Science.gov (United States)

    Meredith, Anna L

    2013-09-01

    This article describes the viral skin diseases affecting the domestic rabbit, the most important being myxomatosis. Transmission and pathogenesis, clinical signs, diagnosis, treatment, and control are described and the article will be of interest to veterinary practitioners who treat rabbits. Shope fibroma virus, Shope papilloma virus, and rabbitpox are also discussed. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Microbial oceanography: Viral strategies at sea

    Science.gov (United States)

    Thingstad, T. Frede; Bratbak, Gunnar

    2016-03-01

    The finding that marine environments with high levels of host microbes have fewer viruses per host than when host abundance is low challenges a theory on the relative roles of lysogenic and lytic viral-survival strategies. See Article p.466

  7. Viral immune evasion: a masterpiece of evolution

    NARCIS (Netherlands)

    Vossen, Mireille T. M.; Westerhout, Ellen M.; Söderberg-Nauclér, Cécilia; Wiertz, Emmanuel J. H. J.

    2002-01-01

    Coexistence of viruses and their hosts imposes an evolutionary pressure on both the virus and the host immune system. On the one hand, the host has developed an immune system able to attack viruses and virally infected cells, whereas on the other hand, viruses have developed an array of immune

  8. Vaccination of cattle against bovine viral diarrhoea

    NARCIS (Netherlands)

    Oirschot, van J.T.; Bruschke, C.J.M.; Rijn, van P.A.

    1999-01-01

    This brief review describes types and quality (efficacy and safety) of bovine viral diarrhoea virus (BVDV) vaccines that are in the market or under development. Both conventional live and killed vaccines are available. The primary aim of vaccination is to prevent congenital infection, but the few

  9. Sanitation of viral haemorrhagic septicaemia (VHS)

    DEFF Research Database (Denmark)

    Olesen, Niels Jørgen

    1998-01-01

    A sanitation programme for stamping-out viral haemorrhagic septicaemia (VHS) was implemented in Denmark in 1965. The programme has resulted in a dramatic reduction in the number of infected rainbow trout farms, from approximate to 400 to 26. The programme is carried out on a voluntary basis...

  10. Meta-analyses on viral hepatitis

    DEFF Research Database (Denmark)

    Gluud, Lise L; Gluud, Christian

    2009-01-01

    This article summarizes the meta-analyses of interventions for viral hepatitis A, B, and C. Some of the interventions assessed are described in small trials with unclear bias control. Other interventions are supported by large, high-quality trials. Although attempts have been made to adjust...

  11. Viral Hepatitis and Thrombosis: A Narrative Review

    NARCIS (Netherlands)

    Squizzato, Alessandro; Gerdes, Victor E. A.

    2012-01-01

    Venous thromboembolism (VTE) is a multicausal disease. Among minor risk factors, acute infections in general are associated with a transient increased risk of VTE. However, acute hepatitis is usually not reported as a potential risk factor for VTE. Recent studies suggest a possible role of viral

  12. The laboratory diagnosis of acute viral hepatitis

    African Journals Online (AJOL)

    defined level and is thus indicative of recent infection as IgM anti-HBc may persist in low titres for a prolonged period. SAMJ. ARTICLES. Detection of HBeAg in the serum is important in the clinical evaluation of a patient with HBV infection as it usually correlates with viral replication, active liver damage and infectivity.3 ...

  13. Institute of Medicine's Report on Viral Hepatitis

    Centers for Disease Control (CDC) Podcasts

    In this podcast, Dr. John Ward, Director of CDC’s Division of Viral Hepatitis, discusses the 2010 report, Hepatitis and Liver Cancer: A National Strategy for Prevention and Control of Hepatitis B and C, from the Institute of Medicine.

  14. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  15. Stability of the resistance to the thiosemicarbazone derived from 5,6-Dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus

    OpenAIRE

    Castro, Eliana Florencia; Campos, Rodolfo Hector; Cavallaro, Lucía Vicenta

    2017-01-01

    Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1–5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of...

  16. Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

    Energy Technology Data Exchange (ETDEWEB)

    Hipp, Katharina, E-mail: katharina.hipp@bio.uni-stuttgart.de [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany); Rau, Peter; Schäfer, Benjamin [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany); Pfannstiel, Jens [University of Hohenheim, Mass Spectrometry Core Facility, August-von-Hartmann-Straße 3, 70599 Stuttgart (Germany); Jeske, Holger [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany)

    2016-11-15

    Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.

  17. Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

    International Nuclear Information System (INIS)

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Pfannstiel, Jens; Jeske, Holger

    2016-01-01

    Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.

  18. Psoriasis Patients Are Enriched for Genetic Variants That Protect against HIV-1 Disease

    Science.gov (United States)

    Chen, Haoyan; Hayashi, Genki; Lai, Olivia Y.; Dilthey, Alexander; Kuebler, Peter J.; Wong, Tami V.; Martin, Maureen P.; Fernandez Vina, Marcelo A.; McVean, Gil; Wabl, Matthias; Leslie, Kieron S.; Maurer, Toby; Martin, Jeffrey N.; Deeks, Steven G.; Carrington, Mary; Bowcock, Anne M.; Nixon, Douglas F.; Liao, Wilson

    2012-01-01

    An important paradigm in evolutionary genetics is that of a delicate balance between genetic variants that favorably boost host control of infection but which may unfavorably increase susceptibility to autoimmune disease. Here, we investigated whether patients with psoriasis, a common immune-mediated disease of the skin, are enriched for genetic variants that limit the ability of HIV-1 virus to replicate after infection. We analyzed the HLA class I and class II alleles of 1,727 Caucasian psoriasis cases and 3,581 controls and found that psoriasis patients are significantly more likely than controls to have gene variants that are protective against HIV-1 disease. This includes several HLA class I alleles associated with HIV-1 control; amino acid residues at HLA-B positions 67, 70, and 97 that mediate HIV-1 peptide binding; and the deletion polymorphism rs67384697 associated with high surface expression of HLA-C. We also found that the compound genotype KIR3DS1 plus HLA-B Bw4-80I, which respectively encode a natural killer cell activating receptor and its putative ligand, significantly increased psoriasis susceptibility. This compound genotype has also been associated with delay of progression to AIDS. Together, our results suggest that genetic variants that contribute to anti-viral immunity may predispose to the development of psoriasis. PMID:22577363

  19. Developing consistent pronunciation models for phonemic variants

    CSIR Research Space (South Africa)

    Davel, M

    2006-09-01

    Full Text Available Pronunciation lexicons often contain pronunciation variants. This can create two problems: It can be difficult to define these variants in an internally consistent way and it can also be difficult to extract generalised grapheme-to-phoneme rule sets...

  20. Semantic prioritization of novel causative genomic variants

    KAUST Repository

    Boudellioua, Imene

    2017-04-17

    Discriminating the causative disease variant(s) for individuals with inherited or de novo mutations presents one of the main challenges faced by the clinical genetics community today. Computational approaches for variant prioritization include machine learning methods utilizing a large number of features, including molecular information, interaction networks, or phenotypes. Here, we demonstrate the PhenomeNET Variant Predictor (PVP) system that exploits semantic technologies and automated reasoning over genotype-phenotype relations to filter and prioritize variants in whole exome and whole genome sequencing datasets. We demonstrate the performance of PVP in identifying causative variants on a large number of synthetic whole exome and whole genome sequences, covering a wide range of diseases and syndromes. In a retrospective study, we further illustrate the application of PVP for the interpretation of whole exome sequencing data in patients suffering from congenital hypothyroidism. We find that PVP accurately identifies causative variants in whole exome and whole genome sequencing datasets and provides a powerful resource for the discovery of causal variants.

  1. Semantic prioritization of novel causative genomic variants

    KAUST Repository

    Boudellioua, Imene; Mohamad Razali, Rozaimi; Kulmanov, Maxat; Hashish, Yasmeen; Bajic, Vladimir B.; Goncalves-Serra, Eva; Schoenmakers, Nadia; Gkoutos, Georgios V.; Schofield, Paul N.; Hoehndorf, Robert

    2017-01-01

    Discriminating the causative disease variant(s) for individuals with inherited or de novo mutations presents one of the main challenges faced by the clinical genetics community today. Computational approaches for variant prioritization include machine learning methods utilizing a large number of features, including molecular information, interaction networks, or phenotypes. Here, we demonstrate the PhenomeNET Variant Predictor (PVP) system that exploits semantic technologies and automated reasoning over genotype-phenotype relations to filter and prioritize variants in whole exome and whole genome sequencing datasets. We demonstrate the performance of PVP in identifying causative variants on a large number of synthetic whole exome and whole genome sequences, covering a wide range of diseases and syndromes. In a retrospective study, we further illustrate the application of PVP for the interpretation of whole exome sequencing data in patients suffering from congenital hypothyroidism. We find that PVP accurately identifies causative variants in whole exome and whole genome sequencing datasets and provides a powerful resource for the discovery of causal variants.

  2. Fundamental Characteristics of Industrial Variant Specification Systems

    DEFF Research Database (Denmark)

    Hansen, Benjamin Loer; Hvam, Lars

    2004-01-01

    fundamental concepts related to this task, which are relevant to understand for academia and practitioners working with the subject. This is done through a description of variant specification tasks and typical aspects of system solutions. To support the description of variant specification tasks and systems...

  3. Characterization of form variants of Xenorhabdus luminescens.

    NARCIS (Netherlands)

    Gerritsen, L.J.M.; Raay, de G.; Smits, P.H.

    1992-01-01

    From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in

  4. CLEVER: Clique-Enumerating Variant Finder

    NARCIS (Netherlands)

    Marschall, T.; Costa, I.; Canzar, S.; bauer, m; Klau, G.W.; Schliep, A.; Schönhuth, A.

    2012-01-01

    Motivation: Next-generation sequencing techniques have facilitated a large-scale analysis of human genetic variation. Despite the advances in sequencing speed, the computational discovery of structural variants is not yet standard. It is likely that many variants have remained undiscovered in most

  5. KSHV Rta promoter specification and viral reactivation

    Directory of Open Access Journals (Sweden)

    Jonathan eGuito

    2012-02-01

    Full Text Available Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent KSHV into the lytic infection cycle is called K-Rta. Rta is a transcriptional activator that specifies promoters by binding direct DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation.. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic CANT DNA repeats in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.

  6. Common Genetic Variants Found in HLA and KIR Immune Genes in Autism Spectrum Disorder

    Directory of Open Access Journals (Sweden)

    Anthony R Torres

    2016-10-01

    Full Text Available The common variant - common disease hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased versus matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the common variant—common disease hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics.Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14bp-indel frequencies are significantly increased by more than 5% over control populations (Table2. The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2. Three activating KIR genes: 3DS1, 2DS1 and 2DS2 have increased frequencies of 15%, 22% and 14% in autism populations, respectively. There is a 6% increase in total activating KIR

  7. Contagious Content: Viral Video Ads Identification of Content Characteristics that Help Online Video Advertisements Go Viral

    Directory of Open Access Journals (Sweden)

    Yentl Knossenburg

    2016-12-01

    Full Text Available Why do some online video advertisements go viral while others remain unnoticed? What kind of video content keeps the viewer interested and motivated to share? Many companies have realized the need to innovate their marketing strategies and have embraced the newest ways of using technology, as the Internet, to their advantage as in the example of virality. Yet few marketers actually understand how, and academic literature on this topic is still in development. This study investigated which content characteristics distinguish successful from non-successful online viral video advertisements by analyzing 641 cases using Structural Equation Modeling. Results show that Engagement and Surprise are two main content characteristics that significantly increase the chance of online video advertisements to go viral.  

  8. Variant Review with the Integrative Genomics Viewer.

    Science.gov (United States)

    Robinson, James T; Thorvaldsdóttir, Helga; Wenger, Aaron M; Zehir, Ahmet; Mesirov, Jill P

    2017-11-01

    Manual review of aligned reads for confirmation and interpretation of variant calls is an important step in many variant calling pipelines for next-generation sequencing (NGS) data. Visual inspection can greatly increase the confidence in calls, reduce the risk of false positives, and help characterize complex events. The Integrative Genomics Viewer (IGV) was one of the first tools to provide NGS data visualization, and it currently provides a rich set of tools for inspection, validation, and interpretation of NGS datasets, as well as other types of genomic data. Here, we present a short overview of IGV's variant review features for both single-nucleotide variants and structural variants, with examples from both cancer and germline datasets. IGV is freely available at https://www.igv.org Cancer Res; 77(21); e31-34. ©2017 AACR . ©2017 American Association for Cancer Research.

  9. Local binary patterns new variants and applications

    CERN Document Server

    Jain, Lakhmi; Nanni, Loris; Lumini, Alessandra

    2014-01-01

    This book introduces Local Binary Patterns (LBP), arguably one of the most powerful texture descriptors, and LBP variants. This volume provides the latest reviews of the literature and a presentation of some of the best LBP variants by researchers at the forefront of textual analysis research and research on LBP descriptors and variants. The value of LBP variants is illustrated with reported experiments using many databases representing a diversity of computer vision applications in medicine, biometrics, and other areas. There is also a chapter that provides an excellent theoretical foundation for texture analysis and LBP in particular. A special section focuses on LBP and LBP variants in the area of face recognition, including thermal face recognition. This book will be of value to anyone already in the field as well as to those interested in learning more about this powerful family of texture descriptors.

