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Sample records for adeno-associated virus serotype

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

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

    2017-01-09

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

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

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    Daniel J. Schuster

    2014-08-01

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

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

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    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.

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

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

    2011-11-01

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

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

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

    2012-05-24

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

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

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

    2012-02-05

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

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

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

    2012-04-01

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

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

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

    2012-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

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

    2013-11-01

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

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

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

    2012-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  13. Adeno-Associated Virus Gene Therapy for Liver Disease

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

    2016-01-01

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

  14. Adeno-associated virus for cystic fibrosis gene therapy

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    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.

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

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    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.

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

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    Ayşegül Akbay Yarpuzlu

    2009-06-01

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

  17. Partial correction of the CNS lysosomal storage defect in a mouse model of juvenile neuronal ceroid lipofuscinosis by neonatal CNS administration of an adeno-associated virus serotype rh.10 vector expressing the human CLN3 gene.

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

    2014-03-01

    Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

  18. Biosafety of recombinant adeno-associated virus vectors.

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

    2013-12-01

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

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

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    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.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

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    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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

    observed in the sub-epithelial layer and the lamina propria.CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice,but were absent in the Tregitope 167 treated group.Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167.Furthermore,the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5 (cytomegalovirus promoter-Tregitope 167),as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice (P < 0.05 and P < 0.001,respectively).CONCLUSION:This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce longterm remission in inflammatory bowel disease.

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

    Directory of Open Access Journals (Sweden)

    Dejia Li

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2014-05-01

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

  8. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

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

    OpenAIRE

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    樊东升; 沈扬

    2000-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    XU ZengHu; ZHOU XiuMei; SHI WenFang; QIAN QiJun

    2008-01-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  16. 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.

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

    Science.gov (United States)

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

    2003-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Ke SONG; Nianjing RAO; Meiling CHEN; Yingguang CAO

    2008-01-01

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

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

    Science.gov (United States)

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

    2007-02-01

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

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

    Science.gov (United States)

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

    1996-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Arpiar eSaunders

    2012-07-01

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

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

    Science.gov (United States)

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

    2011-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Science.gov (United States)

    Diao, Yong; Xu, Ruian

    2012-05-04

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

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

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    2001-01-01

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

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

    Science.gov (United States)

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

    2006-08-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1979-04-15

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  11. 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.

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

    Institute of Scientific and Technical Information of China (English)

    陈德杰; 谭最; 谢友利; 刘芳

    2004-01-01

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

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

    OpenAIRE

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Hastie, Eric; Samulski, R Jude

    2015-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

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

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

    Science.gov (United States)

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

    2017-02-01

    The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10(14) vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10(15) VG in crude cell harvests and an average of 3.5 × 10(14) total VG of purified rAAV9 material, resulting in greater than 1 × 10(5) VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

  18. 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.

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

    Science.gov (United States)

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

    2017-02-24

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

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

    Science.gov (United States)

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

    2014-11-28

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

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

    Science.gov (United States)

    Laredj, Leila N; Beard, Peter

    2011-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    徐卫国; 陈安民; 张衣北; 易成腊

    2004-01-01

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

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

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

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    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.

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

    OpenAIRE

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

    2000-01-01

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

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

    OpenAIRE

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2009-07-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

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

    2006-01-01

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

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

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    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.

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

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    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

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

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    Marrero Luis

    2003-09-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

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    Choi Jin-Ok

    2010-04-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2005-07-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Stefan Michelfelder

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

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

    Science.gov (United States)

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

    1995-01-01

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

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

    Science.gov (United States)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2004-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Mette Christensen

    2010-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lina Li

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2008-01-01

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

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

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    Amin Hajitou

    2013-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

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

  5. 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.

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2007-07-01

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

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2007-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    石亮; 张晨; 向志光; 邓仪晨; 苏静芬; 刘云波

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    高凯; 吴勇杰; 吴小兵; 饶春明; 王军志

    2004-01-01

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

    Science.gov (United States)

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

    2003-12-10

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

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

    Directory of Open Access Journals (Sweden)

    Fengqin Zhu

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

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

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2007-01-01

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

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

    Science.gov (United States)

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

    2005-08-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  19. Adeno-Associated Virus Transfer of a Gene Encoding SNAP-25 Resistant to Botulinum Toxin A Attenuates Neuromuscular Paralysis Associated with Botulism

    Science.gov (United States)

    2008-04-02

    refinement of the technology for application to genetic disorders of motor neurons. 15. SUBJECT TERMS Clostridium botulinum, neurotoxiin, serotype...peripherally transported transgenic protein successfully substituted for native cleavage-susceptible S25, as demonstrated by enhanced retention of nerve

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  1. 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.

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

    Science.gov (United States)

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

    2015-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 王卓然

    2009-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    杨红霞; 邱一果

    2013-01-01

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

  6. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    高凯; 王军志; 饶春明; 吴小兵

    2003-01-01

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

  9. Evaluation of cross-protection of bluetongue virus serotype 4 with other serotypes in sheep

    Directory of Open Access Journals (Sweden)

    Gcwalisile B. Zulu

    2014-02-01

    Full Text Available Bluetongue (BT is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV. Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control, 1 (unrelated serotype, 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes.

  10. Why are dengue virus serotypes so distantly related? Enhancement and limiting serotype similarity between dengue virus strains.

    OpenAIRE

    Kawaguchi, Isao; Sasaki, Akira; Boots, Michael

    2003-01-01

    Dengue virus, the causative agent of dengue fever, has four major serotypes characterized by large genetic and immunological distances. We propose that the unusually large distances between the serotypes can be explained in the light of a process of antibody-dependent enhancement (ADE) leading to increased mortality. Antibody-dependent enhancement results from a new infection with a particular serotype in an individual with acquired immunity to a different serotype. Classical dengue fever cau...

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

    Science.gov (United States)

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

    2016-05-01

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

  12. Dengue virus serotype in Aceh Province

    Directory of Open Access Journals (Sweden)

    Paisal

    2015-06-01

    Full Text Available WHO estimated 50 million dengue infections happen every year in the world. In Indonesia, there were 90,245 DHF cases on 2012 with 816 deaths. In the Province of Aceh, 2,269 cases happened in the same year. This study aimed to identify dengue virus serotype in Aceh. Sampling was done in Kota Banda Aceh Hospital, Kota Lhokseumawe Hospital, Kabupaten Aceh Tamiang Hospital, Kabupaten Aceh Barat Hospital, and Kabupaten Simeulue Hospital between May to December 2012. This was a clinical laboratory research with observation design using cross sectional approach. Research’s population was sample from patients with dengue clinical symptom. Using purposive sampling technique, we have collected 100 samples from the five hospitals (20 samples from each hospital. From RT-PCR, we found 16 positive samples (9 samples were DENV-4, 3 samples were DENV-1, 2 samples were DENV-2, and 2 samples were DENV-3.

  13. Dengue viruses cluster antigenically but not as discrete serotypes

    NARCIS (Netherlands)

    L. Katzelnick (Leah); J.M. Fonville (Judith); G.D. Gromowski (Gregory D.); J.B. Arriaga (Jose Bustos); A. Green (Angela); S.L. James (Sarah ); L. Lau (Louis); M. Montoya (Magelda); C. Wang (Chunling); L.A. Van Blargan (Laura A.); C.A. Russell (Colin); H.M. Thu (Hlaing Myat); T.C. Pierson (Theodore C.); P. Buchy (Philippe); J.G. Aaskov (John G.); J.L. Muñoz-Jordán (Jorge L.); N. Vasilakis (Nikos); R.V. Gibbons (Robert V.); R.B. Tesh (Robert B.); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); A. Durbin (Anna); C.P. Simmons (Cameron P.); E.C. Holmes (Edward C.); E. Harris (Eva); S.S. Whitehead (Stephen S.); D.J. Smith (Derek James)

    2015-01-01

    textabstractThe four genetically divergent dengue virus (DENV) types are traditionally classified as serotypes. Antigenic and genetic differences among the DENV types influence disease outcome, vaccine-induced protection, epidemic magnitude, and viral evolution.We scharacterized antigenic diversity

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

    Institute of Scientific and Technical Information of China (English)

    杨硕; 刘磊; 裴晗; 万幸; 陆朵朵; 胡维琨; 李斌

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 张苏明; 徐金枝

    2007-01-01

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

  17. Urban epidemic of dengue virus serotype 3 infection, Senegal, 2009.

    Science.gov (United States)

    Faye, Ousmane; Ba, Yamar; Faye, Oumar; Talla, Cheikh; Diallo, Diawo; Chen, Rubing; Mondo, Mireille; Ba, Rouguiétou; Macondo, Edgard; Siby, Tidiane; Weaver, Scott C; Diallo, Mawlouth; Sall, Amadou Alpha

    2014-03-01

    An urban epidemic of dengue in Senegal during 2009 affected 196 persons and included 5 cases of dengue hemorrhagic fever and 1 fatal case of dengue shock syndrome. Dengue virus serotype 3 was identified from all patients, and Aedes aegypti mosquitoes were identified as the primary vector of the virus.

  18. Influence of serotype and virus strain on synergism between Marek's disease vaccine viruses.

    Science.gov (United States)

    Witter, R L

    1992-12-01

    The enhanced protective effect (synergism) when certain Marek's disease (MD) vaccine viruses are combined has been widely used in the development of improved vaccines, but the mechanism is poorly understood. To better characterize the basis for synergism among MD vaccine viruses, three vaccine viruses from each of the three MD viral serotypes were evaluated alone and in various combinations for protection against early challenge with very virulent MD viruses in four replicate trials. Synergism seemed to be influenced by viral serotype because significant enhancement occurred frequently between viruses of serotypes 2 and 3 (five of nine bivalent vaccines positive), but rarely between viruses of serotypes 1 and 3 (one of nine bivalent vaccines positive) and 1 and 2 (one of nine bivalent vaccines positive), and was not detectable between viruses of the same serotype (none of nine bivalent vaccines positive). With some exceptions, the degree of synergism tended to vary inversely with the mean protective efficacy of the most protective component virus. Little effect of virus dose, virus dose ratio or type and route of viral challenge was noted. The combination of strains 281MI/1 (serotype 2) and WTHV-1/1 (serotype 3), both poorly protective as monovalent vaccines, consistently demonstrated high levels of synergism (over 300%) in antibody-positive chickens challenged 5 days post-vaccination with Md5 virus. This protocol may be a useful model system for further studies on mechanisms of synergism. However, mixtures that optimize synergism are not necessarily as protective as commercial vaccines.

  19. Rapid Subtyping of Dengue Virus Serotypes 1 and 4 by Restriction Site-Specific PCR

    OpenAIRE

    Marize P Miagostovich; dos Santos, Flavia B.; Gutiérrez, C. Milena; Riley, Lee W.; Harris, Eva

    2000-01-01

    We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to...

  20. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    OpenAIRE

    Alvaro Díaz-Badillo; María de Lourdes Muñoz; Gerardo Perez-Ramirez; Victor Altuzar; Juan Burgueño; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Alejandro Cisneros; Joel Navarrete-Espinosa; Feliciano Sanchez-Sinencio

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybrid...

  1. Intracranial Injection of Adeno-associated Viral Vectors

    OpenAIRE

    Lowery, Rebecca L.; Ania K Majewska

    2010-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    白艳霞; 闫利英; 马清涌; 杨广笑; 王全颖

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

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

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

    Science.gov (United States)

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

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

  6. 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...

  7. Identification of a natural human serotype 3 parainfluenza virus

    Directory of Open Access Journals (Sweden)

    Wang Xiao-Jing

    2011-02-01

    Full Text Available Abstract Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3 from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV.

  8. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  9. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Science.gov (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  10. The use of serotype 1-and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

    DEFF Research Database (Denmark)

    Handberg, Kurt; Nielsen, Ole L.; Jørgensen, Poul Henrik

    2001-01-01

    A serotype 1- and serotype 3-specific detection of Marek's disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from...

  11. Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil

    Science.gov (United States)

    Marinho, Paula Eillanny Silva; Bretas de Oliveira, Danilo; Candiani, Talitah Michel Sanchez; Crispim, Ana Paula Correia; Alvarenga, Pedro Paulo Martins; Castro, Fabrizia Cristina dos Santos; Abrahão, Jonatas Santos; Rios, Maria; Coimbra, Roney Santos

    2017-01-01

    To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014–2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2. PMID:27983492

  12. [Subtypes of dengue virus serotypes 2, 3 and 4 isolated in Santander District, Colombia].