  10. Congenital anomalies and normal skeletal variants

    International Nuclear Information System (INIS)

    Guebert, G.M.; Yochum, T.R.; Rowe, L.J.

    1987-01-01

    Congenital anomalies and normal skeletal variants are a common occurrence in clinical practice. In this chapter a large number of skeletal anomalies of the spine and pelvis are reviewed. Some of the more common skeletal anomalies of the extremities are also presented. The second section of this chapter deals with normal skeletal variants. Some of these variants may simulate certain disease processes. In some instances there are no clear-cut distinctions between skeletal variants and anomalies; therefore, there may be some overlap of material. The congenital anomalies are presented initially with accompanying text, photos, and references, beginning with the skull and proceeding caudally through the spine to then include the pelvis and extremities. The normal skeletal variants section is presented in an anatomical atlas format without text or references

  11. Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

    Directory of Open Access Journals (Sweden)

    Samuel Campbell

    2018-04-01

    Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

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

    OpenAIRE

    Weinstein, David A.; Correia, Catherine E.; Conlon, Thomas; Specht, Andrew; Verstegen, John; Onclin-Verstegen, Karine; Campbell-Thompson, Martha; Dhaliwal, Gurmeet; Mirian, Layla; Cossette, Holly; Falk, Darin J.; Germain, Sean; Clement, Nathalie; Porvasnik, Stacy; Fiske, Laurie

    2010-01-01

    This study by the groups of Drs. Barry Byrne and Cathryn Mah at the University of Florida examines the safety and efficacy of AAV-mediated gene delivery in a canine model of glycogen storage disease type Ia (GSDIa). The authors find that intraportal delivery of AAV8 encoding glucose-6-phosphatase-α (G6Pase) followed 20 weeks later by intraportal administration of AAV1 encoding G6Pase led to significant correction of the GSDIa phenotype.

  13. Adeno-associated virus transformation into the normal miniature pig and the normal guinea pigs cochlea via scala tympani.

    Science.gov (United States)

    Shi, Xunbei; Wu, Nan; Zhang, Yue; Guo, Weiwei; Lin, Chang; Yang, Shiming

    2017-09-01

    To investigate the expression of the miniature pig cochlea after AAV1 transfect into the cochlea via round window membrane (RWM). Twenty miniature pigs are equally divided into four experimental groups. Twelve miniature pigs are equally divided into four control groups. Each pig was transfected with the AAV1 in the experimental group via RWM and each pig was transduced with the artificial perilymph in the control group. The expression of green fluorescent protein (GFP) was observed at 2 weeks, 3 weeks and 4 weeks, respectively. Likewise, AAV1 was delivered into the guinea pigs cochleas using the same method, and the results were compared with that of the miniature pigs. The expression was mainly in the inner hair cells of the miniature pig. The expression of GFP began to appear at 2 weeks, reached the peak at 3 weeks. It also expressed in Hensen's cells, inner pillar cells, outer pillar cells, spiral limbus, and spiral ligament. In the meanwhile, AAV1 was delivered into guinea pig cochlea via the same method, and AAV1 was also expressed in the inner hair cells. But the expression peaked at 2 weeks, and the efficiency of the inner hair cell transfection was higher than that of the pig. AAV1 can be transformed into miniature pig cochlea via scala tympani by the RWM method efficiently.

  14. Kinetics of viral shedding provide insights into the epidemiology of viral hemorrhagic septicemia in Pacific herring

    Science.gov (United States)

    Hershberger, Paul K.; Gregg, Jacob L.; Winton, James R.; Grady, Courtney; Collins, Rachael

    2010-01-01

    Losses from infectious diseases are an important component of natural mortality among marine fish species, but factors controlling the ecology of these diseases and their potential responses to anthropogenic changes are poorly understood. We used viral hemorrhagic septicemia virus (VHSV) and a laboratory stock of Pacific herring Clupea pallasii to investigate the kinetics of viral shedding and its effect on disease transmission and host mortality. Outbreaks of acute disease, accompanied by mortality and viral shedding, were initiated after waterborne exposure of herring to concentrations of VHSV as low as 101 plaque-forming units (pfu) ml–1. Shed virus in flow-through tanks was first detected 4 to 5 d post-exposure, peaked after 6 to 10 d, and was no longer detected after 16 d. Shedding rates, calculated from density, flow and waterborne virus titer reached 1.8 to 5.0 × 108 pfu fish–1 d–1. Onset of viral shedding was dose-dependent and preceded initial mortality by 2 d. At 21 d, cumulative mortality in treatment groups ranged from 81 to 100% and was dependent not on challenge dose, but on the kinetics and level of viral shedding by infected fish in the tank. Possible consequences of the viral shedding and disease kinetics are discussed in the context of epizootic initiation and perpetuation among populations of wild Pacific herring.

  15. CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shawkat; Tashkandi, Manal; Mahfouz, Magdy M.

    2015-01-01

    These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

  16. Disparities in HIV/AIDS, Viral Hepatitis, STDs, and TB

    Science.gov (United States)

    ... Search The CDC Health Disparities in HIV/AIDS, Viral Hepatitis, STDs, and TB Note: Javascript is disabled or ... Other Pacific Islanders MMWR Publications HIV and AIDS Viral Hepatitis STDs Tuberculosis Training and Networking Resources Call for ...

  17. Bim nuclear translocation and inactivation by viral interferon regulatory factor.

    Directory of Open Access Journals (Sweden)

    Young Bong Choi

    2010-08-01

    Full Text Available Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8 uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1-4, which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFbeta receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control

  18. Recombinant hybrid infectious hematopoietic necrosis virus (IHNV) carrying viral haemorrhagic septicaemia virus (VHSV) G or NV genes show different virulence properities

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Biacchesi, S.; Stegmann, Anders

    . By a reverse genetics approach using the related novirrhabdovirus infectious hematopoietic necrosis virus (IHNV) as basis, four hybrid IHNV-VHSV variants were generated. These chimeric variants included substitution of the IHNV glyco(G) or nonstrutrual (Nv) protein with the corresponding G or Nv-protein from......Viral haemorrhagic septicaemia virus (VHSV) is the economically most important viral disease in European rainbow trout farming. The virus was introduced to fresh water farms in the 1950ies from a reservoir of VHSV in the marine environment. Isolates from wild marine fish and fresh water farms...... are difficult to distinguish serologically but they show different virulence profiles: marine isolates typically cause little or no mortality in rainbow trout fry following experimental waterborne challenge, while freshwater isolates often kill the majority of the fish. Genetic analysis reveal that the change...

  19. Variantes de lesões intra-epiteliais escamosas: relato de quatro casos Variants of intraepithelial squamous lesions: report of four cases

    Directory of Open Access Journals (Sweden)

    Álvaro P. Pinto

    2005-04-01

    Full Text Available Entre a rotina de biópsias e produtos cirúrgicos provenientes do colo uterino, um número significativo de lesões intra-epiteliais escamosas (LIE pode causar dificuldade quanto a caracterização e graduação histológica. Tais lesões têm sido identificadas e descritas isoladamente por artigos científicos como variantes histológicas de LIE cervicais. São elas a metaplasia papilar imatura atípica (MPIA e as variantes de neoplasia intra-epitelial cervical graus II/III: queratinizante, com padrão metaplásico imaturo de crescimento e escamomucinosa. Neste artigo são exemplificados quatro casos representativos das entidades citadas acima, provenientes das rotinas do Programa de Prevenção do Câncer Ginecológico do Estado do Paraná e de um laboratório privado especializado em patologia ginecológica de Curitiba, o Laboratório de Citopatologia e Anatomia Patológica Annalab. Os principais critérios diagnósticos são descritos, assim como a correlação citológica e molecular relacionada à presença e à localização do ácido nucleico viral (papilomavírus humano [HPV] nas lesões.In routine basis, among biopsies and surgical specimens derived from uterine cervix, a significant number of squamous intraepithelial lesions (SIL may be difficult to diagnose and grade. Some of these lesions were identified, isolated and reported in scientific articles as histological variants of SIL. They are: metaplastic papillary immature atypia (MPIA and the following grade II/III cervical intraepithelial neoplasia variants: keratinized, immature metaplastic-like proliferation and mucin-producing. In this article four cases representative of these variants are described. They were retrieved from the routines of a large scale gynecological cancer screening program and a private laboratory specialized on gynecological pathology, both from Paraná State, Brazil. The main histological criteria for diagnosis are described, as well cytological and

  20. Somatic cancer variant curation and harmonization through consensus minimum variant level data

    Directory of Open Access Journals (Sweden)

    Deborah I. Ritter

    2016-11-01

    Full Text Available Abstract Background To truly achieve personalized medicine in oncology, it is critical to catalog and curate cancer sequence variants for their clinical relevance. The Somatic Working Group (WG of the Clinical Genome Resource (ClinGen, in cooperation with ClinVar and multiple cancer variant curation stakeholders, has developed a consensus set of minimal variant level data (MVLD. MVLD is a framework of standardized data elements to curate cancer variants for clinical utility. With implementation of MVLD standards, and in a working partnership with ClinVar, we aim to streamline the somatic variant curation efforts in the community and reduce redundancy and time burden for the interpretation of cancer variants in clinical practice. Methods We developed MVLD through a consensus approach by i reviewing clinical actionability interpretations from institutions participating in the WG, ii conducting extensive literature search of clinical somatic interpretation schemas, and iii survey of cancer variant web portals. A forthcoming guideline on cancer variant interpretation, from the Association of Molecular Pathology (AMP, can be incorporated into MVLD. Results Along with harmonizing standardized terminology for allele interpretive and descriptive fields that are collected by many databases, the MVLD includes unique fields for cancer variants such as Biomarker Class, Therapeutic Context and Effect. In addition, MVLD includes recommendations for controlled semantics and ontologies. The Somatic WG is collaborating with ClinVar to evaluate MVLD use for somatic variant submissions. ClinVar is an open and centralized repository where sequencing laboratories can report summary-level variant data with clinical significance, and ClinVar accepts cancer variant data. Conclusions We expect the use of the MVLD to streamline clinical interpretation of cancer variants, enhance interoperability among multiple redundant curation efforts, and increase submission of

  1. IL-10 and IL-28B gene variants as predictors of sustained response to peginterferon and ribavirin therapy in chronic HCV infection.

    Science.gov (United States)

    Sghaier, Ikram; Mouelhi, Leila; Rabia, Noor A; Ghazoueni, Ezzedine; Almawi, Wassim Y; Loueslati, Besma Yacoubi

    2017-04-05

    Interleukin-10 (IL-10) plays an important role in the immunity to hepatitis C virus (HCV). Insofar as IL-10 variants are associated with altered levels of IL-10, previous studies that examined the association of IL-10 polymorphisms with the susceptibility to and progression of chronic HCV, and response to anti-viral treatment were inconsistent. We investigated the association between common IL-10 variants in the intron and the promotor region with HCV and associated features. Study subjects comprised 120 patients infected with HCV-1b, and treated with Peg-IFN/RBV. Genotyping of six IL-10 promoter variants in the intron region (rs1878672, rs1554286, rs1518111) and promotor region (rs1800872, rs1800871, rs1800896) were done by real-time PCR. Compared to G/G, carriage of IL-10 rs1800896 (-1082A/G) A/A genotype was more frequent in patients with sustained virological response (SVR). The decline in viral load over the first 12weeks of treatment was more pronounced in rs1800896 A/A genotype carriers, compared to G/G genotype carriers, and was irrespective of the treatment dosage. Carriage of rs1800896 A/A genotype was positively associated with improvement in viral load decline, which was simultaneous, with and without carriage of the common favourable IL-28B variant. Carriage of both IL-10 rs1800896 G/G and IL-28B non-favourable genotype was associated with twice the risk of getting slow decline of viral load during treatment. Haploview analysis identified ACGCTA and CCGCTG haplotypes to be linked with excellent PegIFN/RBV cure rate, and complete HCV eradication. On the other hand, ACGCTG and CCGCTA haplotypes were associated with resistance to PegIFN/RBV treatment. IL-10 rs1800896 variant markedly influences the clinical outcome of HCV infection, and is a determinant of the response to HCV treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Von Willebrand Factor Gene Variants Associate with Herpes simplex Encephalitis.

    Directory of Open Access Journals (Sweden)

    Nada Abdelmagid

    Full Text Available Herpes simplex encephalitis (HSE is a rare complication of Herpes simplex virus type-1 infection. It results in severe parenchymal damage in the brain. Although viral latency in neurons is very common in the population, it remains unclear why certain individuals develop HSE. Here we explore potential host genetic variants predisposing to HSE. In order to investigate this we used a rat HSE model comparing the HSE susceptible SHR (Spontaneously Hypertensive Rats with the asymptomatic infection of BN (Brown Norway. Notably, both strains have HSV-1 spread to the CNS at four days after infection. A genome wide linkage analysis of 29 infected HXB/BXH RILs (recombinant inbred lines-generated from the prior two strains, displayed variable susceptibility to HSE enabling the definition of a significant QTL (quantitative trait locus named Hse6 towards the end of chromosome 4 (160.89-174Mb containing the Vwf (von Willebrand factor gene. This was the only gene in the QTL with both cis-regulation in the brain and included several non-synonymous SNPs (single nucleotide polymorphism. Intriguingly, in human chromosome 12 several SNPs within the intronic region between exon 43 and 44 of the VWF gene were associated with human HSE pathogenesis. In particular, rs917859 is nominally associated with an odds ratio of 1.5 (95% CI 1.11-2.02; p-value = 0.008 after genotyping in 115 HSE cases and 428 controls. Although there are possibly several genetic and environmental factors involved in development of HSE, our study identifies variants of the VWF gene as candidates for susceptibility in experimental and human HSE.

  3. Virale commercials: de consument als marketeer. Onderzoek naar de redenen waarom consumenten virale commercials doorsturen: hun motieven, de inhoudskenmerken van viral commercials en de mediumcontext waarin virale commercials verschijnen

    NARCIS (Netherlands)

    Ketelaar, P.E.; Lucassen, P.; Kregting, G.H.J.

    2010-01-01

    Research into the reasons why consumers pass along viral commercials: their motives, the content characteristics of viral commercials and the medium context in which viral commercials appear. Based on the uses and gratifications perspective this study has determined which motives of consumers,

  4. Synthesis of spatially variant lattices.

    Science.gov (United States)

    Rumpf, Raymond C; Pazos, Javier

    2012-07-02

    It is often desired to functionally grade and/or spatially vary a periodic structure like a photonic crystal or metamaterial, yet no general method for doing this has been offered in the literature. A straightforward procedure is described here that allows many properties of the lattice to be spatially varied at the same time while producing a final lattice that is still smooth and continuous. Properties include unit cell orientation, lattice spacing, fill fraction, and more. This adds many degrees of freedom to a design such as spatially varying the orientation to exploit directional phenomena. The method is not a coordinate transformation technique so it can more easily produce complicated and arbitrary spatial variance. To demonstrate, the algorithm is used to synthesize a spatially variant self-collimating photonic crystal to flow a Gaussian beam around a 90° bend. The performance of the structure was confirmed through simulation and it showed virtually no scattering around the bend that would have arisen if the lattice had defects or discontinuities.

  5. Different Variants of Fundamental Portfolio

    Directory of Open Access Journals (Sweden)

    Tarczyński Waldemar

    2014-06-01

    Full Text Available The paper proposes the fundamental portfolio of securities. This portfolio is an alternative for the classic Markowitz model, which combines fundamental analysis with portfolio analysis. The method’s main idea is based on the use of the TMAI1 synthetic measure and, in limiting conditions, the use of risk and the portfolio’s rate of return in the objective function. Different variants of fundamental portfolio have been considered under an empirical study. The effectiveness of the proposed solutions has been related to the classic portfolio constructed with the help of the Markowitz model and the WIG20 market index’s rate of return. All portfolios were constructed with data on rates of return for 2005. Their effectiveness in 2006- 2013 was then evaluated. The studied period comprises the end of the bull market, the 2007-2009 crisis, the 2010 bull market and the 2011 crisis. This allows for the evaluation of the solutions’ flexibility in various extreme situations. For the construction of the fundamental portfolio’s objective function and the TMAI, the study made use of financial and economic data on selected indicators retrieved from Notoria Serwis for 2005.

  6. Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

    Science.gov (United States)

    van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

    2017-10-01

    Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is

  7. Variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus.

    Science.gov (United States)

    Volle, Romain; Bailly, Jean-Luc; Mirand, Audrey; Pereira, Bruno; Marque-Juillet, Stéphanie; Chambon, Martine; Regagnon, Christel; Brebion, Amélie; Henquell, Cécile; Peigue-Lafeuille, Hélène; Archimbaud, Christine

    2014-08-15

    Acute enterovirus (EV) meningitis is a major cause of hospitalization among adults and children. It is caused by multiple EV genotypes assigned to 4 species (EV-A, EV-B, EV-C, and EV-D). We determined viral loads in the cerebrospinal fluid (CSF) of 156 patients of all ages with EV meningitis during a 5-year observational prospective study. The virus strains were genotyped, and their time origin was determined with Bayesian phylogenetic methods. The CSF viral loads ranged between 3.4 and 7.5 log10 copies/mL (median, 4.9 log10 copies/mL). They were higher in neonates than in infants and children (P = .02) but were comparable in adults. Viral loads were associated with EV genotypes (P < .001). The EV strains were identified in 152 of 156 patients and assigned to 23 genotypes within the EV-A and EV-B species. The most frequent genotypes, echoviruses 6 and 30, were associated with different viral loads (P < .001). The highest viral loads were in meningitis cases caused by coxsackievirus A9, B4, and B5 genotypes. Most patients infected by a same genotype were infected by a major virus variant of recent emergence. The variations in CSF viral loads in patients at the onset of EV meningitis are related to genotypic differences in the virus strains involved. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Viral subversion of the immune system

    International Nuclear Information System (INIS)

    Gillet, L.; Vanderplasschen, A.

    2005-01-01

    The continuous interactions between host and viruses during their co-evolution have shaped not only the immune system but also the countermeasures used by viruses. Studies in the last decade have described the diverse arrays of pathways and molecular targets that are used by viruses to elude immune detection or destruction, or both. These include targeting of pathways for major histocompatibility complex class I and class II antigen presentation, natural killer cell recognition, apoptosis, cytokine signalling, and complement activation. This paper provides an overview of the viral immune-evasion mechanisms described to date. It highlights the contribution of this field to our understanding of the immune system, and the importance of understanding this aspect of the biology of viral infection to develop efficacious and safe vaccines. (author)

  9. ASTHMA AND VIRAL INFECTIONS IN CHILDREN

    Directory of Open Access Journals (Sweden)

    D. Sh. Macharadze

    2014-01-01

    Full Text Available Viruses are the most common pathogens of acute respiratory diseases — most often causing mild symptoms of common cold: cough, runny nose, temperature increases. At the same time, 1/3 of children have the following symptoms of lower respiratory tract disorders: shortness of breath, wheezing, coughing, respiratory failure. Virus-induced wheezing are risk factors for development of asthma in childhood. Recent clinical and scientific data suggest: the more difficult are viral respiratory infections in young children, the higher their risk of asthma later on. Another feature is that children with allergic diseases are much more likely to have viral respiratory infections(and with longer clinical course, compared with children without atopy. The use of ibuprofen is safe for children over 3 months, including suffering from bronchial asthma.

  10. US findings in acute viral hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Corsi, M; Lorenzon, G; Mesaglio, S

    1988-01-01

    Reports on colecystic alterations during acute viral hepatitis are more and more frequent; the pathogenesis and clinical meaning of these alterations are still debated. Consensual periportal lymphnode enlargment has been not yet reported. The authors describe four cases of acute viral hepatites in whichUS showed alterations of colecystic walls and/or contents; in two cases enlarged periportal lymphnodes were demonstrated too. Later US exams showed a complete regression of both colecystic and lymphnodal lesions. Clinical findings and laboratory out-comes are evaluated; the connection of US results with hepatitis and its meaning are discussed. The causes of colecystic alterations are still questionable; they might be related to blood disorders or to an increased portal pressure, or else they might be considered as phlogistic lesions. The authors conclude that both colecystic and lymphnodal alterations have a phlogistic nature; moreover, they are not related to a particulary evolution of hepatitis. The importance of distinguishing colecystic alterations from different pathology is stressed.

  11. Annotation of selection strengths in viral genomes

    DEFF Research Database (Denmark)

    McCauley, Stephen; de Groot, Saskia; Mailund, Thomas

    2007-01-01

    Motivation: Viral genomes tend to code in overlapping reading frames to maximize information content. This may result in atypical codon bias and particular evolutionary constraints. Due to the fast mutation rate of viruses, there is additional strong evidence for varying selection between intra......- and intergenomic regions. The presence of multiple coding regions complicates the concept of Ka/Ks ratio, and thus begs for an alternative approach when investigating selection strengths. Building on the paper by McCauley & Hein (2006), we develop a method for annotating a viral genome coding in overlapping...... may thus achieve an annotation both of coding regions as well as selection strengths, allowing us to investigate different selection patterns and hypotheses. Results: We illustrate our method by applying it to a multiple alignment of four HIV2 sequences, as well as four Hepatitis B sequences. We...

  12. US findings in acute viral hepatitis

    International Nuclear Information System (INIS)

    Corsi, M.; Lorenzon, G.; Mesaglio, S.

    1988-01-01

    Reports on colecystic alterations during acute viral hepatitis are more and more frequent; the pathogenesis and clinical meaning of these alterations are still debated. Consensual periportal lymphnode enlargment has been not yet reported. The authors describe four cases of acute viral hepatites in whichUS showed alterations of colecystic walls and/or contents; in two cases enlarged periportal lymphnodes were demonstrated too. Later US exams showed a complete regression of both colecystic and lymphnodal lesions. Clinical findings and laboratory out-comes are evaluated; the connection of US results with hepatitis and its meaning are discussed. The causes of colecystic alterations are still questionable; they might be related to blood disorders or to an increased portal pressure, or else they might be considered as phlogistic lesions. The authors conclude that both colecystic and lymphnodal alterations have a phlogistic nature; moreover, they are not related to a particulary evolution of hepatitis. The importance of distinguishing colecystic alterations from different pathology is stressed

  13. Multiplexing Short Primers for Viral Family PCR

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

    2008-06-26

    We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

  14. Immunological features underlying viral hemorrhagic fevers.

    Science.gov (United States)

    Messaoudi, Ilhem; Basler, Christopher F

    2015-10-01

    Several enveloped RNA viruses of the arenavirus, bunyavirus, filovirus and flavivirus families are associated with a syndrome known as viral hemorrhagic fever (VHF). VHF is characterized by fever, vascular leakage, coagulation defects and multi organ system failure. VHF is currently viewed as a disease precipitated by viral suppression of innate immunity, which promotes systemic virus replication and excessive proinflammatory cytokine responses that trigger the manifestations of severe disease. However, the mechanisms by which immune dysregulation contributes to disease remain poorly understood. Infection of nonhuman primates closely recapitulates human VHF, notably Ebola and yellow fever, thereby providing excellent models to better define the immunological basis for this syndrome. Here we review the current state of our knowledge and suggest future directions that will better define the immunological mechanisms underlying VHF. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Global issues related to enteric viral infections.

    Science.gov (United States)

    Desselberger, Ulrich

    2014-01-01

    Acute viral gastroenteritis is a major health issue worldwide and is associated with high annual mortality, particularly in children of developing countries. Rotaviruses, caliciviruses and astroviruses are the main causes. Accurate diagnoses are possible by recently developed molecular techniques. In many setups, zoonotic transmission is an important epidemiological factor. Treatment consists of rehydration and is otherwise symptomatic. The worldwide introduction of universal rotavirus vaccination of infants has significantly reduced rotavirus disease and mortality.

  16. Vaginal microbiota and viral sexually transmitted diseases.

    Science.gov (United States)

    Nardis, C; Mosca, L; Mastromarino, P

    2013-01-01

    Healthy vaginal microbiota is an important biological barrier to pathogenic microorganisms. When this predominantly Lactobacillus community is disrupted, decreased in abundance and replaced by different anaerobes, bacterial vaginosis (BV) may occur. BV is associated with prevalence and incidence of several sexually transmitted infections. This review provides background on BV, discusses the epidemiologic data to support a role of altered vaginal microbiota for acquisition of sexually transmitted diseases and analyzes mechanisms by which lactobacilli could counteract sexually transmitted viral infections.

  17. Bacterial, Fungal, Parasitic, and Viral Myositis

    OpenAIRE

    Crum-Cianflone, Nancy F.

    2008-01-01

    Infectious myositis may be caused by a broad range of bacterial, fungal, parasitic, and viral agents. Infectious myositis is overall uncommon given the relative resistance of the musculature to infection. For example, inciting events, including trauma, surgery, or the presence of foreign bodies or devitalized tissue, are often present in cases of bacterial myositis. Bacterial causes are categorized by clinical presentation, anatomic location, and causative organisms into the categories of pyo...