    Science.gov (United States)

    Cortés, Fabián M; Gómez, Sergio Y; Ocazionez, Raquel E

    2007-01-01

    Virus serotypes 2, 3 and 4 that had circulated in Santander District, Colombia in the period 1998-2004 were analyzed. Identifying the subtype of a dengue virus serotype is a useful tool for surveillance of severe risk factors because the strain potential to cause hemorrhagic dengue makes the difference among them. Simultaneous sequence amplification technique known as restriction site specific-polymerase chain reaction (RSS-PCR) was used to determine the subtype by comparing the electrophoretic pattern of the local isolate to the reference virus. Virus serotype 2 corresponded to subtype A similar to the one isolated in Thailand (1996) and to the other isolated in Porto Rico (1986); virus serotypes 3 were of subtype C like the virus found in Sri Lanka (1990), Honduras (1995) and Porto Rico (2000); virus serotypes 4 were a variant of subtype B similar to a virus from Porto Rico (1987) and to another virus from Tahiti (1985). The study confirmed the presence in Colombia of dengue virus subtypes circulating now in the Americas.

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

    Institute of Scientific and Technical Information of China (English)

    王刚; 尉迟捷; 董小岩; 田文洪; 吴小兵

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王坚; 林茂; 吴平; 何惠娟; 刘新垣

    2012-01-01

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

  15. Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II.

    Science.gov (United States)

    Takano, Tomomi; Nakano, Kenta; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2015-05-01

    Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed "viroporins". Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4'-disothiocyano-2,2'-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II.

  16. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India

    OpenAIRE

    Guleria Randeep; Dar Lalit; Diddi Kavita; Pandey Anubhav; Chahar Harendra S; Bharaj Preeti; Kabra Sushil K; Broor Shobha

    2008-01-01

    Abstract Background Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating d...

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    陈雅宁; 王春荣; 杨宗灿; 姬朝霞; 张延瑞; 韩双印

    2012-01-01

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

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

    黄向辉; 时志斌; 王坤正; 党晓谦; 杨佩; 余鹏博

    2008-01-01

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

  1. Foot-and-mouth disease virus serotype SAT 3 in long-horned Ankole calf, Uganda.

    Science.gov (United States)

    Dhikusooka, Moses Tefula; Tjørnehøj, Kirsten; Ayebazibwe, Chrisostom; Namatovu, Alice; Ruhweza, Simon; Siegismund, Hans Redlef; Wekesa, Sabenzia Nabalayo; Normann, Preben; Belsham, Graham J

    2015-01-01

    After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest relatives isolated previously from buffalo in Uganda.

  2. Foot-and-Mouth Disease Virus Serotype SAT 3 in Long-Horned Ankole Calf, Uganda

    DEFF Research Database (Denmark)

    Dhikusooka, Moses Tefula; Tjørnehøj, Kirsten; Ayebazibwe, Chrisostom;

    2015-01-01

    After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closes...

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

    Institute of Scientific and Technical Information of China (English)

    王俊红; 问姣; 董鹏; 张静; 焦杨; 郭永红

    2015-01-01

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

  4. Dengue virus serotype 4 and chikungunya virus coinfection in a traveller returning from Luanda, Angola, January 2014.

    Science.gov (United States)

    Parreira, R; Centeno-Lima, S; Lopes, A; Portugal-Calisto, D; Constantino, A; Nina, J

    2014-03-13

    A concurrent dengue virus serotype 4 and chikungunya virus infection was detected in a woman in her early 50s returning to Portugal from Luanda, Angola, in January 2014. The clinical, laboratory and molecular findings, involving phylogenetic analyses of partial viral genomic sequences amplified by RT-PCR, are described. Although the circulation of both dengue and chikungunya viruses in Angola has been previously reported, to our knowledge this is the first time coinfection with both viruses has been detected there.

  5. Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus

    Science.gov (United States)

    Chaudhury, Sidhartha; Gromowski, Gregory D.; Ripoll, Daniel R.; Khavrutskii, Ilja V.; Desai, Valmik; Wallqvist, Anders

    2017-01-01

    Background A majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response. Methods/Principal findings We hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III. Conclusions/Significance Our results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response

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

    Science.gov (United States)

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

    1998-11-01

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

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

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    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.

  8. El Niño-Southern Oscillation, local weather and occurrences of dengue virus serotypes

    Science.gov (United States)

    Huang, Xiaodong; Clements, Archie C. A.; Williams, Gail; Devine, Gregor; Tong, Shilu; Hu, Wenbiao

    2015-11-01

    Severe dengue fever is usually associated with secondary infection by a dengue virus (DENV) serotype (1 to 4) that is different to the serotype of the primary infection. Dengue outbreaks only occur following importations of DENV in Cairns, Australia. However, the majority of imported cases do not result in autochthonous transmission in Cairns. Although DENV transmission is strongly associated with the El Niño-Southern Oscillation (ENSO) climate cycle and local weather conditions, the frequency and potential risk factors of infections with the different DENV serotypes, including whether or not they differ, is unknown. This study used a classification tree model to identify the hierarchical interactions between Southern Oscillation Index (SOI), local weather factors, the presence of imported serotypes and the occurrence of the four autochthonous DENV serotypes from January 2000-December 2009 in Cairns. We found that the 12-week moving average of SOI and the 2-week moving average of maximum temperature were the most important factors influencing the variation in the weekly occurrence of the four DENV serotypes, the likelihoods of the occurrence of the four DENV serotypes may be unequal under the same environmental conditions, and occurrence may be influenced by changes in global and local environmental conditions in Cairns.

  9. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India

    Directory of Open Access Journals (Sweden)

    Guleria Randeep

    2008-01-01

    Full Text Available Abstract Background Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak. Results Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5% were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19% dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified. Conclusion This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.

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

    Institute of Scientific and Technical Information of China (English)

    杜文贞; 于天霞

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    董长征; 董秀芳; 李文玲; 岳向勇; 郭韬; 梁传栋; 赵文清

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王艳娇; 马厚勋; 李宝善; 吴平

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    徐波; 朱光辉; 夏金堂; 翁杰锋; 李书华; 李雯

    2008-01-01

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

  14. Proteomic analysis of an Aedes albopictus cell line infected with Dengue serotypes 1 and 3 viruses

    Directory of Open Access Journals (Sweden)

    Thomas Frédéric

    2011-07-01

    Full Text Available Abstract Background Proteomic analysis was performed to identify proteins regulated during infection by Dengue serotypes 1 and 3 in an Aedes albopictus cell line. The potential of these viruses to cause severe disease at primary infection is of interest although few studies have been performed with these two Dengue serotypes. Results The most relevant observation of our study is the significant overexpression of proteins involved in the cellular stress response and the glycolysis pathway after 48 hours of infection. Viral infection activates the translation of some host genes, which may result in stress due to responses involving unfolded proteins. Conclusions Therefore, the oxidation reduction and glycolytic mechanisms could participate in the antiviral response against Dengue virus. The results of our study should help to improve our knowledge of the virus-mosquito interaction at a cellular level with the aim of designing efficient strategies for the control of Dengue virus.

  15. Genome Sequence of Bluetongue virus Serotype 17 Isolated in Brazil in 2014.

    Science.gov (United States)

    Matos, Ana Carolina Diniz; Rosa, Júlio César Câmara; Nomikou, Kyriaki; Guimarães, Lorena Lima Barbosa; Costa, Érica Azevedo; Guedes, Maria Isabel Maldonado Coelho; Driemeier, David; Lobato, Zélia Inês Portela; Mertens, Peter Paul Clement

    2016-10-27

    The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.

  16. Correlation of Serotype-Specific Dengue Virus Infection with Clinical Manifestations

    Science.gov (United States)

    Halsey, Eric S.; Marks, Morgan A.; Gotuzzo, Eduardo; Fiestas, Victor; Suarez, Luis; Vargas, Jorge; Aguayo, Nicolas; Madrid, Cesar; Vimos, Carlos; Kochel, Tadeusz J.; Laguna-Torres, V. Alberto

    2012-01-01

    Background Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype. Methodology and Principal Findings Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations. Conclusions/Significance Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype. PMID:22563516

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

    Science.gov (United States)

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

    2010-06-10

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

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

    Science.gov (United States)

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

    2016-02-01

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

  19. Studies of the Antigenic relationships between Bluetongue virus serotypes 2, 9 AND 15 isolated in Andhra Pradesh, India

    Directory of Open Access Journals (Sweden)

    Sreenivasulu Daggupati

    Full Text Available The presence of multiple serotypes of the midge-borne bluetongue virus and lack of effective vaccine are the major impediments in controlling bluetongue in sheep. Attempts are being made to develop a vaccine employing the available serotypes to control the disease in the state. Hence, it is essential to identify the antigenic relationships among the serotypes to identify the candidate strains to be incorporated in the preparation of vaccine. To understand the antigenic relationships between Bluetongue virus -2, 9 and 15 serotypes, the viruses were propagated in BHK21 cell lines, purified using PEG precipitation method and purified virus used to raise hyper immune serum in rabbits. Neutralizing antibodies for the BTV serotypes were detected by day 21 PI. Reciprocal cross neutralization test was employed to determine the R% values between BTV-2, 9 and 15 which indicated the extent of antigenic relationships among the serotypes. R% value between BTV-2 and BTV-9 was recorded as 2.8. R% value of 3.53 and 2.8 were observed between BTV-2 & 15 and BTV-9 & 15 respectively. The R% values recorded in the present study revealed a weak antigenic relationship between the BTV serotypes,indicating that the serotypes are highly divergent. [Vet. World 2011; 4(10.000: 444-448

  20. Effect of Culicoides sonorensis salivary proteins on clinical disease outcome in experimental Bleutongue virus serotype 8 infection of Dorset sheep

    NARCIS (Netherlands)

    Drolet, B.S.; Reister, L.M.; Lehiy, C.J.; Rijn, van P.A.; Bowen, R.A.

    2015-01-01

    The severity of Bluetongue clinical disease in ruminants varies greatly depending on the outbreak serotype/strain, animal species/breed, and immune status of the herd. To predict disease risk from any of the 26 Bluetongue virus (BTV) serotypes identified to date, experimental animal susceptibility s

  1. Dengue Virus Serotypes Circulating in Khyber Pakhtunkhwa Province, Pakistan, 2013-2015.

    Science.gov (United States)

    Suleman, Muhammad; Faryal, Rani; Alam, Muhammad Masroor; Sharif, Salmaan; Shaukat, Shahzad; Aamir, Uzma Bashir; Khurshid, Adnan; Angez, Mehar; Umair, Massab; Sufian, Mian Muhammad; Arshad, Yasir; Zaidi, Syed Sohail Zahoor

    2017-03-01

    From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.

  2. Spatial Trend of Foot and Mouth Disease Virus (FMDV) Serotypes in Cattle and Buffaloes, Pakistan

    Institute of Scientific and Technical Information of China (English)

    Muhammad Abubakar; Muhammad Javed Arshed; Qurban Ali; Manzoor Hussain

    2012-01-01

    The present study describes the frequency of Foot and Mouth Disease (FMD) virus serotypes (O,A and Asia-1) in major regions (all provinces) of Pakistan using Indirect Sandwich ELISA.Also,spatial distribution of various FMD serotypes and their comparison is discussed.A total of 590 samples (Epithelial tissue) have been analyzed during a period of five years (2005-2009).Out of 590 samples,180 were found positive,giving an overall confirmation of FMDV about 33.2 %.Of the prevalent serotypes,FMDV ‘O’ serotype caused most outbreaks (20.7 %),followed by serotype A (6.6 %) and serotype Asia-1 (4.6 %) while there was no positive case of type ‘C’.The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas.Based on the data of 590 samples (>50 outbreaks),the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %,while in cattle alone,it was 37.1%,higher than in buffalo (28.7 %).There were eight cases of mixed serotypes infection,indicating the presence of endemic state of disease.Another significant feature was the change over time.In phase-I (2005-2007),there was an overall prevalence of 29.4 %,while the occurrence of the serotype O,A and Asia-1 was 20.4 %,2.9 % and 4.7 %,respectively.During phase-II (2008-2009),the overall prevalence was 59.21%,while those of serotype O,A and Asia-1 were 22.4 %,31.6 % and 4.0 %,respectively.This clearly indicated a shift from serotype O to A,which may help to explain the occurrence of more severe outbreaks,despite vaccination.

  3. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  4. Dengue virus serotype 2 from a sylvatic lineage isolated from a patient with dengue hemorrhagic fever.