  18. The epidemiology of viral hepatitis in Qatar

    Directory of Open Access Journals (Sweden)

    Bener Abdulbari

    2009-01-01

    Full Text Available Viral hepatitis is a major public health problem in many countries all over the world and especially in Middle East, Asia, East-Europe, and Africa. The aim of our study was to assess the incidence of viral hepatitis A, B and C in Qatar and compare it with other countries. This is a retrospective cohort study, which was conducted at Hamad General Hospital, State of Qatar from 2002-2006. Patients who were screened and diagnosed with viral hepatitis were included in this study. The diagnostic classification of definite viral hepatitis was made in accordance with criteria based on the International Classification of Disease tenth revision (ICD-10. A total of 527 cases of hepatitis C, 396 cases of hepatitis B, 162 cases of hepatitis A and 108 cases of unspecified were reported during the year 2006. Reported incidence rate per 10,000 populations during the year 2006 for hepatitis A was 1.9, hepatitis B 4.7, and Hepatitis C 6.3. The proportion of hepatitis B and C was significantly higher in male population than females across the years (2002-2006. Hepatitis A was more prevalent in children below 15 years (72.3%, hepatitis B in adults aged above 15 years, and hepatitis C in the population above 35 years of age. The incidence of hepatitis A has been declining in Qataris and increasing in expatriates. There was a significant relationship in gender and age group of the patients with hepatitis A, B and C. We conclude that hepatitis has become a national health issue in Qatar. The incidence rate of hepatitis in Qatar is comparable to its neighboring countries, United Arab Emirates and Saudi Arabia. There is a need for further research on hepatitis and the associated risk factors.

  19. Ultrasonographic imaging of papillary thyroid carcinoma variants

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jung Hee [Dept. of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2017-04-15

    Ultrasonography (US) is routinely used to evaluate thyroid nodules. The US features of papillary thyroid carcinoma (PTC), the most common thyroid malignancy, include hypoechogenicity, spiculated/microlobulated margins, microcalcifications, and a nonparallel orientation. However, many PTC variants have been identified, some of which differ from the classic type of PTC in terms of biological behavior and clinical outcomes. This review describes the US features and clinical implications of the variants of PTC. With the introduction of active surveillance replacing immediate biopsy or surgical treatment of indolent, small PTCs, an understanding of the US characteristics of PTC variants will facilitate the individualized management of patients with PTC.

  20. Biological species in the viral world.

    Science.gov (United States)

    Bobay, Louis-Marie; Ochman, Howard

    2018-06-05

    Due to their dependence on cellular organisms for metabolism and replication, viruses are typically named and assigned to species according to their genome structure and the original host that they infect. But because viruses often infect multiple hosts and the numbers of distinct lineages within a host can be vast, their delineation into species is often dictated by arbitrary sequence thresholds, which are highly inconsistent across lineages. Here we apply an approach to determine the boundaries of viral species based on the detection of gene flow within populations, thereby defining viral species according to the biological species concept (BSC). Despite the potential for gene transfer between highly divergent genomes, viruses, like the cellular organisms they infect, assort into reproductively isolated groups and can be organized into biological species. This approach revealed that BSC-defined viral species are often congruent with the taxonomic partitioning based on shared gene contents and host tropism, and that bacteriophages can similarly be classified in biological species. These results open the possibility to use a single, universal definition of species that is applicable across cellular and acellular lifeforms.

  1. Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard

    2004-01-01

    Induction of a monospecific antiviral CD8+ T cell response may pose a risk to the host due to the narrow T cell response induced. At the individual level, this may result in selection of CD8+ T cell escape variants, particularly during chronic viral infection. Second, prior immunization toward a ...... with escape variants. These findings underscore that a monospecific vaccine may induce efficient protective immunity given the right set of circumstances....... of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral...... variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after...

  2. eCD4-Ig variants that more potently neutralize HIV-1.

    Science.gov (United States)

    Fetzer, Ina; Gardner, Matthew R; Davis-Gardner, Meredith E; Prasad, Neha R; Alfant, Barnett; Weber, Jesse A; Farzan, Michael

    2018-03-28

    The HIV-1 entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralizes all HIV-1, HIV-2 and SIV isolates that it has been tested against, suggesting that it may be useful in clinical settings where antibody escape is a concern. Here we characterize three new eCD4-Ig variants, each with different architectures and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig, and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications. IMPORTANCE HIV-1 bNAbs have properties different from antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral lifecycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the

  3. Role of viral coinfections in asthma development.

    Directory of Open Access Journals (Sweden)

    Maria Luz Garcia-Garcia

    Full Text Available Viral respiratory infections, especially acute bronchiolitis, play a key role in the development of asthma in childhood. However, most studies have focused on respiratory syncytial virus or rhinovirus infections and none of them have compared the long-term evolution of single versus double or multiple viral infections.Our aim was to compare the frequency of asthma development at 6-8 years in children with previous admission for bronchiolitis associated with single versus double or multiple viral infection.A cross-sectional study was performed in 244 children currently aged 6-8 years, previously admitted due to bronchiolitis between September 2008 and December 2011. A structured clinical interview and the ISAAC questionnaire for asthma symptoms for 6-7-year-old children, were answered by parents by telephone. Specimens of nasopharyngeal aspirate for virological study (polymerase chain reaction and clinical data were prospectively taken during admission for bronchiolitis.Median current age at follow-up was 7.3 years (IQR: 6.7-8.1. The rate of recurrent wheezing was 82.7% in the coinfection group and 69.7% in the single-infection group, p = 0.06. The number of wheezing-related admissions was twice as high in coinfections than in single infections, p = 0.004. Regarding the ISAAC questionnaire, 30.8% of coinfections versus 15% of single infections, p = 0.01, presented "wheezing in the last 12 months", data that strongly correlate with current prevalence of asthma. "Dry cough at night" was also reported more frequently in coinfections than in single infections, p = 0.02. The strongest independent risk factors for asthma at 6-8 years of age were: age > 9 months at admission for bronchiolitis (OR: 3.484; CI95%: 1.459-8.317, p:0.005, allergic rhinitis (OR: 5.910; 95%CI: 2.622-13.318, p<0.001, and viral coinfection-bronchiolitis (OR: 3.374; CI95%: 1.542-7.386, p:0.01.Asthma at 6-8 years is more frequent and severe in those children previously hospitalized

  4. RAGE splicing variants in mammals.

    Science.gov (United States)

    Sterenczak, Katharina Anna; Nolte, Ingo; Murua Escobar, Hugo

    2013-01-01

    The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimer's disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

  5. Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity.

    Directory of Open Access Journals (Sweden)

    Katja Pfafferott

    2011-02-01

    Full Text Available Cellular immune responses during acute Hepatitis C virus (HCV and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%. The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

  6. Isolation of a variant of Candida albicans.

    Science.gov (United States)

    Buckley, H R; Price, M R; Daneo-Moore, L

    1982-01-01

    During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell. Images PMID:6752021

  7. Isolation of a variant of Candida albicans.

    Science.gov (United States)

    Buckley, H R; Price, M R; Daneo-Moore, L

    1982-09-01

    During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell.

  8. Genetic variants of ghrelin in metabolic disorders.

    Science.gov (United States)

    Ukkola, Olavi

    2011-11-01

    An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual's risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In cross-sectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. TREM2 Variants in Alzheimer's Disease

    Science.gov (United States)

    Guerreiro, Rita; Wojtas, Aleksandra; Bras, Jose; Carrasquillo, Minerva; Rogaeva, Ekaterina; Majounie, Elisa; Cruchaga, Carlos; Sassi, Celeste; Kauwe, John S.K.; Younkin, Steven; Hazrati, Lilinaz; Collinge, John; Pocock, Jennifer; Lashley, Tammaryn; Williams, Julie; Lambert, Jean-Charles; Amouyel, Philippe; Goate, Alison; Rademakers, Rosa; Morgan, Kevin; Powell, John; St. George-Hyslop, Peter; Singleton, Andrew; Hardy, John

    2013-01-01

    BACKGROUND Homozygous loss-of-function mutations in TREM2, encoding the triggering receptor expressed on myeloid cells 2 protein, have previously been associated with an autosomal recessive form of early-onset dementia. METHODS We used genome, exome, and Sanger sequencing to analyze the genetic variability in TREM2 in a series of 1092 patients with Alzheimer's disease and 1107 controls (the discovery set). We then performed a meta-analysis on imputed data for the TREM2 variant rs75932628 (predicted to cause a R47H substitution) from three genomewide association studies of Alzheimer's disease and tested for the association of the variant with disease. We genotyped the R47H variant in an additional 1887 cases and 4061 controls. We then assayed the expression of TREM2 across different regions of the human brain and identified genes that are differentially expressed in a mouse model of Alzheimer's disease and in control mice. RESULTS We found significantly more variants in exon 2 of TREM2 in patients with Alzheimer's disease than in controls in the discovery set (P = 0.02). There were 22 variant alleles in 1092 patients with Alzheimer's disease and 5 variant alleles in 1107 controls (P<0.001). The most commonly associated variant, rs75932628 (encoding R47H), showed highly significant association with Alzheimer's disease (P<0.001). Meta-analysis of rs75932628 genotypes imputed from genomewide association studies confirmed this association (P = 0.002), as did direct genotyping of an additional series of 1887 patients with Alzheimer's disease and 4061 controls (P<0.001). Trem2 expression differed between control mice and a mouse model of Alzheimer's disease. CONCLUSIONS Heterozygous rare variants in TREM2 are associated with a significant increase in the risk of Alzheimer's disease. (Funded by Alzheimer's Research UK and others.) PMID:23150934

  10. ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees

    Directory of Open Access Journals (Sweden)

    Jessica L. Kevill

    2017-10-01

    Full Text Available Deformed wing virus (DWV is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL 2006–2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

  11. Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

    Directory of Open Access Journals (Sweden)

    Duborjal Hervé

    2008-02-01

    Full Text Available Abstract Background Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus. Results Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population. Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1. Conclusion This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized

  12. Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma

    Directory of Open Access Journals (Sweden)

    Bauer-Pham Kim

    2010-02-01

    Full Text Available Abstract Background In a cat that had ostensibly recovered from feline leukemia virus (FeLV infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. Results The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1. In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. Conclusions Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

  13. Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir.

    Directory of Open Access Journals (Sweden)

    Xiomara V Thomas

    Full Text Available BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103 were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.

  14. VIRAL ETIOLOGY OF RECURRENT URINARY TRACT INFECTIONS

    Directory of Open Access Journals (Sweden)

    H. S. Ibishev

    2017-01-01

    Full Text Available Introduction. Recurrent urinary tract infection is an actual problem of modern urology.Objective. Complex investigation of urinary tract infections including viral etiology for chronic recurrent cystitis in womenMaterials and methods. The study included 31 women with recurrent infection of urinary tract. Inclusion criteria were the presence of lower urinary tract symptoms caused by infection, severe recurrent course, the lack of anatomical and functional disorders of the urinary tract, the absence of bacterial pathogens during the study, taking into account the culture of aerobic and anaerobic culturing techniques.Results. The analysis of the clinical manifestations, the dominant in the study group were pain and urgency to urinate at 100% and 90% of women surveyed, respectively, and less frequent urination were recorded in 16.1% of patients. In general clinical examination of urine in all cases identified leukocyturia and 90% of the hematuria. By using a polymerase chain reaction (PCR in midstream urine of all examined was verified 10 types of human papilloma virus (HPV with the predominance of 16 and 18 types . Considering the presence of recurrent infectious and inflammatory processes of the urinary tract, cystoscopy with bladder biopsy was performed for all patients. When histomorphological biopsies of all patients surveyed noted the presence of the specific characteristics of HPV: papillary hyperplasia with squamous koilocytosis, pale cytoplasm and shrunken kernels. When analyzing the results of PCR biopsy data corresponded with the results of PCR in midstream urine in all biopsies was detected HPV.Conclusions. Human papillomavirus infection may be involved in the development of viral cystitis. In the etiological structure of viral cystitis, both highly oncogenic and low oncogenic HPV types can act.

  15. Beta-glucosidase variants and polynucleotides encoding same

    Science.gov (United States)

    Wogulis, Mark; Harris, Paul; Osborn, David

    2017-06-27

    The present invention relates to beta-glucosidase variants, e.g. beta-glucosidase variants of a parent Family GH3A beta-glucosidase from Aspergillus fumigatus. The present invention also relates to polynucleotides encoding the beta-glucosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the beta-glucosidase variants.

  16. Flavonoids: promising natural compounds against viral infections.

    Science.gov (United States)

    Zakaryan, Hovakim; Arabyan, Erik; Oo, Adrian; Zandi, Keivan

    2017-09-01

    Flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. Different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. This review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. We also present future perspectives on therapeutic applications of flavonoids against viral infections.

  17. [Decimeter-wave physiotherapy in viral hepatitis].

    Science.gov (United States)

    Kents, V V; Mavrodiĭ, V M

    1995-01-01

    Effectiveness was evaluated of magnetotherapy, inductothermy, UNF electric field and electromagnetic waves of decimetric wave band (460 MHz) on the projection of the liver, adrenals and thyroid gland in controlled trials enrolling a total of 835 patients with viral hepatitis (type A, B, associated forms). A conclusion is reached that optimum effectiveness of decimetric field on the projection of the adrenals and thyroid gland can be achieved through the application of minimum power and everyday alternation of exposures. It has been estimated that as many as 69 percent of the patients derive benefit from the above treatment.

  18. Content Recommendation for Viral Social Influence

    DEFF Research Database (Denmark)

    Ivanov, Sergei; Theocharidis, Konstantinos; Terrovitis, Manolis

    2017-01-01

    How do we create content that will become viral in a whole network after we share it with friends or followers? Significant research activity has been dedicated to the problem of strategically selecting a seed set of initial adopters so as to maximize a meme’s spread in a network. This line of work...... assumes that the success of such a campaign depends solely on the choice of a tunable seed set of adopters, while the way users perceive the propagated meme is fixed. Yet, in many real-world settings, the opposite holds: a meme’s propagation depends on users’ perceptions of its tunable characteristics...

  19. Functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma.

    Directory of Open Access Journals (Sweden)

    Lisa J Martin

    Full Text Available Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated.Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP.We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively. In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05. We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls.Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

  20. Within-Host Variations of Human Papillomavirus Reveal APOBEC-Signature Mutagenesis in the Viral Genome.

    Science.gov (United States)

    Hirose, Yusuke; Onuki, Mamiko; Tenjimbayashi, Yuri; Mori, Seiichiro; Ishii, Yoshiyuki; Takeuchi, Takamasa; Tasaka, Nobutaka; Satoh, Toyomi; Morisada, Tohru; Iwata, Takashi; Miyamoto, Shingo; Matsumoto, Koji; Sekizawa, Akihiko; Kukimoto, Iwao

    2018-03-28

    Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied with the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here we explored within-host genetic diversity of HPV by performing deep sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52 and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), and were deep-sequenced. After constructing a reference vial genome sequence for each specimen, nucleotide positions showing changes with > 0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with varying numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the tri-nucleotides context encompassing substituted bases revealed that Tp C pN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions. IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep sequencing analyses, we show for the first time a comprehensive snapshot of the "within

  1. Persistent viral infections and immune aging.

    Science.gov (United States)

    Brunner, Stefan; Herndler-Brandstetter, Dietmar; Weinberger, Birgit; Grubeck-Loebenstein, Beatrix

    2011-07-01

    Immunosenescence comprises a set of dynamic changes occurring to both, the innate as well as the adaptive immune system that accompany human aging and result in complex manifestations of still poorly defined deficiencies in the elderly population. One of the most prominent alterations during aging is the continuous involution of the thymus gland which is almost complete by the age of 50. Consequently, the output of naïve T cells is greatly diminished in elderly individuals which puts pressure on homeostatic forces to maintain a steady T cell pool for most of adulthood. In a great proportion of the human population, this fragile balance is challenged by persistent viral infections, especially Cytomegalovirus (CMV), that oblige certain T cell clones to monoclonally expand repeatedly over a lifetime which then occupy space within the T cell pool. Eventually, these inflated memory T cell clones become exhausted and their extensive accumulation accelerates the age-dependent decline of the diversity of the T cell pool. As a consequence, infectious diseases are more frequent and severe in elderly persons and immunological protection following vaccination is reduced. This review therefore aims to shed light on how various types of persistent viral infections, especially CMV, influence the aging of the immune system and highlight potential measures to prevent the age-related decline in immune function. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. [Viral encephalitis virus, a new bioterrorist menace].

    Science.gov (United States)

    Rigaudeau, Sophie; Micol, Romain; Bricaire, François; Bossi, Philippe

    2005-01-29

    Often responsible for little known infections, today viral encephalitis viruses appear as a new bioterrorist menace, because of their easy production and their great pathogenic potential. Spraying is the best way to permit the rapid diffusion of certain encephalitis viruses. Diagnosis of viral encephalitis, predominating in tropical surroundings, is difficult. In the majority of cases, symptoms differ little from those of common flu. With supplementary examinations, the biological abnormalities are usually non-specific. There are no characteristic images on scans or MRI. Identification of the virus in the nasopharynx, blood or cerebrospinal fluid, in serology, PCR or RT-PCR permits confirmation of the virus. Treatment is essentially symptomatic and relies on appropriate reanimation measures. Ribavirin can be indicated in some cases such as the Rift Valley fever, but is formally contraindicated in West Nile encephalitis. The aim of terrorist groups who would use this type of weapon is more to provoke panic and disorganisation than to kill as many people as possible.

  3. Endogenous viral elements in animal genomes.

    Directory of Open Access Journals (Sweden)

    Aris Katzourakis

    2010-11-01

    Full Text Available Integration into the nuclear genome of germ line cells can lead to vertical inheritance of retroviral genes as host alleles. For other viruses, germ line integration has only rarely been documented. Nonetheless, we identified endogenous viral elements (EVEs derived from ten non-retroviral families by systematic in silico screening of animal genomes, including the first endogenous representatives of double-stranded RNA, reverse-transcribing DNA, and segmented RNA viruses, and the first endogenous DNA viruses in mammalian genomes. Phylogenetic and genomic analysis of EVEs across multiple host species revealed novel information about the origin and evolution of diverse virus groups. Furthermore, several of the elements identified here encode intact open reading frames or are expressed as mRNA. For one element in the primate lineage, we provide statistically robust evidence for exaptation. Our findings establish that genetic material derived from all known viral genome types and replication strategies can enter the animal germ line, greatly broadening the scope of paleovirological studies and indicating a more significant evolutionary role for gene flow from virus to animal genomes than has previously been recognized.

  4. Detection of viral hemorrhagic septicemia virus

    Science.gov (United States)

    Winton, James; Kurath, Gael; Batts, William

    2007-01-01

    Viral hemorrhagic septicemia virus (VHSV) is considered to be one of the most important viral pathogens of finfish and is listed as reportable by many nations and international organizations (Office International des Epizooties 2006). Prior to 1988, VHSV was thought to be limited to Europe (Wolf 1988; Smail 1999). Subsequently, it was shown that the virus is endemic among many marine and anadromous fish species in both the Pacific and Atlantic Oceans (Meyers and Winton 1995; Skall et al. 2005). Genetic analysis reveals that isolates of VHSV can be divided into four genotypes that generally correlate with geographic location with the North American isolates generally falling into VHSV Genotype IV (Snow et al. 2004). In 2005-2006, reports from the Great Lakes region indicated that wild fish had experienced disease or, in some cases, very large die-offs from VHSV (Elsayed et al. 2006, Lumsden et al. 2007). The new strain from the Great Lakes, now identified as VHSV Genotype IVb, appears most closely related to isolates of VHSV from mortalities that occurred during 2000-2004 in rivers and near-shore areas of New Brunswick and Nova Scotia, Canada (Gagne et al. 2007). The type IVb isolate found in the Great Lakes region is the only strain outside of Europe that has been associated with significant mortality in freshwater species.

  5. Viral asthma: implications for clinical practice

    Directory of Open Access Journals (Sweden)

    Roger Menendez

    2010-07-01

    Full Text Available Roger Menendez1, Michael D Goldman21Allergy and Asthma Center of El Paso, El Paso, TX, USA; 2Pulmonary Division, UCLA Gaffen School of Medicine, Los Angeles, CA, USAAbstract: The natural history of asthma appears to be driven primarily by the timing and duration of viral respiratory infections. From the very high rate of infections in childhood, to the more sporadic pattern seen in adults, the cycle of acute injury followed by an inefficient repair process helps explain the clinical patterns of asthma severity currently recognized by asthma guidelines. Why the asthmatic host responds to viral injury in a particular way is largely a mystery and the subject of intense investigation. The role of viruses in asthma extends not just to intermittent but to persistent disease, and to both the atopic as well as nonatopic phenotypes. Future therapeutic strategies should include primary prevention via the development of antiviral innate immunity-enhancing vaccines, as well as secondary prevention via the use of antiviral agents, or immunomodulators designed to boost the antiviral response or interrupt the proinflammatory cascade.Keywords: asthma, rhinoviruses, exacerbations, epidemiology, phenotypes, clinical trials

  6. Viral respiratory diseases: vaccines and antivirals.

    Science.gov (United States)

    Lennette, E H

    1981-01-01

    Acute respiratory diseases, most of which are generally attributed to viruses, account for about 6% of all deaths and for about 60% of the deaths associated with all respiratory disease. The huge cost attributable to viral respiratory infections as a result of absenteeism and the disruption of business and the burden of medical care makes control of these diseases an important objective. The viruses that infect the respiratory tract fall taxonomically into five viral families. Although immunoprophylaxis would appear to be the logical approach, the development of suitable vaccines has been confronted with numerous obstacles, including antigenic drift and shift in the influenzaviruses, the large number of antigenically distinct immunotypes among rhinoviruses, the occurrence after immunization of rare cases of a severe form of the disease following subsequent natural infection with respiratory syncytial virus, and the risk of oncogenicity of adenoviruses for man. Considerable expenditure on the development of new antiviral drugs has so far resulted in only three compounds that are at present officially approved and licensed for use in the USA. Efforts to improve the tools available for control should continue and imaginative and inventive approaches are called for. However, creativity and ingenuity must operate within the constraints imposed by economic, political, ethical, and legal considerations.

  7. Word Variant Identification in Old French

    Directory of Open Access Journals (Sweden)

    Peter Willett

    1997-01-01

    Full Text Available Increasing numbers of historical texts are available in machine-readable form, which retain the original spelling, which can be very different from the modern-day equivalents due to the natural evolution of a language, and because the concept of standardisation in spelling is comparatively modern. Among medieval vernacular writers, the same word could be spelled in different ways and the same author (or scribe might even use several alternative spellings in the same passage. Thus, we do not know,a priori, how many variant forms of a particular word there are in such texts, let alone what these variants might be. Searching on the modern equivalent, or even the commonest historical variant, of a particular word may thus fail to retrieve an appreciable number of occurrences unless the searcher already has an extensive knowledge of the language of the documents. Moreover, even specialist scholars may be unaware of some idiosyncratic variants. Here, we consider the use of computer methods to retrieve variant historical spellings.

  8. Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

    DEFF Research Database (Denmark)

    Ahlborn, Lise B; Dandanell, Mette; Steffensen, Ane Y

    2015-01-01

    by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c......Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently...... needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined...

  9. The Ins and Outs of Viral Infection: Keystone Meeting Review

    Directory of Open Access Journals (Sweden)

    Sara W. Bird

    2014-09-01

    Full Text Available Newly observed mechanisms for viral entry, assembly, and exit are challenging our current understanding of the replication cycle of different viruses. To address and better understand these mechanisms, a Keystone Symposium was organized in the snowy mountains of Colorado (“The Ins and Outs of Viral Infection: Entry, Assembly, Exit, and Spread”; 30 March–4 April 2014, Beaver Run Resort, Breckenridge, Colorado, organized by Karla Kirkegaard, Mavis Agbandje-McKenna, and Eric O. Freed. The meeting served to bring together cell biologists, structural biologists, geneticists, and scientists expert in viral pathogenesis to discuss emerging mechanisms of viral ins and outs. The conference was organized around different phases of the viral replication cycle, including cell entry, viral assembly and post-assembly maturation, virus structure, cell exit, and virus spread. This review aims to highlight important topics and themes that emerged during the conference.

  10. APOBEC3 Interference during Replication of Viral Genomes

    Directory of Open Access Journals (Sweden)

    Luc Willems

    2015-06-01

    Full Text Available Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  11. Molecular Evolution of the VP1 Gene in Human Norovirus GII.4 Variants in 1974–2015

    Directory of Open Access Journals (Sweden)

    Takumi Motoya

    2017-12-01

    Full Text Available Human norovirus (HuNoV is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1 gene sequences (466 strains to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10−3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1, shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly

  12. Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

    Science.gov (United States)

    Hoover, Spencer; Striker, Rob

    2008-04-01

    The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

  13. Characterization of the Copy Number and Variants of Deformed Wing Virus (DWV in the Pairs of Honey Bee Pupa and Infesting Varroa destructor or Tropilaelaps mercedesae

    Directory of Open Access Journals (Sweden)

    Yunfei Wu

    2017-08-01

    Full Text Available Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV. Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor. These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication.

  14. Sensitive detection of viral transcripts in human tumor transcriptomes.

    Directory of Open Access Journals (Sweden)

    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  15. TCR stimulation strength is inversely associated with establishment of functional brain-resident memory CD8 T cells during persistent viral infection.

    Science.gov (United States)

    Maru, Saumya; Jin, Ge; Schell, Todd D; Lukacher, Aron E

    2017-04-01

    Establishing functional tissue-resident memory (TRM) cells at sites of infection is a newfound objective of T cell vaccine design. To directly assess the impact of antigen stimulation strength on memory CD8 T cell formation and function during a persistent viral infection, we created a library of mouse polyomavirus (MuPyV) variants with substitutions in a subdominant CD8 T cell epitope that exhibit a broad range of efficiency in stimulating TCR transgenic CD8 T cells. By altering a subdominant epitope in a nonstructural viral protein and monitoring memory differentiation of donor monoclonal CD8 T cells in immunocompetent mice, we circumvented potentially confounding changes in viral infection levels, virus-associated inflammation, size of the immunodominant virus-specific CD8 T cell response, and shifts in TCR affinity that may accompany temporal recruitment of endogenous polyclonal cells. Using this strategy, we found that antigen stimulation strength was inversely associated with the function of memory CD8 T cells during a persistent viral infection. We further show that CD8 TRM cells recruited to the brain following systemic infection with viruses expressing epitopes with suboptimal stimulation strength respond more efficiently to challenge CNS infection with virus expressing cognate antigen. These data demonstrate that the strength of antigenic stimulation during recruitment of CD8 T cells influences the functional integrity of TRM cells in a persistent viral infection.