    Directory of Open Access Journals (Sweden)

    Jane Cardosa

    Full Text Available Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF with thrombocytopenia (12,000/ul and a raised hematocrit (29.5% above baseline in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.

  5. Different serotypes of dengue viruses differently regulate the expression of the host cell antigen processing machinery.

    Science.gov (United States)

    Gan, Chye Sheng; Yusof, Rohana; Othman, Shatrah

    2015-09-01

    Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

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

    Science.gov (United States)

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

    2016-10-01

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

  7. Analysis of Recent Serotype O Foot‐and‐Mouth Disease Viruses from Livestock in Kenya: Evidence of Four Independently Evolving Lineages

    DEFF Research Database (Denmark)

    Wekesa, S. N.; Muwanika, V. B.; Siegismund, H. R.;

    2015-01-01

    Foot‐and‐mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these virus...

  8. Rabbit and human hepatitis E virus strains belong to a single serotype.

    Science.gov (United States)

    Wang, Song; Cheng, Xianfeng; Dai, Xing; Dong, Chen; Xu, Mingjie; Liang, Jiuhong; Dong, Min; Purdy, Michael A; Meng, Jihong

    2013-09-01

    Hepatitis E virus (HEV) is a zoonotic pathogen and all four established genotypes of HEV belong to a single serotype. The recently identified rabbit HEV is antigenically and genetically related to human HEV. It is unclear whether rabbit HEV belongs to the same serotype as human HEV. The purpose of this study was to determine the serotypic relationship between rabbit and human HEVs. HEV ORF2 recombinant capsid protein p166 (amino acids 452-617) of four known HEV genotypes and rabbit HEV were used to induce immune serum, which were evaluated for their ability to neutralize human HEV genotype 1, 4, and rabbit HEV strains by an in vitro PCR-based HEV neutralization assay. Immune sera of five kinds of p166 proteins were all found to neutralize or cross-neutralize the three different HEV strains, suggesting a common neutralization epitope(s) existing between human and rabbit HEV. Rabbit models of a second-passage rabbit HEV strain, JS204-2, and a genotype 4 human HEV strain, NJ703, were established as evidenced by fecal virus shedding, viremia and anti-HEV IgG seroconversion. Six rabbits, recovered from JS204 infection, were challenged with NJ703, and another six recovered from NJ703 infection were challenged with JS204-2. After challenge, viremia was not detected, shorter fecal virus shedding durations and obvious early stage declines in anti-HEV IgG values were observed. The results from this study indicate that rabbit HEV belongs to the same serotype as human HEV.

  9. Changing pattern of dengue virus serotypes circulating during 2008-2012 and reappearance of dengue serotype 3 may cause outbreak in Kolkata, India.

    Science.gov (United States)

    Saha, Kallol; Ghosh, Monika; Firdaus, Rushna; Biswas, Aritra; Seth, Bikash; Bhattacharya, Debojyoti; Mukherjee, Kheya; Sadhukhan, Provash Chandra

    2016-10-01

    Dengue virus infection is a major cause of morbidity within the endemic tropical and subtropical regions of the world. Dengue virus has four distinct serotypes with specific clinical manifestations. In this study, we observed the changing pattern of dengue serotypes, age-wise dengue infection and useful sero-detection methods needed in a dengue endemic region. We identified dengue serotypes during a period of 5 years among patients with dengue symptoms visiting one of the largest tertiary care infectious disease hospitals of eastern India in Kolkata. A total of 433 dengue RNA positive samples were isolated from 712 acute dengue suspected cases. Age wise distribution highlighted the susceptible age group being >21 years (24.02%) followed by 11-15 years (21.71%) and 5-10 years (21.02%) of the total infected population. Higher numbers of infected cases were found within females as they are involved in more indoor works. The period of study experienced two dengue outbreaks one in 2008 and another in 2012. For early dengue detection, NS1 was found to be more confirmatory than IgM ELISA regarding sensitivity and specificity. DENV-1, 2, and 4 serotypes were the common circulating strains from 2008 until 2010, after which DENV-3 serotype infections rise and led to a massive dengue outbreak in Kolkata with increased numbers of DHF and DSS cases in 2012. The finding within our study emphasizes the public health importance of such prospective surveillance programs with respect to the changing dengue viral etiology and serotypes. J. Med. Virol. 88:1697-1702, 2016. © 2016 Wiley Periodicals, Inc.

  10. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Science.gov (United States)

    Mattia, Kimberly; Puffer, Bridget A; Williams, Katherine L; Gonzalez, Ritela; Murray, Meredith; Sluzas, Emily; Pagano, Dan; Ajith, Sandya; Bower, Megan; Berdougo, Eli; Harris, Eva; Doranz, Benjamin J

    2011-01-01

    The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

  11. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Directory of Open Access Journals (Sweden)

    Kimberly Mattia

    Full Text Available The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

  12. Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India.

    Directory of Open Access Journals (Sweden)

    Nazia Afreen

    2016-03-01

    Full Text Available Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011-2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011-2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

  13. Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India.

    Science.gov (United States)

    Afreen, Nazia; Naqvi, Irshad H; Broor, Shobha; Ahmed, Anwar; Kazim, Syed Naqui; Dohare, Ravins; Kumar, Manoj; Parveen, Shama

    2016-03-01

    Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011-2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011-2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

  14. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

    Science.gov (United States)

    In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

  15. Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002–2009

    DEFF Research Database (Denmark)

    Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia;

    2011-01-01

    of FMDV serotype A in the region. The A22/Iraq FMDV vaccine is antigenically distinct from the A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino...

  16. Bluetongue virus serotype 6 in Europe in 2008 - Emergence and disappearance of an unexpected non-virulent BTV

    NARCIS (Netherlands)

    Rijn, van P.A.; Geurts, Y.; Spek, van der A.N.; Veldman, D.; Gennip, van H.G.P.

    2012-01-01

    Bluetongue viruses (BTVs) could invade N-W Europe similar to BTV serotype 8 (BTV8/net06), since the source and route of introduction of this virus has not been solved. Therefore, the Dutch survey for Bluetongue by PCR testing was extended by further analysis of PCR positives to identify the involved

  17. Natural strain variation and antibody neutralization of dengue serotype 3 viruses.

    Directory of Open Access Journals (Sweden)

    Wahala M P B Wahala

    2010-03-01

    Full Text Available Dengue viruses (DENVs are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII. It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.

  18. Vector competence of Aedes albopictus from Houston, Texas, for dengue serotypes 1 to 4, yellow fever and Ross River viruses.

    Science.gov (United States)

    Mitchell, C J; Miller, B R; Gubler, D J

    1987-09-01

    A combination of virus infection and transmission experiments showed that a Houston, Texas strain of Aedes albopictus is a competent vector for dengue (DEN), yellow fever (YF) and Ross River (RR) viruses. However, at 14 days incubation, DEN virus infection rates in a Puerto Rican strain of Aedes aegypti were significantly higher for each of the four DEN serotypes, except DEN-1, than in Houston Ae. albopictus fed simultaneously on the same virus suspensions. The degree of correlation between disseminated DEN infection rates in Houston Ae. albopictus and transmission to an in vitro system ranged from 42 to 88% for the four DEN serotypes. No significant difference was noted in YF virus infection rates or transmission rates in the two mosquito species fed on the same virus suspensions and incubated for the same time period. Also, RR virus infection and transmission rates in Houston and Hawaiian strains of Ae. albopictus were generally comparable.

  19. Improved Serotype-Specific Dengue Virus Detection in Trinidad and Tobago using a Multiplex, Real-Time RT-PCR

    Science.gov (United States)

    Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.

    2014-01-01

    Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614

  20. Exploiting serological data to understand the epidemiology of foot-and-mouth disease virus serotypes circulating in Libya.

    Science.gov (United States)

    Eldaghayes, Ibrahim; Dayhum, Abdunaser; Kammon, Abdulwahab; Sharif, Monier; Ferrari, Giancarlo; Bartels, Christianus; Sumption, Keith; King, Donald P; Grazioli, Santina; Brocchi, Emiliana

    2017-01-01

    Sporadic outbreaks of foot-and-mouth disease (FMD) have occurred in Libya for almost fifty years. During the spring of 2013, a countrywide serosurvey was undertaken to assess the level of FMD virus circulation and identify FMD virus serotypes in the country. A total of 4221 sera were collected, comprising samples from large ruminants (LR; n=1428 samples from 357 farms) and small ruminants (SR; n=2793 samples from 141 farms). FMD sero-prevalence of NSP antibodies determined by ELISA were 19.0% (271/1428) with 95% CI (16.9 - 21.0) and 13.5% (378/2793) with 95% CI (12.3 - 14.8) for LR and SR samples, respectively. The sero-prevalence of NSP antibodies in LR was 12.3% and 19.8% for age group 2 year, respectively (X(2)= 118.1, P= 0.000). These observed NSP serologic profiles support the hypothesis of an endemic level of FMD circulation in Libya. All positive sera were tested for SP antibodies for O, A and SAT-2 FMD virus serotypes. Serotype O was the dominant circulating serotype followed by serotype A, while evidence of SAT-2 was not found. These data provide an insight into the wider epidemiology of FMD in Libya, and contribute to field and laboratory investigations that during 2013 serotype O (O/ME-SA/Ind-2001 lineage) was isolated from clinical samples collected from the country.

  1. Exploiting serological data to understand the epidemiology of foot-and-mouth disease virus serotypes circulating in Libya

    Science.gov (United States)

    Eldaghayes, Ibrahim; Dayhum, Abdunaser; Kammon, Abdulwahab; Sharif, Monier; Ferrari, Giancarlo; Bartels, Christianus; Sumption, Keith; King, Donald P.; Grazioli, Santina; Brocchi, Emiliana

    2017-01-01

    Sporadic outbreaks of foot-and-mouth disease (FMD) have occurred in Libya for almost fifty years. During the spring of 2013, a countrywide serosurvey was undertaken to assess the level of FMD virus circulation and identify FMD virus serotypes in the country. A total of 4221 sera were collected, comprising samples from large ruminants (LR; n=1428 samples from 357 farms) and small ruminants (SR; n=2793 samples from 141 farms). FMD sero-prevalence of NSP antibodies determined by ELISA were 19.0% (271/1428) with 95% CI (16.9 – 21.0) and 13.5% (378/2793) with 95% CI (12.3 – 14.8) for LR and SR samples, respectively. The sero-prevalence of NSP antibodies in LR was 12.3% and 19.8% for age group 2 year, respectively (X2= 118.1, P= 0.000). These observed NSP serologic profiles support the hypothesis of an endemic level of FMD circulation in Libya. All positive sera were tested for SP antibodies for O, A and SAT-2 FMD virus serotypes. Serotype O was the dominant circulating serotype followed by serotype A, while evidence of SAT-2 was not found. These data provide an insight into the wider epidemiology of FMD in Libya, and contribute to field and laboratory investigations that during 2013 serotype O (O/ME-SA/Ind-2001 lineage) was isolated from clinical samples collected from the country. PMID:28180094

  2. Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

    Energy Technology Data Exchange (ETDEWEB)

    Chandramouli, Sumana; Joseph, Jeremiah S.; Daudenarde, Sophie; Gatchalian, Jovylyn; Cornillez-Ty, Cromwell; Kuhn, Peter (Scripps)

    2010-03-04

    With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-{angstrom} crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.

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

    Institute of Scientific and Technical Information of China (English)

    景阳; 郑国玺; 祝康; 刘晖; 张文

    2015-01-01

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

  4. Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination

    DEFF Research Database (Denmark)

    Balinda, Sheila; Siegismund, Hans; Muwanika, Vincent;

    2010-01-01

    Background Foot-and-mouth disease (FMD) is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutio...

  5. Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR 2 Primers

    Directory of Open Access Journals (Sweden)

    Abdul Rahman Siregar

    2015-11-01

    Full Text Available Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3. This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

  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. Role of endogenous avian leukosis virus and serotype 2 Marek’s disease virus in enhancement of spontaneous lymphoid-leukosis-like tumors in chickens

    Science.gov (United States)

    The influence of endogenous subgroup E avian Leukosis virus (ALV-E) and strain SB-1 of serotype 2 Marek’s disease virus (MDV) on the enhancement of spontaneous lymphoid leukosis (LL)-like tumors was studied in chickens of Avian Disease and Oncology Laboratory (ADOL) line named 0.TVB*S1, or RFS. This...

  8. Spontaenous Avian Leukosis Virus-like lymphomas in specific-pathogen-free chickens inoculated with serotype 2 Marek’s disease virus

    Science.gov (United States)

    Chickens of Avian Disease and Oncology Laboratory (ADOL) line alv6, known to develop spontaneous avian leukosis virus (ALV)-like lymphomas at two years of age or older, were inoculated either in-ovo, or at 1 day of age with strain SB-1 of serotype 2 Marek’s disease virus (MDV). Inoculated and uninoc...

  9. Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype

    Directory of Open Access Journals (Sweden)

    Kimberly A. Dowd

    2016-08-01

    Full Text Available Recent epidemics of Zika virus (ZIKV have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

  10. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone.

    Science.gov (United States)

    Osorio, Jorge E; Wallace, Derek; Stinchcomb, Dan T

    2016-01-01

    Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever.

  11. Molecular epidemiology of dengue virus serotypes 2 and 3 in Paraguay during 2001-2006: the association of viral clade introductions with shifting serotype dominance.

    Science.gov (United States)

    Aquino, Jose D J Diaz; Tang, Wei-Feng; Ishii, Ryoichi; Ono, Tetsuro; Eshita, Yuki; Aono, Hiroshi; Makino, Yoshihiro

    2008-11-01

    To determine the genetic variability of dengue viruses (DENVs) in Paraguay, the complete envelope gene was sequenced for 4 DENV-2 and 22 DENV-3 strains isolated from 2001 to 2006. The sequence data were used in Bayesian phylogenetic analyses, which revealed that Paraguayan DENV-2 strains fell into two distinct clades within the American/Asian genotype, thus suggesting that the introduction of a new DENV-2 clade was likely associated with the shift of dominant serotype from DENV-3 to DENV-2 in 2005 and might have caused an outbreak of DENV-2. This study also indicated that DENV-3 strains fell into genotype III, of which, several 2006 isolates varied from the remaining isolates in their tree locations. The introduction of this new clade was likely associated with the shift of dominant serotype from DENV-2 to DENV-3 in 2006 and might have caused an epidemic of DENV-3. More data are needed to test this hypothesis.

  12. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, J.L.; Langeveld, J.P.M.; Venteo, A.;

    1999-01-01

    African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological...... immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule...

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

    Science.gov (United States)

    2004-08-09

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

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

    Science.gov (United States)

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

    2016-01-01

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

  15. Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1

    Science.gov (United States)

    Messer, William B.; Yount, Boyd L.; Royal, Scott R.; de Alwis, Ruklanthi; Widman, Douglas G.; Smith, Scott A.; Crowe, James E.; Pfaff, Jennifer M.; Kahle, Kristen M.; Doranz, Benjamin J.; Ibarra, Kristie D.; Harris, Eva

    2016-01-01

    ABSTRACT The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they

  16. Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes.

    Science.gov (United States)

    Lee, Lucy F; Silva, Robert F; Cui, Xiaoping; Zhang, Huanmin; Heidari, Mohammad; Reddy, Sanjay M

    2007-12-01

    The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6DeltaLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5DeltaLORF11 plaques were evident at 12-13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5DeltaLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.

  17. Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

    NARCIS (Netherlands)

    Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.

    2009-01-01

    Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven ou

  18. Genetic diversity of foot-and-mouth disease virus serotype O in Pakistan and Afghanistan, 1997–2009

    DEFF Research Database (Denmark)

    Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia;

    2011-01-01

    Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan; serotypes O, A and Asia-1 of the virus are responsible for the outbreaks in these countries with FMDV type O usually being the most common. In the present study, the nucleotide sequences encoding the FMDV capsid protein VP1 from...

  19. Diversity and transboundary mobility of serotype O foot-and-mouth disease virus in East Africa: Implications for vaccination policies

    DEFF Research Database (Denmark)

    Balinda, Sheila; Sangula, Abraham; Heller, Rasmus;

    2010-01-01

    Foot-and-mouth disease (FMD) virus serotype O has been responsible for most reported outbreaks of the disease in East Africa. A sustained campaign for the past 40 years to control FMD mainly by vaccination, combined with quarantine and zoosanitary measures has been undertaken with limited success...

  20. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita;

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed...

  1. A label-free optical biosensor for serotyping "unknown" influenza viruses

    Science.gov (United States)

    Zhang, Hanyuan; Henry Dunand, Carole; Wilson, Patrick; Miller, Benjamin L.

    2016-05-01

    The ability to accurately classify influenza viruses is critical to understanding patterns of infection, vaccine efficacy, and to the process of developing new vaccines. Unfortunately, this task is hampered both by the virus' ability to undergo antigenic drift and shift (rendering it a "previously unknown" strain), and by technological limitations. In an effort to overcome these challenges, we have developed a label-free human monoclonal antibody array for flu serology, using a pattern recognition approach to assign virus serotype. The array is built on the Arrayed Imaging Reflectometry (AIR) platform. AIR relies on the creation of a near-perfect antireflective condition on the surface of a silicon chip. When this antireflective condition is perturbed because of binding to an antibody spot (or other immobilized probe molecule), binding may be sensitively and quantitatively detected as an increase in reflected light. We describe fabrication and characterization of the array, and preliminary testing with isolated influenza hemagglutinin. We anticipate that this approach may be extended to other viruses by expansion of the array.

  2. Recombinant Bivalent Vaccine against Foot-and-Mouth Disease Virus Serotype O/A Infection in Guinea Pig

    Institute of Scientific and Technical Information of China (English)

    Jian-Zhong YI; Ming-Qiu LIU; Cai-Zhu ZHU; Qiang ZHANG; Zu-Tian SHENG; Qing-Yun DU; Wei-Yao YAN; Zhao-Xin ZHENG

    2004-01-01

    In this study, two DNA fragments encoding amino acid (141-160)-(21-40)-(141-160) of the VP 1 of FMDV (foot-and-mouth disease virus) serotype O and (138-160)-(21-40)-( 138-160) of the serotype A FMDV were chemically synthesized. These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A. The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovineIgG heavy chain coding sequence. Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses. FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 μg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively. 70% and 57%of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.

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

    Science.gov (United States)

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

    2010-11-01

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

  4. Two dimensional VOPBA reveals laminin receptor (LAMR1 interaction with dengue virus serotypes 1, 2 and 3

    Directory of Open Access Journals (Sweden)

    Cardosa Mary

    2005-03-01

    Full Text Available Abstract Background The search for the dengue virus receptor has generated many candidates often identified only by molecular mass. The wide host range of the viruses in vitro combined with multiple approaches to identifying the receptor(s has led to the notion that many receptors or attachment proteins may be involved and that the different dengue virus serotypes may utilize different receptors on the same cells as well as on different cell types. Results In this study we used sequential extraction of PS Clone D cell monolayers with the detergent β-octylglucopyranoside followed by sodium deoxycholate to prepare a cell membrane-rich fraction. We then used 2 dimensional (2D gel electrophoresis to separate the membrane proteins and applied a modified virus overlay protein binding assay (VOPBA to show that dengue virus serotypes 1, 2 and 3 all interact with the 37 kDa/67 kDa laminin receptor (LAMR1, a common non-integrin surface protein on many cell types. Conclusion At least 3 of the 4 dengue serotypes interact with the 37 kDa/67 kDa laminin receptor, LAMR1, which may be a common player in dengue virus-cell surface interaction.

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

    Institute of Scientific and Technical Information of China (English)

    赛佳明; 张慧琴

    2006-01-01

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

  6. Bovine adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry.

    Science.gov (United States)

    Li, Xiaoxin; Bangari, Dinesh S; Sharma, Anurag; Mittal, Suresh K

    2009-09-30

    Bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin-Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized alpha(2,3)-linked and alpha(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.

  7. Genetic diversity of Chikungunya virus, India 2006-2010: evolutionary dynamics and serotype analyses.

    Science.gov (United States)

    Sumathy, K; Ella, Krishna M

    2012-03-01

    The genetic diversity of Chikungunya virus (CHIKV) causing recurring outbreaks in India since 2006 was studied. The 2006 epidemic was caused by a virus strain of the East, Central and South African (ECSA) genotype with 226A in the E1 glycoprotein. The variant strain with E1-A226V mutation caused outbreaks since 2007 in the state of Kerala where Aedes albopictus is the abundant mosquito vector. Molecular epidemiology data since 2007 is scarce from other regions of the country. RT-PCR, sequencing and phylogenetic analyses of CHIKV isolates from the 2009 to 2010 epidemics in the States of Tamil Nadu and Andhra Pradesh placed them in a separate clade within the ECSA lineage. The isolates of the study had 226A in the E1 glycoprotein. The isolates had a novel E1-K211E mutation that was under significant positive selection. E1-211E is highly conserved in the Asian genotype of the virus circulated by Aedes aegypti. Unique mutations in E2 glycoprotein were identified. The two sub-lineages of ECSA genotype circulating in India parallel the abundance of Ae. albopictus and Ae. aegypti. Novel mutations in the envelope glycoproteins suggest adaptive evolution of the virus to local vector abundance. Cross neutralization of the virus isolates from recurring Indian epidemics indicated that no distinct serotypes had evolved. The study has provided insights into the origin, distribution and evolutionary adaptation of the virus to local vector abundance in the region that has reportedly, the highest incidence of CHIKV infection in the world.

  8. Molecular characterization of serotype Asia-1 foot-and-mouth disease viruses in Pakistan and Afghanistan; emergence of a new genetic Group and evidence for a novel recombinant virus

    DEFF Research Database (Denmark)

    Jamal, Syed Muhammad; Ferrari, Giancarlo; Ahmed, Safia;

    2011-01-01

    Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate...... appropriate vaccine selection and tracing of outbreaks.The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998–2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1...

  9. Some epitopes conservation in non structural 3 protein dengue virus serotype 4

    Directory of Open Access Journals (Sweden)

    Tegar A. P. Siregar

    2016-03-01

    conservation ofT and B cell epitope in NS3 protein among DENV-4 strains and four serotypes DENV of Indonesia strains.Methods: Research was held at the Department of Microbiology, Faculty of Medicine, UniversitasIndonesia, June 2013 to April 2014. NS3 amino acid sequence of DENV-4 081 strain was obtained afterNS3 gene of DENV-4 081 PCR products were sequenced. T and B cell epitopes of NS3 protein of DENV-4081 strain were analysed and compared to NS3 proteins of 124 DENV-4 strains around the world and fourserotypes of Indonesia strains. World strains were isolated from America (i.e. Venezuela, Colombia, etc.and Asia (i.e. China, Singapore, etc.. For the comparison, T and B cell epitope positions of NS3 proteinwere obtained from published report.Results: Eight positions of T cell epitopes and two positions of B cell epitopes of NS3 DENV-4 081 wereidentical and conserved to NS3 protein of 124 DENV-4 strains around the world. B cell epitope of NS3 DENV-4 081 protein at aa 537-544 was found identical and conserved to four serotypes DENV of Indonesia strains.Conclusion: This wide conservation of T and B epitopes in almost all DENV-4 strains around the worldand all serotypes of Indonesia strains. (Health Science Journal of Indonesia 2015;6:126-31Keywords: dengue virus, NS3 protein, T cell epitope, B cell epitope

  10. 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…

  11. Antigenic site variation in foot-and-mouth disease virus serotype O grown under vaccinal serum antibodies in vitro.

    Science.gov (United States)

    Sarangi, Laxmi N; Mohapatra, Jajati K; Subramaniam, Saravanan; Sanyal, Aniket; Pattnaik, Bramhadev

    2013-09-01

    Foot-and-mouth disease virus (FMDV) is constantly evolving under neutralizing antibody pressure in either naturally infected or vaccinated animals. This study was carried out to understand the dynamics of evolution of antigenic sites. Neutralizing antibody-resistant populations of three strains of FMDV serotype O (INDR2/1975, IND120/2002 and IND271/2001) were isolated by serial propagation in BHK-21 cells in the presence of sub-neutralizing level of bovine vaccinal sera (BVS). In the partial neutralization escape variants, fixation of aa substitutions were observed at critical residues of all established antigenic sites of serotype O {144 of VP1 (site 1), 45 and 48 of VP1 (site 3), 72 and 134 of VP2 (Site 2)} except site 4 and 5. In majority of the variant populations, site 3 was found to be substituted and therefore immunodominance may not be associated with a particular site, rather it appears to be a virus strain and infected host specific affair. Substitutions were also observed in proximity to the identified residues {41 and 51 (βB-βC loop), 133, 140 and 143 (βG-βH loop), 201, 204 and 209 (C terminus) of VP1, 71 and 75 (βB-βC loop), 131 (βE-αB region), 174 and 179 (βG-βH loop) and 219 (C terminus) of VP3} within antigenic sites of serotype O or other serotypes which could be significant in terms of neutralizing antibody binding and immune escape. Presence of similar residues in the Indian field viruses as selected in the variants supports the importance of these sites in antigenic diversification of serotype O FMD virus.