  16. TCR stimulation strength is inversely associated with establishment of functional brain-resident memory CD8 T cells during persistent viral infection.

    Directory of Open Access Journals (Sweden)

    Saumya Maru

    2017-04-01

    Full Text Available Establishing functional tissue-resident memory (TRM cells at sites of infection is a newfound objective of T cell vaccine design. To directly assess the impact of antigen stimulation strength on memory CD8 T cell formation and function during a persistent viral infection, we created a library of mouse polyomavirus (MuPyV variants with substitutions in a subdominant CD8 T cell epitope that exhibit a broad range of efficiency in stimulating TCR transgenic CD8 T cells. By altering a subdominant epitope in a nonstructural viral protein and monitoring memory differentiation of donor monoclonal CD8 T cells in immunocompetent mice, we circumvented potentially confounding changes in viral infection levels, virus-associated inflammation, size of the immunodominant virus-specific CD8 T cell response, and shifts in TCR affinity that may accompany temporal recruitment of endogenous polyclonal cells. Using this strategy, we found that antigen stimulation strength was inversely associated with the function of memory CD8 T cells during a persistent viral infection. We further show that CD8 TRM cells recruited to the brain following systemic infection with viruses expressing epitopes with suboptimal stimulation strength respond more efficiently to challenge CNS infection with virus expressing cognate antigen. These data demonstrate that the strength of antigenic stimulation during recruitment of CD8 T cells influences the functional integrity of TRM cells in a persistent viral infection.

  17. Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle.

    Directory of Open Access Journals (Sweden)

    Natalie Dow

    Full Text Available Bovine viral diarrhea virus (BVDV, a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

  18. Genetics in psychiatry: common variant association studies

    Directory of Open Access Journals (Sweden)

    Buxbaum Joseph D

    2010-03-01

    Full Text Available Abstract Many psychiatric conditions and traits are associated with significant heritability. Genetic risk for psychiatric conditions encompass rare variants, identified due to major effect, as well as common variants, the latter analyzed by association analyses. We review guidelines for common variant association analyses, undertaking after assessing evidence of heritability. We highlight the importance of: suitably large sample sizes; an experimental design that controls for ancestry; careful data cleaning; correction for multiple testing; small P values for positive findings; assessment of effect size for positive findings; and, inclusion of an independent replication sample. We also note the importance of a critical discussion of any prior findings, biological follow-up where possible, and a means of accessing the raw data.

  19. Hemoglobin Variants: Biochemical Properties and Clinical Correlates

    Science.gov (United States)

    Thom, Christopher S.; Dickson, Claire F.; Gell, David A.; Weiss, Mitchell J.

    2013-01-01

    Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples. PMID:23388674

  20. Viral Vectors for Use in the Development of Biodefense Vaccines

    National Research Council Canada - National Science Library

    Lee, John S; Hadjipanayis, Angela G; Parker, Michael D

    2005-01-01

    .... DNA vectors, live-attenuated viruses and bacteria, recombinant proteins combined with adjuvant, and viral- or bacterial-vectored vaccines have been developed as countermeasures against many potential...

  1. Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins

    International Nuclear Information System (INIS)

    Backovic, Marija; Leser, George P.; Lamb, Robert A.; Longnecker, Richard; Jardetzky, Theodore S.

    2007-01-01

    To gain insight into Epstein-Barr virus (EBV) glycoprotein B (gB), recombinant, secreted variants were generated. The role of putative transmembrane regions, the proteolytic processing and the oligomerization state of the gB variants were investigated. Constructs containing 2 of 3 C-terminal hydrophobic regions were secreted, indicating that these do not act as transmembrane anchors. The efficiency of cleavage of the gB furin site was found to depend on the nature of C-terminus. All of the gB constructs formed rosette structures reminiscent of the postfusion aggregates formed by other viral fusion proteins. However, substitution of putative fusion loop residues, WY 112-113 and WLIY 193-196 , with less hydrophobic amino acids from HSV-1 gB, produced trimeric protein and abrogated the ability of the EBV gB ectodomains to form rosettes. These data demonstrate biochemical features of EBV gB that are characteristic of other class I and class II viral fusion proteins, but not of HSV-1 gB

  2. [Clinico-pathogenetic variants of chronic gastritis].

    Science.gov (United States)

    Chernin, V V; Dzhulaĭ, G S

    2004-01-01

    To evaluate specific features of the course of chronic gastritis (CG), morphofunctional condition of gastric mucosa, vegetative regulation, adrenergic and cholinergic shifts, histamine metabolism and effects of exogenic and endogenic risk factors in CG patients; to study clinicopathogenetic variants of CG. A total of 311 CG patients aged from 16 to 72 years were studied. They were divided into three groups by their gastric mucosa condition. The control group consisted of 30 healthy donors. The following parameters were studied: visual and histological condition of gastric mucosa, total acidity, the levels of free hydrochloric acid, pepsin, bioelectric gastric activity, general autonomic tonicity, cholinesterase activity. Three clinicopathogenetic variants of the disease have been identified. Variant 1 was characterized by a recurrent course, subjective manifestation of the disease only in exacerbation, surface (primarily antral) mucosal affection, normal or enhanced secretory and motor functions of the stomach, adequate reaction of acid production to caffeine and histamine stimulation, parasympathicotonia, absolute hyperhistaminemia, relative hypoacetylcholinemia, subnormal urinary excretion of adrenalin. Variant 2 manifested with rare recurrences, longer and more severe exacerbations, frequent spontaneous and provoked aggravations, moderate focal atrophy of the mucosa, secretory insufficiency with adequate reaction to histamine and minor to caffeine stimuli, hypomotor gastric dyskinesia, vegetative eutonia, normohistaminemia, absolute hypoacetylcholinemia, subnormal urinary excretion of noradrenaline. Variant 3 runs without definite remissions and exacerbations, with continuous abdominal pain and dyspepsia, frequent spontaneous aggravations, marked extended mucosal atrophy with secretory insufficiency up to achlorhydria, no stimulation of acid production in response to caffeine and histamine, gastric hypomotility, sympathicotonia, absolute hypohistaminemia

  3. Phylogenetic analysis consistent with a clinical history of sexual transmission of HIV-1 from a single donor reveals transmission of highly distinct variants

    Directory of Open Access Journals (Sweden)

    McClure Myra

    2011-07-01

    Full Text Available Abstract Background To combat the pandemic of human immunodeficiency virus 1 (HIV-1, a successful vaccine will need to cope with the variability of transmissible viruses. Human hosts infected with HIV-1 potentially harbour many viral variants but very little is known about viruses that are likely to be transmitted, or even if there are viral characteristics that predict enhanced transmission in vivo. We show for the first time that genetic divergence consistent with a single transmission event in vivo can represent several years of pre-transmission evolution. Results We describe a highly unusual case consistent with a single donor transmitting highly related but distinct HIV-1 variants to two individuals on the same evening. We confirm that the clustering of viral genetic sequences, present within each recipient, is consistent with the history of a single donor across the viral env, gag and pol genes by maximum likelihood and Bayesian Markov Chain Monte Carlo based phylogenetic analyses. Based on an uncorrelated, lognormal relaxed clock of env gene evolution calibrated with other datasets, the time since the most recent common ancestor is estimated as 2.86 years prior to transmission (95% confidence interval 1.28 to 4.54 years. Conclusion Our results show that an effective design for a preventative vaccine will need to anticipate extensive HIV-1 diversity within an individual donor as well as diversity at the population level.

  4. Normal variants of skin in neonates

    Directory of Open Access Journals (Sweden)

    Kulkarni M

    1996-01-01

    Full Text Available 2221 consecutive live births taking place between March 1994 and February 1995 were evaluated for a minimum period of 5 days to note for the occurrence of various normal anatomical variants specially those of skin. Birth weight, gestational age, maternal age, socio-economic status and consanguinity were carefully recorded in all the cases. Mongolian spots (72%, Epstein pearls (43.8%, Milia (26.2% and Erythema toxicum (25.2%, were the common dermatological variants noted. Maturity of the babies and possibly genetic factors (consanguinity are important factors in their causation as ordered in our study.

  5. The curation of genetic variants: difficulties and possible solutions.

    Science.gov (United States)

    Pandey, Kapil Raj; Maden, Narendra; Poudel, Barsha; Pradhananga, Sailendra; Sharma, Amit Kumar

    2012-12-01

    The curation of genetic variants from biomedical articles is required for various clinical and research purposes. Nowadays, establishment of variant databases that include overall information about variants is becoming quite popular. These databases have immense utility, serving as a user-friendly information storehouse of variants for information seekers. While manual curation is the gold standard method for curation of variants, it can turn out to be time-consuming on a large scale thus necessitating the need for automation. Curation of variants described in biomedical literature may not be straightforward mainly due to various nomenclature and expression issues. Though current trends in paper writing on variants is inclined to the standard nomenclature such that variants can easily be retrieved, we have a massive store of variants in the literature that are present as non-standard names and the online search engines that are predominantly used may not be capable of finding them. For effective curation of variants, knowledge about the overall process of curation, nature and types of difficulties in curation, and ways to tackle the difficulties during the task are crucial. Only by effective curation, can variants be correctly interpreted. This paper presents the process and difficulties of curation of genetic variants with possible solutions and suggestions from our work experience in the field including literature support. The paper also highlights aspects of interpretation of genetic variants and the importance of writing papers on variants following standard and retrievable methods. Copyright © 2012. Published by Elsevier Ltd.

  6. Molecular characterization of atypical antigenic variants of canine rabies virus reveals its reintroduction by wildlife vectors in southeastern Mexico.

    Science.gov (United States)

    Garcés-Ayala, Fabiola; Aréchiga-Ceballos, Nidia; Ortiz-Alcántara, Joanna M; González-Durán, Elizabeth; Pérez-Agüeros, Sandra I; Méndez-Tenorio, Alfonso; Torres-Longoria, Belem; López-Martínez, Irma; Hernández-Rivas, Lucía; Díaz-Quiñonez, José Alberto; Ramírez-González, José Ernesto

    2017-12-01

    Rabies is an infectious viral disease that is practically always fatal following the onset of clinical signs. In Mexico, the last case of human rabies transmitted by dogs was reported in 2006 and canine rabies has declined significantly due to vaccination campaigns implemented in the country. Here we report on the molecular characterization of six rabies virus strains found in Yucatan and Chiapas, remarkably, four of them showed an atypical reaction pattern when antigenic characterization with a reduced panel of eight monoclonal antibodies was performed. Phylogenetic analyses on the RNA sequences unveiled that the three atypical strains from Yucatan are associated with skunks. Analysis using the virus entire genome showed that they belong to a different lineage distinct from the variants described for this animal species in Mexico. The Chiapas atypical strain was grouped in a lineage that was considered extinct, while the others are clustered within classic dog variants.

  7. Viral Contamination Source in Clinical Microbiology Laboratory.

    Science.gov (United States)

    Wang, Xin Ling; Song, Juan; Song, Qin Qin; Yu, Jie; Luo, Xiao Nuan; Wu, Gui Zhen; Han, Jun

    2016-08-01

    To understand the potential causes of laboratory-acquired infections and to provide possible solutions that would protect laboratory personnel, samples from a viral laboratory were screened to determine the main sources of contamination with six subtypes of Rhinovirus. Rhinovirus contamination was found in the gloves, cuffs of protective wear, inner surface of biological safety cabinet (BSC) windows, and trash handles. Remarkably, high contamination was found on the inner walls of the centrifuge and the inner surface of centrifuge tube casing in the rotor. Spilling infectious medium on the surface of centrifuge tubes was found to contribute to contamination of centrifuge surfaces. Exposure to sodium hypochlorite containing no less than 0.2 g/L available chlorine decontaminated the surface of the centrifuge tubes from Rhinovirus after 2 min. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  8. Update on viral community-acquired pneumonia

    OpenAIRE

    Rocha Neto, Ozéas Galeno da; Leite, Ricardo Ferreira; Baldi, Bruno Guedes

    2013-01-01

    A pneumonia de origem viral é uma causa prevalente de infecção respiratória em adultos imunocompetentes. Tem apresentação variada, ocasionando desde formas leves a quadros graves de insuficiência respiratória com necessidade de ventilação mecânica. Contudo, em nosso país, há poucos estudos a respeito da apresentação clínica e diagnóstico dessa infecção. Dessa forma, os autores do presente artigo têm por objetivo revisar os principais agentes virais causadores de pneumonia na comunidade e disc...

  9. Viral gastrointestinal syndrome in our environment

    Directory of Open Access Journals (Sweden)

    Patić A.

    2014-01-01

    Full Text Available Viral gastrointestinal syndrome is a cause of morbidity and death worldwide. Infection is spread through contact with an infected person, as well as through contaminated food and water. A lethal outcome is possible in infants and young children due to dehydration and electrolyte imbalance. The study included 141 patients with gastroenteritis from Vojvodina. Real-Time PCR method in stool samples was used to determine the presence of rota-, noro-, and astrovirus nucleic acid. Out of 141 patients with gastroenteritis, 60.3% were confirmed to have one of the three viruses. Rotavirus was significantly more common in children up to 3 years of age (43.3%. Norovirus was more frequently detected in patients older than 20 (50%. These infections started in collectives. Astrovirus was detected in four patients (2.8%. The results confirm the necessity to implement PCR in routine diagnostics for the proper treatment of patients.