  12. Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States

    Science.gov (United States)

    Bluetongue is caused by an arbovirus which produces widespread edema and tissue necrosis in domestic and wild ruminants that can be fatal. Bluetongue virus serotypes 10, 11, 13, and 17 are typically found throughout the United States (US), while serotype 2 was previously only detected in the southea...

  13. Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes

    Science.gov (United States)

    Lulla, Valeria; Lulla, Aleksei; Wernike, Kerstin; Aebischer, Andrea; Beer, Martin

    2016-01-01

    ABSTRACT African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic

  14. Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens

    Science.gov (United States)

    Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek'...

  15. An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.

    Science.gov (United States)

    El Garch, H; Crafford, J E; Amouyal, P; Durand, P Y; Edlund Toulemonde, C; Lemaitre, L; Cozette, V; Guthrie, A; Minke, J M

    2012-09-15

    A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

  16. Evidence for transmission of bluetongue virus serotype 26 through direct contact.

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    Carrie Batten

    Full Text Available The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26 in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.

  17. Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

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    Alam SM

    2013-08-01

    Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

  18. Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

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    Cisneros Alejandro

    2009-12-01

    Full Text Available Abstract Background Dengue (DEN is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating. Results To study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91-prM-E-NS1(2400 structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype. Conclusions This is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for

  19. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

    Science.gov (United States)

    Kochinger, Stefanie; Renevey, Nathalie; Hofmann, Martin A; Zimmer, Gert

    2014-06-13

    Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

  20. Comparison of test methodologies for foot-and-mouth disease virus serotype A vaccine matching.

    Science.gov (United States)

    Tekleghiorghis, Tesfaalem; Weerdmeester, Klaas; van Hemert-Kluitenberg, Froukje; Moormann, Rob J M; Dekker, Aldo

    2014-05-01

    Vaccination has been one of the most important interventions in disease prevention and control. The impact of vaccination largely depends on the quality and suitability of the chosen vaccine. To determine the suitability of a vaccine strain, antigenic matching is usually studied by in vitro analysis. In this study, we performed three in vitro test methods to determine which one gives the lowest variability and the highest discriminatory capacity. Binary ethylenimine inactivated vaccines, prepared from 10 different foot-and-mouth disease (FMD) virus serotype A strains, were used to vaccinate cattle (5 animals for each strain). The antibody titers in blood serum samples 3 weeks postvaccination (w.p.v.) were determined by a virus neutralization test, neutralization index test, and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). The titers were then used to calculate relationship coefficient (r1) values. These r1 values were compared to the genetic lineage using receiver operating characteristic (ROC) analysis. In the two neutralization test methods, the median titers observed against the test strains differed considerably, and the sera of the vaccinated animals did not always show the highest titers against their respective homologous virus strains. When the titers were corrected for test strain effect (scaling), the variability (standard error of the mean per vaccinated group) increased because the results were on a different scale, but the discriminatory capacity improved. An ROC analysis of the r1 value calculated on both observed and scaled titers showed that only r1 values of the liquid-phase blocking ELISA gave a consistent statistically significant result. Under the conditions of the present study, the liquid-phase blocking ELISA showed less variation and still had a higher discriminatory capacity than the other tests.

  1. Complete Genome Sequence of Pig-Originated Foot-and-Mouth Disease Virus Serotype O from Bangladesh

    Science.gov (United States)

    Ali, M. Rahmat; Ullah, Huzzat; Siddique, Mohammad Anwar

    2016-01-01

    In this article, we document the first pig-isolated complete genome sequence of foot-and-mouth disease virus type O in Bangladesh. The complete viral genome revealed a potential serotypic recombination at the 5′ untranslated region (UTR). Conventional amino acid deletion was lacking in 3A region, and antigenic heterogeneity to circulatory type O existed within the VP1 region. PMID:27789636

  2. Survey for antibodies to infectious bursal disease virus serotype 2 in wild turkeys and Sandhill Cranes of Florida, USA.

    Science.gov (United States)

    Candelora, Kristen L; Spalding, Marilyn G; Sellers, Holly S

    2010-07-01

    Captive-reared Whooping Cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997-98 and 2001-02 release seasons. Exposure to infectious bursal disease virus (IBDV) serotype 2 as evidenced by seroconversion was suspected to be the factor that precipitated these mortality events. Very little is known about the incidence of IBD in wild bird populations. Before this study, natural exposure had not been documented in wild birds of North America having no contact with captive-reared cranes, and the prevalence and transmission mechanisms of the virus in wild birds were unknown. Sentinel chickens (Gallus gallus) monitored on two Whooping Crane release sites in central Florida, USA, during the 2003-04 and 2004-05 release seasons seroconverted, demonstrating natural exposure to IBDV serotype 2. Blood samples collected from Wild Turkeys (Meleagris gallopavo) and Sandhill Cranes (Grus canadensis) in eight of 21 counties in Florida, USA, and one of two counties in southern Georgia, USA, were antibody-positive for IBDV serotype 2, indicating that exposure from wild birds sharing habitat with Whooping Cranes is possible. The presence of this virus in wild birds in these areas is a concern for the resident flock of Whooping Cranes because they nest and raise their chicks in Florida, USA. However, passively transferred antibodies may protect them at this otherwise vulnerable period in their lives.

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

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    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.

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

    NARCIS (Netherlands)

    Majowicz, Anna

    2014-01-01

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

  5. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    Science.gov (United States)

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection.

  6. Development and Evaluation of a Rapid Antigen Detection and Serotyping Lateral Flow Antigen Detection System for Foot-and-Mouth Disease Virus.

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    Kazuki Morioka

    Full Text Available We developed a lateral flow strip using monoclonal antibodies (MAbs which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV. This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 10(3 to 10(4 of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden, which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.

  7. Induction of neutralizing antibodies against four serotypes of dengue viruses by MixBiEDIII, a tetravalent dengue vaccine.

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    Hui Zhao

    Full Text Available The worldwide expansion of four serotypes of dengue virus (DENV poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII was proposed. Tandem EDIIIs of two serotypes (type 1-2 and type 3-4 of DENV connected by a Gly-Ser linker ((Gly4Ser3 were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.

  8. Characterization of foot-and-mouth disease viruses (FMDVs) from Ugandan cattle outbreaks during 2012-2013: evidence for circulation of multiple serotypes.

    Science.gov (United States)

    Namatovu, Alice; Tjørnehøj, Kirsten; Belsham, Graham J; Dhikusooka, Moses T; Wekesa, Sabenzia N; Muwanika, Vincent B; Siegismund, Hans R; Ayebazibwe, Chrisostom

    2015-01-01

    To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda's cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012-2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.

  9. Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera.

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    Ruklanthi de Alwis

    2014-10-01

    Full Text Available Dengue viruses (DENV are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to

  10. Quantitative profiling of the shedding rate of the three Marek's disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal viruses.

    Science.gov (United States)

    Islam, Aminul; Walkden-Brown, Stephen W

    2007-08-01

    The shedding profile of Marek's disease virus serotype 1 (MDV1, virulent), serotype 2 (MDV2, vaccinal) and herpesvirus of turkeys (HVT, vaccinal) in commercial broiler chickens was determined by measuring the daily rate of production of feather dander from chickens housed in isolators and by quantifying the viral load of each of these serotypes in the dander using quantitative real-time PCR (qPCR). MDV1 and HVT viruses were detectable in dander filtered from isolator exhaust air from day 7 and MDV2 from day 12 after infection and thereafter until the end of the experiment at 61 days of age of the chickens. There was no difference in shedding rate among the three MDV1 isolates. Daily shedding of MDV1 increased sharply between days 7 and 28 and stabilized thereafter at about 10(9) virus copies per chicken per day, irrespective of vaccination status. Challenge with the three different MDV1 isolates markedly increased shedding of the vaccinal viruses HVT and MDV2 in dander by 38- and 75-fold, respectively. These results demonstrate the utility of qPCR for the differentiation and quantification of different MDV serotypes in feather dander and have significant implications for the routine monitoring of Marek's disease using qPCR assays of dust, for epidemiological modelling of the behaviour and spread of MDVs in chicken populations and for studies into the evolution of virulence in MDV1 in the face of blanket vaccination with imperfect vaccines that ameliorate disease but do not prevent infection and replication of virulent virus.

  11. Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates

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    Shyamal G

    2005-01-01

    Full Text Available Purpose: To standardize and apply a polymerase chain reaction (PCR on the glycoprotein D gene to differentiate Herpes simplex virus (HSV 1 & 2 serotypes in culture negative intraocular specimens. Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV and varicella zoster virus (VZV by nucleic acid amplification methods. Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens, and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

  12. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences

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    Natália Oliveira

    2015-07-01

    Full Text Available Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF and dengue shock syndrome (DSS. Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3. DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM. Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

  13. A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences.

    Science.gov (United States)

    Oliveira, Natália; Souza, Elaine; Ferreira, Danielly; Zanforlin, Deborah; Bezerra, Wessulla; Borba, Maria Amélia; Arruda, Mariana; Lopes, Kennya; Nascimento, Gustavo; Martins, Danyelly; Cordeiro, Marli; Lima-Filho, José

    2015-07-01

    Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3). DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV) was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM). Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

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

    Science.gov (United States)

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

    2017-01-01

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

  15. Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination

    Directory of Open Access Journals (Sweden)

    Balinda Sheila N

    2010-08-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced. Results Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L, the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965 and serotype O viral isolate (K/117/1999 revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O. Conclusions Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2, a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A

  16. Evolutionary analysis of structural protein gene VP1 of foot-and-mouth disease virus serotype Asia 1.

    Science.gov (United States)

    Zhang, Qingxun; Liu, Xinsheng; Fang, Yuzhen; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Ding, Yaozhong; Chen, Haotai; Shao, Junjun; Zhao, Furong; Lin, Tong; Chang, Huiyun; Zhang, Jie; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I-VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10(-3) substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown.

  17. Molecular surveillance of dengue in Semarang, Indonesia revealed the circulation of an old genotype of dengue virus serotype-1.

    Directory of Open Access Journals (Sweden)

    Sukmal Fahri

    Full Text Available Dengue disease is currently a major health problem in Indonesia and affects all provinces in the country, including Semarang Municipality, Central Java province. While dengue is endemic in this region, only limited data on the disease epidemiology is available. To understand the dynamics of dengue in Semarang, we conducted clinical, virological, and demographical surveillance of dengue in Semarang and its surrounding regions in 2012. Dengue cases were detected in both urban and rural areas located in various geographical features, including the coastal and highland areas. During an eight months' study, a total of 120 febrile patients were recruited, of which 66 were serologically confirmed for dengue infection using IgG/IgM ELISA and/or NS1 tests. The cases occurred both in dry and wet seasons. Majority of patients were under 10 years old. Most patients were diagnosed as dengue hemorrhagic fever, followed by dengue shock syndrome and dengue fever. Serotyping was performed in 31 patients, and we observed the co-circulation of all four dengue virus (DENV serotypes. When the serotypes were correlated with the severity of the disease, no direct correlation was observed. Phylogenetic analysis of DENV based on Envelope gene sequence revealed the circulation of DENV-2 Cosmopolitan genotype and DENV-3 Genotype I. A striking finding was observed for DENV-1, in which we found the co-circulation of Genotype I with an old Genotype II. The Genotype II was represented by a virus strain that has a very slow mutation rate and is very closely related to the DENV strain from Thailand, isolated in 1964 and never reported in other countries in the last three decades. Moreover, this virus was discovered in a cool highland area with an elevation of 1,001 meters above the sea level. The discovery of this old DENV strain may suggest the silent circulation of old virus strains in Indonesia.

  18. Development and characterization of a reverse genetic system for studying dengue virus serotype 3 strain variation and neutralization.

    Directory of Open Access Journals (Sweden)

    William B Messer

    Full Text Available Dengue viruses (DENV are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I, Thailand 1995 (genotype II, Sri Lanka 1989 and Cuba 2002 (genotype III and Puerto Rico 1977 (genotype IV. We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  20. European bluetongue serotype 8

    NARCIS (Netherlands)

    Drolet, Barbara S.; Reister-Hendricks, Lindsey M.; Podell, Brendan K.; Breitenbach, Jonathan E.; Mcvey, D.S.; Rijn, van Piet A.; Bowen, Richard A.

    2016-01-01

    Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive serotyp

  1. Emergence and Distribution of Foot-and-Mouth Disease Virus Serotype A and O in Bangladesh.

    Science.gov (United States)

    Nandi, S P; Rahman, M Z; Momtaz, S; Sultana, M; Hossain, M A

    2015-06-01

    Foot-and-mouth disease (FMD) is endemic in Bangladesh and is predominantly due to FMDV serotype O. In 2012, FMD outbreaks were identified in five different districts of Bangladesh. Of 56 symptomatic cattle epithelial tissue samples, diagnostic PCR assay based on 5'-URT detected 38 FMDV infections. Viral genotyping targeting VP1-encoding region confirmed emergence of two distinct serotypes, A and O with an abundance of serotype A in Chittagong and Gazipur districts and serotype O in Pabna and Faridpur. Only single lineage of both A and O was retrieved from samples of five different regions. Sequencing and phylogenetic analysis of VP1 sequences revealed that serotype O sequences were closely related to the Ind 2001 sublineage of Middle East-South Asia (ME-SA) topotype that was previously circulating in Bangladesh, and serotype A sequences belonging to the genotype VII that was dominant in India during the last decade. The results suggest that extensive cross-border animal movement from neighbouring countries is the most likely source of FMDV serotypes in Bangladesh.

  2. Evolutionary analysis of foot-and-mouth disease virus serotype SAT 1 isolates from east africa suggests two independent introductions from southern africa

    DEFF Research Database (Denmark)

    Sangula, Abraham K.; Belsham, Graham; Muwanika, Vincent B.

    2010-01-01

    appreciation of the epidemiological status of serotype SAT 1 virus in the region, we inferred its evolutionary and phylogeographic history by means of genealogy-based coalescent methods using 53 VP1 coding sequences covering a sampling period from 1948-2007. Results: The VP1 coding sequence of 11 serotype SAT...... and highlight the importance of a regional approach to trans-boundary animal disease control in order to monitor circulating strains and apply appropriate vaccines....

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

    Science.gov (United States)

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

    2014-01-01

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

  4. The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

    Science.gov (United States)

    Chang, Kyung-Soo; Lee, Sung-Il; Ohashi, Kazuhiko; Ibrahim, Ahmed; Onuma, Misao

    2002-05-01

    In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

  5. Identification of conserved and HLA-A*2402-restricted epitopes in Dengue virus serotype 2.

    Science.gov (United States)

    Duan, Zhi-Liang; Liu, Hui-Fang; Huang, Xi; Wang, Si-Na; Yang, Jin-Lin; Chen, Xin-Yu; Li, De-Zhou; Zhong, Xiao-Zhi; Chen, Bo-Kun; Wen, Jin-Sheng

    2015-01-22

    In this study, we set out to identify dengue virus serotype 2 (DENV-2)-specific HLA-A*2402-restricted epitopes and determine the characteristics of T cells generated to these epitopes. We screened the full-length amino-acid sequence of DENV-2 to find potential epitopes using the SYFPEITHI algorithm. Twelve putative HLA-A*2402-binding peptides conserved in hundreds of DENV-2 strains were synthesized, and the HLA restriction of peptides was tested in HLA-A*2402 transgenic mice. Nine peptides (NS4b(228-237), NS2a(73-81), E(298-306), M(141-149), NS4a(96-105), NS4b(159-168), NS5(475-484), NS1(162-171), and NS5(611-620)) induced high levels of peptide-specific IFN-γ-secreting cells in HLA-A*2402 transgenic mice. Apart from IFN-γ, NS4b(228-237-), NS2a(73-81-) and E(298-306)-specific CD8(+) cells produced TNF-α and IL-6 simultaneously, whereas M(141-149-) and NS5(475-484-) CD8(+) cells produced only IL-6. Moreover, splenic mononuclear cells (SMCs) efficiently recognized and killed peptide-pulsed splenocytes. Furthermore, each of nine peptides could be recognized by splenocytes from DENV-2-infected HLA-A*2402 transgenic mice. The SMCs from HLA-A*2402 transgenic mice immunized with nine immunogenic peptides efficiently killed DENV-2-infected splenic monocytes. The present identified epitopes have the potential to be new diagnostic tools for characterization of T-cell immunity in DENV infection and may serve as part of a universal epitope-based vaccine.

  6. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism.

    Science.gov (United States)

    Thullier, P; Demangel, C; Bedouelle, H; Mégret, F; Jouan, A; Deubel, V; Mazié, J C; Lafaye, P

    2001-08-01

    Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296-400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE(306-314) and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE(296-400) in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE(306-314) synthetic peptide and this reaction was inhibited by DE(296-400). Moreover, DE(306-314) could block dengue virus infection of target cells in an in vitro assay. A three-dimensional model of DE revealed that the three amino acids shared by DE(296-400) and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE(306-314) region. Since 4E11 blocked the binding of DE(296-400) to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE(306-314) with target cell-displayed HSHS. Our data suggest that the DE(306-314) segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE(306-314) to HSHS may be antiviral reagents of therapeutic interest.

  7. Meta-Analysis of Dengue Severity during Infection by Different Dengue Virus Serotypes in Primary and Secondary Infections.

    Directory of Open Access Journals (Sweden)

    Kuan-Meng Soo

    Full Text Available Dengue virus (DENV infection is currently a major cause of morbidity and mortality in the world; it has become more common and virulent over the past half-century and has gained much attention. Thus, this review compared the percentage of severe cases of both primary and secondary infections with different serotypes of dengue virus.Data related to the number of cases involving dengue fever (DF, dengue hemorrhagic fever (DHF, dengue shock syndrome (DSS or severe dengue infections caused by different serotypes of dengue virus were obtained by using the SCOPUS, the PUBMED and the OVID search engines with the keywords "(dengue* OR dengue virus* AND (severe dengue* OR severity of illness index* OR severity* OR DF* OR DHF* OR DSS* AND (serotypes* OR serogroup*", according to the MESH terms suggested by PUBMED and OVID.Approximately 31 studies encompassing 15,741 cases reporting on the dengue serotypes together with their severity were obtained, and meta-analysis was carried out to analyze the data. This study found that DENV-3 from the Southeast Asia (SEA region displayed the greatest percentage of severe cases in primary infection (95% confidence interval (CI, 31.22-53.67, 9 studies, n = 598, I2 = 71.53%, whereas DENV-2, DENV-3, and DENV-4 from the SEA region, as well as DENV-2 and DENV-3 from non-SEA regions, exhibited the greatest percentage of severe cases in secondary infection (95% CI, 11.64-80.89, 4-14 studies, n = 668-3,149, I2 = 14.77-96.20%. Moreover, DENV-2 and DENV-4 from the SEA region had been found to be more highly associated with dengue shock syndrome (DSS (95% CI, 10.47-40.24, 5-8 studies, n = 642-2,530, I2 = 76.93-97.70%, while DENV-3 and DENV-4 from the SEA region were found to be more highly associated with dengue hemorrhagic fever (DHF (95% CI, 31.86-54.58, 9 studies, n = 674-2,278, I2 = 55.74-88.47%, according to the 1997 WHO dengue classification. Finally, DENV-2 and DENV-4 from the SEA region were discovered to be more highly

  8. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

    Directory of Open Access Journals (Sweden)

    Vaishali P. Waman

    2016-08-01

    Full Text Available Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae. There are four serotypes of Dengue Virus (DENV-1 to DENV-4, each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis

  9. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

    Science.gov (United States)

    Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R.; Kale, Mohan M.

    2016-01-01

    Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae). There are four serotypes of Dengue Virus (DENV-1 to DENV-4), each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis revealed that the

  10. Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

    Science.gov (United States)

    Duan, Zhiliang; Guo, Jianglong; Huang, Xi; Liu, Huifang; Chen, Xinyu; Jiang, Minghua; Wen, Jinsheng

    2015-07-01

    Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

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

    OpenAIRE

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

    2008-01-01

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

  12. Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes.

    OpenAIRE

    Lulla, V.; Lulla, A; Wernike, K; Aebischer, A.; Beer, M.; Roy, P.

    2016-01-01

    UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious ca...

  13. Foot-and-mouth disease virus serotypes detected in Tanzania from 2003 to 2010: Conjectured status and future prospects

    Directory of Open Access Journals (Sweden)

    Christopher J. Kasanga

    2012-06-01

    Full Text Available This study was conducted to investigate the presence of foot-and-mouth disease virus (FMDV in different geographic locations of Tanzania. Epithelial tissues and fluids (n = 364 were collected from cattle exhibiting oral and foot vesicular lesions suggestive of FMD and submitted for routine FMD diagnosis. The analysis of these samples collected during the period of 2002 and 2010 was performed by serotype-specific antigen capture ELISA to determine the presence of FMDV. The results of this study indicated that 167 out of 364 (46.1% of the samples contained FMDV antigen. Of the 167 positive samples, 37 (28.4% were type O, 7 (4.1% type A, 45 (21.9% SAT 1 and 79 (45.6% SAT 2. Two FMDV serotypes (O and SAT 2 were widely distributed throughout Tanzania whilst SAT 1 and A types were only found in the Eastern zone. Our findings suggest that serotypes A, O, SAT 1 and SAT 2 prevail in Tanzania and are associated with the recent FMD outbreaks. The lack of comprehensive animal movement records and inconsistent vaccination programmes make it difficult to determine the exact source of FMD outbreaks or to trace the transmission of the disease over time. Therefore, further collection and analysis of samples from domestic and wild animals are being undertaken to investigate the genetic and antigenic characteristics of the circulating strains, so that a rational method to control FMD in Tanzania and the neighbouring countries can be recommended.

  14. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

    DEFF Research Database (Denmark)

    Bachanek-Bankowska, Katarzyna; Mero, Herieth R.; Wadsworth, Jemma

    2016-01-01

    Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping...... the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce...... CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide...

  15. Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes.

    Science.gov (United States)

    Imrie, Allison; Meeks, Janet; Gurary, Alexandra; Sukhbataar, Munkhzul; Kitsutani, Paul; Effler, Paul; Zhao, Zhengshan

    2007-09-01

    Proinflammatory cytokines secreted by memory CD8+ and CD4+ T cells are thought to play a direct role in the pathogenesis of dengue virus infection by increasing vascular permeability and thereby inducing the pathophysiologic events associated with dengue hemorrhagic fever and dengue shock syndrome. Severe disease is frequently observed in the setting of secondary infection with heterologous dengue virus serotypes, suggesting a role for cross-reactive memory T cells in the immunopathogenesis of severe disease. We used a large panel of well-characterized dengue virus-specific CD8+ T-cell clones isolated from Pacific Islanders previously infected with dengue virus 1 to examine effector memory function, focusing on a novel dominant HLA-B*5502-restricted NS5(329-337) epitope, and assessed T-cell responses to stimulation with variant peptides representing heterologous serotypes. Variant peptides were differentially recognized by dengue virus 1-specific effector CD8+ cytotoxic T lymphocytes (CTL) in a heterogeneous and clone-specific manner, in which cytolytic function and cytokine secretion could be enhanced, diminished, or abrogated compared with cognate peptide stimulation. Dengue virus-specific CTL stimulated with cognate and variant peptides demonstrated a cytokine response hierarchy of gamma IFN (IFN-gamma) > tumor necrosis factor alpha (TNF-alpha) > interleukin-2 (IL-2), and a subset of clones also produced IL-4 and IL-6. Individual clones demonstrated greater avidity for variant peptides representing heterologous serotypes, including serotypes previously encountered by the subject, and IFN-gamma and TNF-alpha secretion was enhanced by stimulation with these heterologous peptides. Altered antiviral T-cell responses in response to stimulation with heterologous dengue virus serotypes have implications for control of virus replication and for disease pathogenesis.