  10. Microbial and viral pathogens in colorectal cancer.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2011-05-01

    The heterogenetic and sporadic nature of colorectal cancer has led to many epidemiological associations with causes of this disease. As our understanding of the underlying molecular processes in colorectal-cancer develops, the concept of microbial-epithelial interactions as an oncogenic trigger might provide a plausible hypothesis for the pathogenesis of colorectal cancer. By contrast with other cancers of the gastrointestinal tract (gastric carcinoma, mucosa-associated lymphoid-tissue lymphoma), a direct causal link between microbial infection (bacteria and viruses) and colorectal carcinoma has not been established. Studies support the involvement of these organisms in oncogenesis, however, in colorectal cancer, clinical data are lacking. Here, we discuss current evidence (both in vitro and clinical studies), and focus on a putative role for bacterial and viral pathogens as a cause of colorectal cancer.

  11. Microbial and viral pathogens in colorectal cancer.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2012-02-01

    The heterogenetic and sporadic nature of colorectal cancer has led to many epidemiological associations with causes of this disease. As our understanding of the underlying molecular processes in colorectal-cancer develops, the concept of microbial-epithelial interactions as an oncogenic trigger might provide a plausible hypothesis for the pathogenesis of colorectal cancer. By contrast with other cancers of the gastrointestinal tract (gastric carcinoma, mucosa-associated lymphoid-tissue lymphoma), a direct causal link between microbial infection (bacteria and viruses) and colorectal carcinoma has not been established. Studies support the involvement of these organisms in oncogenesis, however, in colorectal cancer, clinical data are lacking. Here, we discuss current evidence (both in vitro and clinical studies), and focus on a putative role for bacterial and viral pathogens as a cause of colorectal cancer.

  12. MR imaging of acute viral hepatitis

    International Nuclear Information System (INIS)

    Sakai, Toyohiko; Itoh, Hisao; Takahashi, Norio; Kitada, Masahisa; Saito, Masayuki; Ohshiro, Kennwa; Ishimori, Masatoshi; Ishii, Yasushi.

    1991-01-01

    Twenty-three MR studies of 19 patients with acute viral hepatitis were reviewed. The findings of MR imaging including peripotal high intensity (PHI) on T 2 -weighted images and gallbladder wall thickening (GBWT) were compared with the level of serum GOT level and clinical phase which was determined by the interval between the peak of serum GOT level and MR study. PHI was found in 15 out of 23 studies (65%) and GBWT in 7 out of 22 studies (32%). The incidence of these findings were correlated well with the severity of serum GOT level and clinical phase. PHI became less prominent gradually as during the clinical recovery. While GBWT was found in the earlier phase and disappeared immediately. PHI seems to correspond to edema and infiltration of inflammatory cells in the periportal area of the liver. (author)

  13. MR imaging of acute viral hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Toyohiko; Itoh, Hisao; Takahashi, Norio; Kitada, Masahisa; Saito, Masayuki; Ohshiro, Kennwa; Ishimori, Masatoshi [Matsunami General Hospital, Kasamatsu, Gifu (Japan); Ishii, Yasushi

    1991-02-01

    Twenty-three MR studies of 19 patients with acute viral hepatitis were reviewed. The findings of MR imaging including peripotal high intensity (PHI) on T{sub 2}-weighted images and gallbladder wall thickening (GBWT) were compared with the level of serum GOT level and clinical phase which was determined by the interval between the peak of serum GOT level and MR study. PHI was found in 15 out of 23 studies (65%) and GBWT in 7 out of 22 studies (32%). The incidence of these findings were correlated well with the severity of serum GOT level and clinical phase. PHI became less prominent gradually as during the clinical recovery. While GBWT was found in the earlier phase and disappeared immediately. PHI seems to correspond to edema and infiltration of inflammatory cells in the periportal area of the liver. (author).

  14. Papillomaviruses: Viral evolution, cancer and evolutionary medicine.

    Science.gov (United States)

    Bravo, Ignacio G; Félez-Sánchez, Marta

    2015-01-28

    Papillomaviruses (PVs) are a numerous family of small dsDNA viruses infecting virtually all mammals. PVs cause infections without triggering a strong immune response, and natural infection provides only limited protection against reinfection. Most PVs are part and parcel of the skin microbiota. In some cases, infections by certain PVs take diverse clinical presentations from highly productive self-limited warts to invasive cancers. We propose PVs as an excellent model system to study the evolutionary interactions between the immune system and pathogens causing chronic infections: genotypically, PVs are very diverse, with hundreds of different genotypes infecting skin and mucosa; phenotypically, they display extremely broad gradients and trade-offs between key phenotypic traits, namely productivity, immunogenicity, prevalence, oncogenicity and clinical presentation. Public health interventions have been launched to decrease the burden of PV-associated cancers, including massive vaccination against the most oncogenic human PVs, as well as systematic screening for PV chronic anogenital infections. Anti-PVs vaccines elicit protection against infection, induce cross-protection against closely related viruses and result in herd immunity. However, our knowledge on the ecological and intrapatient dynamics of PV infections remains fragmentary. We still need to understand how the novel anthropogenic selection pressures posed by vaccination and screening will affect viral circulation and epidemiology. We present here an overview of PV evolution and the connection between PV genotypes and the phenotypic, clinical manifestations of the diseases they cause. This differential link between viral evolution and the gradient cancer-warts-asymptomatic infections makes PVs a privileged playground for evolutionary medicine research. © The Author(s) 2015. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.

  15. Pathological studies on bovine viral diarrhea

    International Nuclear Information System (INIS)

    Elkady, A.A.M.A.

    2002-01-01

    Bovine viral diarrhea virus (BVDV) is classified as an RNA virus in the family flavin viride and is a member of the genus pest virus (Collet et al 1989). BVDV has a worldwide distribution and infections in cattle populations (Kahrs et al 1970). It was recognized since 50 years ago, the initial description of an acute enteric disease of cattle in North America, which was characterized by outbreaks of diarrhea and erosive of digestive tract (Olafsonp et al 1946). The disease and causative agent were named bovine viral diarrhea (B V D ) and (B V DV), respectively. This virus was subsequently associated with a sporadically occurring and highly fatal enteric disease that was termed mucosal disease (M D), (Ramsey and Chivers 1953). The initial isolate of BVDV did not produce cytopathic effect in cell culture, whereas an isolate from MD did produce cytopathic effects (Lee et al 1957). In vitro characteristic of non cytopathic or sytopathic effects of BVDV is referred to as the biotype of the virus. It has now been established that MD occurs only when xattle that are born immuno tolerant to and persistently infected with a noncyropathic BVDV become super infected with a cytopathic BVDV. The knowledge of the molecular biology. Pathogenesis and epidemiology of BVDV has greatly evolved in the past 10-15 years and has provided a better understanding of this complex infectious agent. Infection with BVDV can result in a wide spectrum of diseases ranging from subclinical infection s to a highly fatal from known as mucosal disease (ND). The clinical response to infection depends on multiple interactive factors. Host factors that influence the clinical outcome of BVDV infection include whether the host is immunocompetent or immuno tolerant to BVDV, pregnancy status, gestational age of the fetus, immune status (passively derived or actively derived from previous infection or vaccination) and concurrent level of environmental stress

  16. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  17. Virus del dengue: estructura y ciclo viral Dengue virus: structure and viral cycle

    Directory of Open Access Journals (Sweden)

    Myriam L Velandia

    2011-03-01

    Full Text Available El virus del dengue (DENV es el agente causal de la enfermedad conocida como dengue, que es la principal enfermedad viral transmitida por artrópodos en el mundo. El DENV es un flavivirus que ingresa por endocitosis y se replica en el citoplasma de la célula infectada, originando tres proteínas estructurales y siete proteínas no estructurales, sobre las cuales se conocen sólo algunas de sus funciones en la replicación viral o en la infección. El ciclo viral que ocurre en las células infectadas hasta ahora está comenzando a aclararse y su conocimiento permitirá en el futuro próximo diseñar racionalmente moléculas que lo intervengan y eviten la replicación del virus. Durante la infección, el individuo puede presentar fiebre indiferenciada o, en otros casos, puede presentar un proceso generalizado de activación de la respuesta inmunitaria innata y adquirida, lo cual provoca la liberación de factores inflamatorios solubles que alteran la fisiología de los tejidos, principalmente el endotelio, conllevando al desarrollo de manifestaciones clínicas graves. Aunque se ha identificado un gran número de factores del individuo asociados al desarrollo de la enfermedad por DENV, queda por identificar el papel de las diferentes proteínas virales en la patogenia de la enfermedad. En la presente revisión, se presenta una breve actualización sobre la estructura y biología del DENV, de su ciclo viral intracelular y, finalmente, se introducen algunos conceptos sobre la inmunopatogenia de la enfermedad producida por este agente.Dengue virus (DENV is responsible for the clinical entity known as dengue that is a great concern for economy and public health of tropical countries. This flavivirus is a single strand RNA virus that after their translation and replication in host cells produces three structural and seven non-structural proteins with specific function in replication or cell binding process that we will describe here. Intracellular

  18. Analysis of Hepatitis C Virus Genotype 1b Resistance Variants in Japanese Patients Treated with Paritaprevir-Ritonavir and Ombitasvir.

    Science.gov (United States)

    Krishnan, Preethi; Schnell, Gretja; Tripathi, Rakesh; Beyer, Jill; Reisch, Thomas; Zhang, Xinyan; Setze, Carolyn; Rodrigues, Lino; Burroughs, Margaret; Redman, Rebecca; Chayama, Kazuaki; Kumada, Hiromitsu; Collins, Christine; Pilot-Matias, Tami

    2016-02-01

    Treatment of HCV genotype 1b (GT1b)-infected Japanese patients with paritaprevir (NS3/4A inhibitor boosted with ritonavir) and ombitasvir (NS5A inhibitor) in studies M12-536 and GIFT-I demonstrated high sustained virologic response (SVR) rates. The virologic failure rate was 3% (13/436) across the two studies. Analyses were conducted to evaluate the impact of baseline resistance-associated variants (RAVs) on treatment outcome and the emergence and persistence of RAVs in patients experiencing virologic failure. Baseline paritaprevir resistance-conferring variants in NS3 were infrequent, while Y93H in NS5A was the most prevalent ombitasvir resistance-conferring variant at baseline. A comparison of baseline prevalence of polymorphisms in Japanese and western patients showed that Q80L and S122G in NS3 and L28M, R30Q, and Y93H in NS5A were significantly more prevalent in Japanese patients. In the GIFT-I study, the prevalence of Y93H in NS5A varied between 13% and 21% depending on the deep-sequencing detection threshold. Among patients with Y93H comprising 40% of their preexisting viral population, the 24-week SVR (SVR24) rates were >99% (276/277), 93% (38/41), and 76% (25/33), respectively, indicating that the prevalence of Y93H within a patient's viral population is a good predictor of treatment response. The predominant RAVs at the time of virologic failure were D168A/V in NS3 and Y93H alone or in combination with other variants in NS5A. While levels of NS3 RAVs declined over time, NS5A RAVs persisted through posttreatment week 48. Results from these analyses are informative in understanding the resistance profile of an ombitasvir- plus paritaprevir/ritonavir-based regimen in Japanese GT1b-infected patients. Copyright © 2016 Krishnan et al.

  19. Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness.

    Directory of Open Access Journals (Sweden)

    Luke Uebelhoer

    2008-09-01

    Full Text Available Mechanisms by which hepatitis C virus (HCV evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA class I-restricted epitopes targeted by CD8(+ T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637, displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC anchor and T cell receptor (TCR contact residues. Only one of these amino acid substitutions at position 9 (P9 of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7 TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily

  20. Magnetic resonance angiography: infrequent anatomic variants

    International Nuclear Information System (INIS)

    Trejo, Mariano; Meli, Francisco; Lambre, Hector; Blessing, Ricardo; Gigy Traynor, Ignacio; Miguez, Victor

    2002-01-01

    We studied through RM angiography (3D TOF) with high magnetic field equipment (1.5 T) different infrequent intracerebral vascular anatomic variants. For their detection we emphasise the value of post-processed images obtained after conventional angiographic sequences. These post-processed images should be included in routine protocols for evaluation of the intracerebral vascular structures. (author)

  1. Report of a rare anatomic variant

    DEFF Research Database (Denmark)

    De Brucker, Y; Ilsen, B; Muylaert, C

    2015-01-01

    We report the CT findings in a case of partial anomalous pulmonary venous return (PAPVR) from the left upper lobe in an adult. PAPVR is an anatomic variant in which one to three pulmonary veins drain into the right atrium or its tributaries, rather than into the left atrium. This results in a left...

  2. Analysis of the energy development variants

    International Nuclear Information System (INIS)

    Tsvetanov, P.