  16. Serotype-specific anti-Dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production.

    Science.gov (United States)

    Masrinoul, Promsin; Omokoko, Magot Diata; Pambudi, Sabar; Ikuta, Kazuyoshi; Kurosu, Takeshi

    2013-08-01

    Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

  17. Serotype-specific identification of Dengue virus by silicon nanowire array biosensor.

    Science.gov (United States)

    Huang, Min Joon; Xie, Hui; Wan, Qiangqiang; Zhang, Li; Ning, Yong; Zhang, Guo-Jun

    2013-06-01

    In this work, we demonstrated a silicon nanowire (SiNW) biosensing platform capable of simultaneously identifying different Dengue serotypes on a single sensing chip. Four peptide nucleic acids (PNAs), specific to each Dengue serotypes (DENV-1 to DENV-4), were spotted on different areas of the SiNW array surface, and the covalently immobilized PNA probes were then interacted with different Dengue serotypes target to establish the specificity of detection. Detection scheme is based on the changes in resistances due to accumulation of negative charges contributed by the hybridized DNA target. The results show that resistance changes only occur in regions where the Dengue target hybridizes with its complementary probe. What is more, a mixture of two different Dengue serotypes obtained from a one-step duplex RT-PCR was applied to the multiplex SiNW surface to validate SiNW capability to identify multiple Dengue serotypes on a single sensing platform. Through this study, we have established the multiplex SiNW biosensor as a promising device to detect multiple Dengue infections with high specificity.

  18. Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus

    Science.gov (United States)

    Hanley, Kathryn A.; Sundararajan, Anitha; Devitt, Nicholas P.; Schilkey, Faye D.; Hansen, Immo A.

    2017-01-01

    Background The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. Methodology/Principal findings We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. Conclusion/Significance Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of

  19. Circulation of Dengue virus-1 (DENV-1 serotype in Delhi, during 2010–11 after Dengue virus-3 (DENV-3 predominance: A single centre hospital-based study

    Directory of Open Access Journals (Sweden)

    Ekta Gupta , Sweta Mohan , Meenu Bajpai , Aashish Choudhary & Gaurav Singh

    2012-06-01

    Full Text Available Background: Delhi, a city in north India, has so far witnessed several reported outbreaks of dengue. Dengue inDelhi from being epidemic is slowly changing towards being endemic and hyper-endemic. Circulating type ofthe virus is also changing over the years. In the absence of an effective vaccine, dengue prevention to a majorextent relies on virological surveillance, and development of effective, locally adapted control programmes. Inthe present study, we tried to identify the between-year non-epidemic serotype of dengue virus circulating inDelhi, during 2010–11.Methods: Acute-phase samples were collected from the patients attending the Institute of Liver & Biliary Sciences,New Delhi, India. Dengue diagnosis was done using WHO case definitions. All the samples were subjected toDengue NS1 Ag ELISA and modified nested RT-PCR.Results: A total of 75 acute-phase samples were received, of which 19 (25.3% were positive for dengue NS1antigen. Dengue RT-PCR was positive in 14.6% (11/75 samples. All the RT-PCR isolates were of DENV-1serotype. No case of concomitant infection with more than one serotype was observed. Median age of involvementwas 23 yr (range10–86. Maximum number of cases were seen in the age group of 21–30 yr. Male to female ratiowas 1.2 : 1. Maximum number of suspected dengue cases (n=79 was seen during September and October.Conclusions: DENV-1 was circulating in Delhi in the year 2010–11 in non-epidemic period following reportedpredominance of DENV-3 and co-circulation of all dengue serotypes in the epidemic years 2003, 2006 and 2007.

  20. First confirmation of foot and mouth disease virus serotype SAT-1 in cattle and small ruminants in Ethiopia in 2007/08.

    Science.gov (United States)

    Legesse, Yoseph; Asfaw, Yilkal; Sahle, Mesfin; Ayelet, Gelagay; Jenberie, Shiferaw; Negussie, Haileleul

    2013-06-01

    The study was conducted in three regional states of Ethiopia: Amhara, Oromia, and the Southern Nations Nationalities and people regional state from August 2007 to April 2008 with the objective of identifying the foot and mouth disease virus (FMDV) serotypes circulating in the region. Two serotypes were recorded from epithelial tissue and oesophageal-pharyngeal (OP) fluid that were taken from outbreaks in study regions of Ethiopia. Serotype O FMDV was identified in Girar Jarso, Yabello, and Ankesha Guagusa districts while SAT-1 was isolated in Surma and Maji districts from tissue samples and this was the first report of the FMDV serotype in Ethiopia. Similarly, the OP fluid samples were found positive for SAT-1 FMDV in Maji and Surma districts.

  1. Detection of Marek's disease virus serotype 1 (MDV1) glycoprotein D in MDV1-infected chick embryo fibroblasts.

    Science.gov (United States)

    Ono, M; Jang, H K; Maeda, K; Kawaguchi, Y; Tohya, Y; Niikura, M; Mikami, T

    1996-08-01

    Chick embryo fibroblasts (CEFs) infected with three strains of Marek's disease virus serotype 1 (MDV1), GA, Md5 and JM, were subjected to indirect immunofluorescence assay with monoclonal antibodies (MAbs) against MDV1 homolog of glycoprotein D (MDV1 gD) of herpes simplex virus. By the MAbs, a number of MDV1 gD-positive cells were detected in CEFs infected with GA, whereas only a few and no positive cells were detected in CEFs infected with Md5 and JM, respectively. The MDV1 gD in GA-infected CEFs was recognized as the band of 64 kDa in immunoblot analysis using one of the MAbs. This is the first report that the MDV1 gD was detected in MDV1-infected cell cultures.

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

    Science.gov (United States)

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

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

  3. Capsid coding region diversity of re-emerging lineage C foot-and-mouth disease virus serotype Asia1 from India.

    Science.gov (United States)

    Subramaniam, Saravanan; Mohapatra, Jajati K; Das, Biswajit; Sharma, Gaurav K; Biswal, Jitendra K; Mahajan, Sonalika; Misri, Jyoti; Dash, Bana B; Pattnaik, Bramhadev

    2015-07-01

    Foot-and-mouth disease virus (FMDV) serotype Asia1 was first reported in India in 1951, where three major genetic lineages (B, C and D) of this serotype have been described until now. In this study, the capsid protein coding region of serotype Asia1 viruses (n = 99) from India were analyzed, giving importance to the viruses circulating since 2007. All of the isolates (n = 50) recovered during 2007-2013 were found to group within the re-emerging cluster of lineage C (designated as sublineage C(R)). The evolutionary rate of sublineage C(R) was estimated to be slightly higher than that of the serotype as a whole, and the time of the most recent common ancestor for this cluster was estimated to be approximately 2001. In comparison to the older isolates of lineage C (1993-2001), the re-emerging viruses showed variation at eight amino acid positions, including substitutions at the antigenically critical residues VP279 and VP2131. However, no direct correlation was found between sequence variations and antigenic relationships. The number of codons under positive selection and the nature of the selection pressure varied widely among the structural proteins, implying a heterogeneous pattern of evolution in serotype Asia1. While episodic diversifying selection appears to play a major role in shaping the evolution of VP1 and VP3, selection pressure acting on codons of VP2 is largely pervasive. Further, episodic positive selection appears to be responsible for the early diversification of lineage C. Recombination events identified in the structural protein coding region indicates its probable role in adaptive evolution of serotype Asia1 viruses.

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

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

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

  5. Evolutionary analysis of foot-and-mouth disease virus serotype SAT 1 isolates from east africa suggests two independent introductions from southern africa

    DEFF Research Database (Denmark)

    Sangula, Abraham K.; Belsham, Graham; Muwanika, Vincent B.;

    2010-01-01

    Background: In East Africa, foot-and-mouth disease virus serotype SAT 1 is responsible for occasional severe outbreaks in livestock and is known to be maintained within the buffalo populations. Little is known about the evolutionary forces underlying its epidemiology in the region. To enhance our...... introductions from southern Africa were identified from a maximum clade credibility tree. One group was exclusive to Uganda while the other was present within Kenya and Tanzania. Conclusions: Our results provide a baseline characterization of the inter-regional spread of SAT 1 in sub-Saharan Africa...... appreciation of the epidemiological status of serotype SAT 1 virus in the region, we inferred its evolutionary and phylogeographic history by means of genealogy-based coalescent methods using 53 VP1 coding sequences covering a sampling period from 1948-2007. Results: The VP1 coding sequence of 11 serotype SAT...

  6. Time-Varying, Serotype-Specific Force of Infection of Dengue Virus

    Science.gov (United States)

    2014-05-20

    Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, GA 30333; jDepartment...2010were analyzed for the presenceof DENV neutralizing antibodies by serotype-specific PRNT (11) in baby hamster kidney BHK21 cells using a carboxymethyl

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

    Science.gov (United States)

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

  8. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J

    2010-01-01

    A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mu...

  9. Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae captured in Manaus, Amazonas

    Directory of Open Access Journals (Sweden)

    Valéria CS Pinheiro

    2005-12-01

    Full Text Available The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1% were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

  10. Characterization of foot-and-mouth disease viruses from Ugandan cattle outbreaks during 2012-2013: Evidence for circulation of multiple serotypes

    DEFF Research Database (Denmark)

    Namatovu, Alice; Tjørnehøj, Kirsten; Belsham, Graham

    2015-01-01

    , 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from...

  11. Epidemiologic characteristics of bluetongue virus serotype 8 laboratory-confirmed outbreaks in The Netherlands in 2007 and a comparison with the situation in 2006

    NARCIS (Netherlands)

    Elbers, A.R.W.; Spek, van der A.N.; Rijn, van P.A.

    2009-01-01

    A major epidemic of bluetongue virus serotype 8 (BTV-8) occurred in Western Europe in 2006. During 2007 it became evident that BTV-8 had survived the winter and a re-emerging epidemic quickly developed. The objective of this study was to describe the severity and clinical impact of the BTV-8 epidemi

  12. Characterization of foot-and-mouth disease viruses from Ugandan cattle outbreaks during 2012-2013: Evidence for circulation of multiple serotypes

    DEFF Research Database (Denmark)

    Namatovu, Alice; Tjørnehøj, Kirsten; Belsham, Graham;

    2015-01-01

    To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012-2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab sampl...

  13. Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.

    Science.gov (United States)

    Bashyam, Hema S; Green, Sharone; Rothman, Alan L

    2006-03-01

    Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.

  14. Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity.

    Science.gov (United States)

    Kamkaew, Maliwan; Chimnaronk, Sarin

    2015-08-01

    The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus, the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent Kd value of 53nM for binding to the 5'-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development.

  15. A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities

    OpenAIRE

    1995-01-01

    It is generally accepted that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus- specific CD8+ CTLs of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine- amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships betw...

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

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

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

  17. Isolation of bluetongue virus serotype 1 from Culicoides vector captured in livestock farms and sequence analysis of the viral genome segment-2.

    Science.gov (United States)

    Dadawala, A I; Biswas, S K; Rehman, W; Chand, K; De, A; Mathapati, B S; Kumar, P; Chauhan, H C; Chandel, B S; Mondal, B

    2012-08-01

    Bluetongue virus serotype-1 (BTV-1) was isolated from Culicoides oxystoma vectors captured on livestock farms in two places of Gujarat, India. The viruses were isolated on BHK-21 cells, which produced characteristic BTV-related cytopathic effects between 24 and 48 h post-infection. Virus antigen was demonstrated in infected cells at different passage by a BTV-specific sandwich ELISA. Further, polyacrylamide gel electrophoresis and silver staining of viral genomic RNA revealed ten double-stranded RNA segments characteristic of BTV. Serotype of the isolates was identified by virus neutralization and PCR coupled with sequencing. The isolates were designated as SKN-7 and SKN-8 and their genome segment-2 (VP2) were sequenced. Phylogenetic analyses revealed very close relationship between them although they are not identical. SKN-8 showed closer relationship with a recently isolated BTV-1 from goat. Bluetongue virus was earlier isolated from Culicoides in adjacent state more than 20 years ago, although the serotype of the virus was not determined.

  18. Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection

    Science.gov (United States)

    Lorca-Oró, Cristina; Pujols, Joan; García-Bocanegra, Ignacio; Mentaberre, Gregorio; Granados, José Enrique; Solanes, David; Fandos, Paulino; Galindo, Iván; Domingo, Mariano; Lavín, Santiago; López-Olvera, Jorge Ramón

    2012-01-01

    Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication. PMID:22666321

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

  20. Genetic diversity of serotype A foot-and-mouth disease viruses in Kenya from 1964 to 2013; implications for control strategies in eastern Africa

    DEFF Research Database (Denmark)

    Wekesa, Sabenzia N.; Sangula, Abraham K.; Belsham, Graham;

    2014-01-01

    between 1964 and 2013 were determined. Coalescent-based methods were used to infer times of divergence of the virus strains and the evolutionary rates alongside 27 other serotype A FMDV sequences from Genbank and the World Reference Laboratory (WRL). This study represents the first comprehensive genetic...... distribution. Genotypes G-III and G-VIII that were first isolated in 1964 are now apparently extinct; G-VII was last recorded in 2005, while G-I (including the new lineage) is currently in widespread circulation. High genetic diversity, widespread distribution and transboundary spread of serotype A FMDVs...