    1990-01-01

    Analysis of the variants of energy development is made as the third stage of a procedure of energy-economy interrelations dynamics study, the other two stages being the scenarios description and the formulation of the variants. This stage includes a research on the dimensions and the dynamics of the resources demands, the general features and the trends of the national energy development. There is a presentation of a comparative analysis of the variants in terms of economic indices and energy values, computed by the model IMPACT-B. A resource evaluation of the development variants is given in terms of investments, requirements (direct, indirect and total) and limited national resources demands of the energy system. The trends of the national energy development discussed are: trends characterizing the changes in the structure of the energy consumption, resulting from changes in the economy; trends of the energy system impact on the productivity of labor; general trends of the proportionality in the industrial, the household and services sector development. 16 refs., 16 figs., 4 tabs. (R.Ts.)

  3. Cellobiohydrolase I gene and improved variants

    Science.gov (United States)

    Adney, William S [Golden, CO; Decker, Stephen R [Berthoud, CO; Mc Carter, Suzanne [San Carlos, CA; Baker, John O [Golden, CO; Nieves, Raphael [Lakewood, CO; Himmel, Michael E [Littleton, CO; Vinzant, Todd B [Golden, CO

    2008-05-20

    The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

  4. XVCL: XML-based Variant Configuration Language

    DEFF Research Database (Denmark)

    Jarzabek, Stan; Basset, Paul; Zhang, Hongyu

    2003-01-01

    XVCL (XML-based Variant Configuration Language) is a meta-programming technique and tool that provides effective reuse mechanisms. XVCL is an open source software developed at the National University of Singapore. Being a modern and versatile version of Bassett's frames, a technology that has...

  5. Glucose 6-phosphate dehydrogenase variants in Japan.

    Science.gov (United States)

    Miwa, S

    1980-01-01

    Fifty-four cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency have so far been reported in Japan. Among them, 21 G6PD variants have been characterized. Nineteen out of the 21 variants were characterized in our laboratory and G6PD Heian and "Kyoto" by others. G6PD Tokyo, Tokushima, Ogikubo, Kurume, Fukushima, Yokohama, Yamaguchi, Wakayama, Akita, Heian and "Kyoto" were classified as Class 1, because all these cases showed chronic hemolytic anemia and severe enzyme deficiency. All these variants showed thermal instability. G6PD Mediterranean-like, Ogori, Gifu and Fukuoka were classified as Class 2, whereas G6PD Hofu, B(-) Chinese, Ube, Konan, Kamiube and Kiwa belonged to Class 3. All the 6 Class 3 variants were found as the results of the screening tests. The incidence of the deficiency in Japanese seems to be 0.1-0.5% but that of the cases which may slow drug-induced hemolysis would be much less. G6PD Ube and Konan appear to be relatively common in Japan.

  6. Genetic variants influencing phenotypic variance heterogeneity.

    Science.gov (United States)

    Ek, Weronica E; Rask-Andersen, Mathias; Karlsson, Torgny; Enroth, Stefan; Gyllensten, Ulf; Johansson, Åsa

    2018-03-01

    Most genetic studies identify genetic variants associated with disease risk or with the mean value of a quantitative trait. More rarely, genetic variants associated with variance heterogeneity are considered. In this study, we have identified such variance single-nucleotide polymorphisms (vSNPs) and examined if these represent biological gene × gene or gene × environment interactions or statistical artifacts caused by multiple linked genetic variants influencing the same phenotype. We have performed a genome-wide study, to identify vSNPs associated with variance heterogeneity in DNA methylation levels. Genotype data from over 10 million single-nucleotide polymorphisms (SNPs), and DNA methylation levels at over 430 000 CpG sites, were analyzed in 729 individuals. We identified vSNPs for 7195 CpG sites (P mean DNA methylation levels. We further showed that variance heterogeneity between genotypes mainly represents additional, often rare, SNPs in linkage disequilibrium (LD) with the respective vSNP and for some vSNPs, multiple low frequency variants co-segregating with one of the vSNP alleles. Therefore, our results suggest that variance heterogeneity of DNA methylation mainly represents phenotypic effects by multiple SNPs, rather than biological interactions. Such effects may also be important for interpreting variance heterogeneity of more complex clinical phenotypes.

  7. Genetic variants associated with lung function

    DEFF Research Database (Denmark)

    Thyagarajan, Bharat; Wojczynski, Mary; Minster, Ryan L

    2014-01-01

    with exceptional longevity have not been identified. METHOD: We conducted a genome wide association study (GWAS) to identify novel genetic variants associated with lung function in the Long Life Family Study (LLFS) (n = 3,899). Replication was performed using data from the CHARGE/SpiroMeta consortia...

  8. Viral tropism and pathology associated with viral hemorrhagic septicemia in larval and juvenile Pacific herring

    Science.gov (United States)

    Lovy, Jan; Lewis, N.L.; Hershberger, P.K.; Bennett, W.; Meyers, T.R.; Garver, K.A.

    2012-01-01

    Viral hemorrhagic septicemia virus (VHSV) genotype IVa causes mass mortality in wild Pacific herring, a species of economic value, in the Northeast Pacific Ocean. Young of the year herring are particularly susceptible and can be carriers of the virus. To understand its pathogenesis, tissue and cellular tropisms of VHSV in larval and juvenile Pacific herring were investigated with immunohistochemistry, transmission electron microscopy, and viral tissue titer. In larval herring, early viral tropism for epithelial tissues (6d post-exposure) was indicated by foci of epidermal thickening that contained heavy concentrations of virus. This was followed by a cellular tropism for fibroblasts within the fin bases and the dermis, but expanded to cells of the kidney, liver, pancreas, gastrointestinal tract and meninges in the brain. Among wild juvenile herring that underwent a VHS epizootic in the laboratory, the disease was characterized by acute and chronic phases of death. Fish that died during the acute phase had systemic infections in tissues including the submucosa of the gastrointestinal tract, spleen, kidney, liver, and meninges. The disease then transitioned into a chronic phase that was characterized by the appearance of neurological signs including erratic and corkscrew swimming and darkening of the dorsal skin. During the chronic phase viral persistence occurred in nervous tissues including meninges and brain parenchymal cells and in one case in peripheral nerves, while virus was mostly cleared from the other tissues. The results demonstrate the varying VHSV tropisms dependent on the timing of infection and the importance of neural tissues for the persistence and perpetuation of chronic infections in Pacific herring.

  9. Plasma Viral miRNAs Indicate a High Prevalence of Occult Viral Infections

    Directory of Open Access Journals (Sweden)

    Enrique Fuentes-Mattei

    2017-06-01

    Full Text Available Prevalence of Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8 varies greatly in different populations. We hypothesized that the actual prevalence of KSHV/HHV8 infection in humans is underestimated by the currently available serological tests. We analyzed four independent patient cohorts with post-surgical or post-chemotherapy sepsis, chronic lymphocytic leukemia and post-surgical patients with abdominal surgical interventions. Levels of specific KSHV-encoded miRNAs were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR, and KSHV/HHV-8 IgG were measured by immunoassay. We also measured specific miRNAs from Epstein Barr Virus (EBV, a virus closely related to KSHV/HHV-8, and determined the EBV serological status by ELISA for Epstein-Barr nuclear antigen 1 (EBNA-1 IgG. Finally, we identified the viral miRNAs by in situ hybridization (ISH in bone marrow cells. In training/validation settings using independent multi-institutional cohorts of 300 plasma samples, we identified in 78.50% of the samples detectable expression of at least one of the three tested KSHV-miRNAs by RT-qPCR, while only 27.57% of samples were found to be seropositive for KSHV/HHV-8 IgG (P < 0.001. The prevalence of KSHV infection based on miRNAs qPCR is significantly higher than the prevalence determined by seropositivity, and this is more obvious for immuno-depressed patients. Plasma viral miRNAs quantification proved that EBV infection is ubiquitous. Measurement of viral miRNAs by qPCR has the potential to become the “gold” standard method to detect certain viral infections in clinical practice.

  10. Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

    Directory of Open Access Journals (Sweden)

    Annika Brinkmann

    2017-11-01

    Full Text Available We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS-based identification of viral hemorrhagic fever (VHF agents and assess the feasibility of this approach in diagnostics.An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients.The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours.Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

  11. Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

    Science.gov (United States)

    Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina; Nitsche, Andreas

    2017-11-01

    We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

  12. A Strong Case for Viral Genetic Factors in HIV Virulence

    Directory of Open Access Journals (Sweden)

    Joshua T. Herbeck

    2011-03-01

    Full Text Available HIV infections show great variation in the rate of progression to disease, and the role of viral genetic factors in this variation had remained poorly characterized until recently. Now a series of four studies [1–4] published within a year has filled this important gap and has demonstrated a robust effect of the viral genotype on HIV virulence.

  13. Experience with Hepatitis B viral load testing in Nigeria | Okwuraiwe ...

    African Journals Online (AJOL)

    Background: Quantification of the viral burden is an important laboratory tool in the management of hepatitis B virus (HBV)-infected patients. However, widespread use of assays is still hampered by the high cost. Treatment reduces viral load to undetectable levels. HBV infected patients tend to have high HBV DNA levels, ...

  14. Core Gene Expression and Association of Genotypes with Viral ...

    African Journals Online (AJOL)

    Purpose: To determine genotypic distribution, ribonucleic acid (RNA) RNA viral load and express core gene from Hepatitis C Virus (HCV) infected patients in Punjab, Pakistan. Methods: A total of 1690 HCV RNA positive patients were included in the study. HCV genotyping was tested by type-specific genotyping assay, viral ...

  15. Anti-viral effect of herbal medicine Korean traditional Cynanchum ...

    African Journals Online (AJOL)

    Background: Pestiviruses in general, and Bovine Viral Diarrhea (BVD) in particular, present several potential targets for directed antiviral therapy. Material and Methods: The antiviral effect of Cynanchum paniculatum (Bge.) Kitag (Dog strangling vine: DS) extract on the bovine viral diarrhea (BVD) virus was tested. First ...

  16. Why pass on viral messages? Because they connect emotionally

    NARCIS (Netherlands)

    Dobele, A.; Lindgreen, A.; Beverland, M.; Vanhamme, J.; Wijk, van R.

    2007-01-01

    In this article, we identify that successful viral marketing campaigns trigger an emotional response in recipients. Working under this premise, we examine the effects of viral messages containing the six primary emotions (surprise, joy, sadness, anger, fear, and disgust) on recipients' emotional

  17. 101 . experience with hepatitis b viral load testing in nigeria

    African Journals Online (AJOL)

    User

    ABSTRACT. Background: Quantification of the viral burden is an important laboratory tool in the management of hepatitis B virus. (HBV)-infected patients. However, widespread use of assays is still hampered by the high cost. Treatment reduces viral load to undetectable levels. HBV infected patients tend to have high HBV ...

  18. Distribution, incidence and severity of viral diseases of yam ...

    African Journals Online (AJOL)

    A survey was conducted in major yam cultivation zones in Côte d'Ivoire in 2009 to determine the incidence, severity of viral diseases, and viruses associated with the infected plants. Incidence and severity of the viral diseases were estimated based on symptoms. Enzyme-linked immunosorbent assay (ELISA) and ...

  19. Short Report Challenges with targeted viral load testing for medical ...

    African Journals Online (AJOL)

    Challenges with targeted viral load testing 179. Malawi Medical ... targeted viral load (VL) testing for patients who have been on ART for at least .... Tuberculosis. 32. Community-acquired pneumonia. 17. Non-typhoidal Salmonella sepsis. 5. Bacterial meningitis. 5. Disseminated Kaposi sarcoma. 4. Cryptococcal meningitis. 4.

  20. Analysis of host genetic diversity and viral entry as sources of between-host variation in viral load

    Science.gov (United States)

    Wargo, Andrew R.; Kell, Alison M.; Scott, Robert J.; Thorgaard, Gary H.; Kurath, Gael

    2012-01-01

    Little is known about the factors that drive the high levels of between-host variation in pathogen burden that are frequently observed in viral infections. Here, two factors thought to impact viral load variability, host genetic diversity and stochastic processes linked with viral entry into the host, were examined. This work was conducted with the aquatic vertebrate virus, Infectious hematopoietic necrosis virus (IHNV), in its natural host, rainbow trout. It was found that in controlled in vivo infections of IHNV, a suggestive trend of reduced between-fish viral load variation was observed in a clonal population of isogenic trout compared to a genetically diverse population of out-bred trout. However, this trend was not statistically significant for any of the four viral genotypes examined, and high levels of fish-to-fish variation persisted even in the isogenic trout population. A decrease in fish-to-fish viral load variation was also observed in virus injection challenges that bypassed the host entry step, compared to fish exposed to the virus through the natural water-borne immersion route of infection. This trend was significant for three of the four virus genotypes examined and suggests host entry may play a role in viral load variability. However, high levels of viral load variation also remained in the injection challenges. Together, these results indicate that although host genetic diversity and viral entry may play some role in between-fish viral load variation, they are not major factors. Other biological and non-biological parameters that may influence viral load variation are discussed.