  1. An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

    OpenAIRE

    Hung, Jan-Jong; Hsieh, Meng-Ti; Young, Ming-Jer; Kao, Chuan-Liang; King, Chwan-Chuen; Chang, Wen

    2004-01-01

    Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin c...

  2. Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East

    DEFF Research Database (Denmark)

    Reid, Scott M.; Mioulet, Valerie; Knowles, Nick J.

    2014-01-01

    field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all...... three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two...

  3. Genetic and antigenic analysis of foot-and-mouth disease virus serotype O responsible for outbreaks in India during 2013.

    Science.gov (United States)

    Subramaniam, Saravanan; Mohapatra, Jajati K; Das, Biswajit; Sanyal, Aniket; Pattnaik, Bramhadev

    2015-03-01

    In recent times, majority of the foot-and-mouth disease (FMD) outbreaks in India are caused by serotype O Ind2001 lineage. The lineage has diverged into four sub-lineages (Ind2001a, b, c and d). We report here the genetic and antigenic analyses of nine Ind2001d isolates that caused outbreaks during April 2013-March 2014 in India. The length of the genomes of outbreak viruses varied between 8153 and 8181 nucleotides without any insertion or deletion in the coding region. Of the nine isolates analyzed antigenically against the currently used Indian vaccine strain INDR2/1975, eight showed good cross serological match (>0.3) indicating optimal antigenic coverage by the vaccine strain. An unprecedented deletion of 22 nucleotides between position 57 and 78 was observed in the 3' untranslated region of one of the isolates without compromising the virus viability, which imply that partial distortion in SL2 of 3'UTR may not have influence on virus viability at least under in-vitro conditions. Recently the Ind2001 lineage has been reported from several countries including Libya and spread of this lineage across a wide geographical area needs to be monitored carefully to avoid any future pandemic.

  4. Comparative histopathology and immunohistochemistry of QX-like, Massachusetts and 793/B serotypes of infectious bronchitis virus infection in chickens.

    Science.gov (United States)

    Benyeda, Zs; Szeredi, L; Mató, T; Süveges, T; Balka, Gy; Abonyi-Tóth, Zs; Rusvai, M; Palya, V

    2010-11-01

    The aim of this study was to compare experimentally the pathogenicity and tissue distribution of the recently emerged QX-like strain of infectious bronchitis virus (IBV) with the widespread M41 and 793/B serotypes of the virus. Histopathological and immunohistochemical methods were employed to define the main sites of virus replication. One-day-old specific pathogen free chickens were inoculated with five different QX-like strains, or with the M41 and 793/B IBV strains and monitored for 42 days post-infection. Tracheal lesions developed in all infected birds, confirming the ability of all of the tested strains to induce respiratory disease. Replication of the isolates in the alimentary tract was detected, but the infection did not cause significant gut lesions. Four of the five QX-like IBV strains induced severe kidney lesions. Dilation of the oviduct with accumulation of serum-like fluid in the lumen of this structure, reported previously from field cases of QX-like IBV infection, was observed following experimental infection with all of the five QX-like strains. Microscopical and immunohistochemical examination of the affected oviducts did not help to elucidate the pathogenesis of this lesion.

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

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

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

  6. Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread.

    Science.gov (United States)

    Ramey, Andrew M; Reeves, Andrew B; Ogawa, Haruko; Ip, Hon S; Imai, Kunitoshi; Bui, Vuong Nghia; Yamaguchi, Emi; Silko, Nikita Y; Afonso, Claudio L

    2013-12-01

    Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

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

    Institute of Scientific and Technical Information of China (English)

    孟飞; 吕成昱; 张海宁; 申成凯; 冯尚祥

    2015-01-01

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

  8. Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States.

    Science.gov (United States)

    Gaudreault, Natasha N; Mayo, Christie E; Jasperson, Dane C; Crossley, Beate M; Breitmeyer, Richard E; Johnson, Donna J; Ostlund, Eileen N; MacLachlan, N James; Wilson, William C

    2014-07-01

    Bluetongue is a potentially fatal arboviral disease of domestic and wild ruminants that is characterized by widespread edema and tissue necrosis. Bluetongue virus (BTV) serotypes 10, 11, 13, and 17 occur throughout much of the United States, whereas serotype 2 (BTV-2) was previously only detected in the southeastern United States. Since 1998, 10 other BTV serotypes have also been isolated from ruminants in the southeastern United States. In 2010, BTV-2 was identified in California for the first time, and preliminary sequence analysis indicated that the virus isolate was closely related to BTV strains circulating in the southeastern United States. In the current study, the whole genome sequence of the California strain of BTV-2 was compared with those of other BTV-2 strains in the Americas. The results of the analysis suggest co-circulation of genetically distinct viruses in the southeastern United States, and further suggest that the 2010 western isolate is closely related to southeastern strains of BTV. Although it remains uncertain as to how this novel virus was translocated to California, the findings of the current study underscore the need for ongoing surveillance of this economically important livestock disease.

  9. Analysis of recombinant, multivalent dengue virus containing envelope (E proteins from serotypes-1, -3 and -4 and expressed in baculovirus

    Directory of Open Access Journals (Sweden)

    Fedik A. Rantam

    2015-01-01

    Full Text Available Dengue virus has four serotypes that cause a public health problem in Indonesia. Currently, there is no preventative vaccine for this disease, but some model vaccines are in development. The envelop (E protein genes from three isolates of dengue virus (DENV-1, -3 and -4 were isolated, cloned into Escherichia coli and then sub-cloned into a baculovirus vector before co-transfection into Sf9 cells. Recombinant E genes were inserted between the Smal and Sacl sites of the plasmid, adjacent to the baculoviral structural gene, polyhedrin. The sequence of recombinant E gene was relatively stable with 97–98% homology, although there were amino acid substitutions in some regions. The recombinant protein was more antigenic when exposed to polyclonal sera from infected humans than sera from immunized mice, but its binding to monoclonal antibodies IgG1a and IgG2b was stronger than other isotopes, including IgM, IgG and Ig1b. Recombinant E protein induced cellular immune responses in immunized mice, as demonstrated by lymphocyte secretion of IL-3. This study indicates that recombinant E protein expressed in a baculovirus system can induce humoral and cellular immune responses.

  10. Physical Factors Affecting in Vitro Replication of Foot and Mouth Disease Virus (Serotype “O”

    Directory of Open Access Journals (Sweden)

    Muhammad Taslim Ghori*, Khushi Muhammad and Masood Rabbani1

    2011-10-01

    Full Text Available Effect of physical factors (temperature, pH and UV light on replicating ability of “O” type of Foot and Mouth Disease (FMD virus on Baby Hamster Kidney (BHK cell line was determined. The freshly grown FMD virus containing 106 units of tissue culture infective dose (TCID50 was divided into aliquots. Each of the 9 virus aliquots was exposed to 37, 57 or 77C for 15, 30 or 45 minutes, respectively. Each of the 5 virus aliquots was mixed with MEM-199 maintenance medium having pH 3, 5, 7, 9, or 11. Similarly, each of the 3 aliquots having 1 mm depth of the medium was exposed to ultraviolet light (252.7 nm wavelength: one foot distance for 15, 30 or 45 minutes. Each of the virus aliquot exposed to either of the temperature, pH or ultraviolet light (UV for either of the interaction time was inoculated to 8 wells of the 96-well cell culture plate containing complete monolayer of BHK cell line. One row of 8 wells served as virus control and other row of 8 wells served as control for monolayer of the BHK-21 cell line. The plates were incubated at 37°C for 48 hours. It was observed that temperature of 57 and 77C inactivated the virus within 15 minutes. The virus when admixed in the MEM-199 maintenance medium having pH 3, 5, 9 or 11, of the medium inactivated the virus while pH 7 did not show any detrimental effect on its survival. The ultraviolet light for 15, 30 or 45 minutes showed undetectable effect on survival of the virus as either of the virus aliquot exposed to the UV light for either of the interaction time showed cytopathogenic effects (CPE. It was concluded that the temperature of 57°C or higher for 15 minutes, acidic pH (below 5 or basic pH (more than 9 may inactivate the FMD virus.

  11. Lower activation-induced T-cell apoptosis is related to the pathological immune response in secondary infection with hetero-serotype dengue virus.

    Science.gov (United States)

    Yang, Wang; Yan, Huacheng; Ma, Yuling; Yu, Tiantian; Guo, Hongxia; Kuang, Yuchan; Ren, Ruiwen; Li, Jintao

    2016-03-01

    The available evidence suggests that dengue virus-specific T lymphocytes and cytokine storm play a pivotal role in the immunopathogenesis of plasma leakage. Investigations are underway to identify the immune profiles associated with increased or decreased risk for severe disease. In this study, CD14+ cells from the peripheral blood mononuclear cells (PBMCs) of patients who recovered from DENV-1 infection were infected with DENV-1 or DENV-2 and co-cultured with memory T cells. We found that secondary infection with DENV-2 suppresses the cell reproductive capacity but forms more cell clones and more functional cells to produce more proinflammatory factors (IFN-γ, TNF-α, IL-6, IL-8, IL-12 and IL-17) and less regulatory cytokines (IL-10, TGF-β) which results in higher viral replication compared to secondary infection with DENV-1. Memory dengue virus-specific T cells which are induced in a primary dengue virus infection are reactivated by the heterologous serotype of dengue virus and antigen-presenting cells (APCs) during a secondary infection. Dramatically, less apoptosis and more continuous activation of T cells in secondary infection with hetero-serotype DENV were observed. This discovery which has not been reported previously may be the reasonable and vital interpretation for the cytokine storm and severe symptoms observed in secondary infection with DENV. In summary, secondary infection with hetero-serotype DENV elicits the relatively pathological immune response while secondary infection with homologous-serotype DENV induces the relatively protective immune response by activation-induced cell death (AICD) of T cells.

  12. Analysis of Recent Serotype O Foot-and-Mouth Disease Viruses from Livestock in Kenya: Evidence of Four Independently Evolving Lineages.

    Science.gov (United States)

    Wekesa, S N; Muwanika, V B; Siegismund, H R; Sangula, A K; Namatovu, A; Dhikusooka, M T; Tjørnehøj, K; Balinda, S N; Wadsworth, J; Knowles, N J; Belsham, G J

    2015-06-01

    Foot-and-mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these viruses were determined to ascertain whether these were independent outbreaks or one single strain spreading throughout the country. The sequences of the complete VP1-coding region were analysed from viruses sampled within different areas of Kenya during 2010 and 2011. The results indicated that the 2010 to 2011 outbreaks in Kenya were caused by four independent strains. By comparison with earlier type O isolates from Eastern Africa, it was apparent that the outbreaks were caused by viruses from three different lineages of topotype EA-2 and a fourth virus strain belonging to topotype EA-4. The topotypes EA-1 and EA-3 were not detected from these outbreaks. Implications of these results for FMD control in Eastern Africa are discussed.

  13. SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

    Science.gov (United States)

    Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

    2013-12-20

    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

  14. Inactivated and adjuvanted vaccine for the control of the African horse sickness virus serotype 9 infection: evaluation of efficacy in horses and guinea-pig model.

    Science.gov (United States)

    Lelli, Rossella; Molini, Umberto; Ronchi, Gaetano Federico; Rossi, Emanuela; Franchi, Paola; Ulisse, Simonetta; Armillotta, Gisella; Capista, Sara; Khaiseb, Siegfried; Di Ventura, Mauro; Pini, Attilio

    2013-01-01

    African horse sickness (AHS) is a non-contagious viral disease of solipeds transmitted by Culicoides. The disease is endemic in most African countries. Past experience has shown that Italy is a country exposed to emerging infectious diseases endemic to Africa; an incursion of AHS virus together with the widespread presence of Culicoides vectors could be the cause of a serious epidemic emergency. A live attenuated vaccine containing seven of the nine viral serotypes, serotype 5 and 9 are excluded, is commercially available from Onderstepoort Biological Products. However, the use of live vaccines is a matter of endless disputes, and therefore inactivated or recombinant alternative products have been investigated over the years. Since research on AHS is hampered by the use of horses to evaluate vaccine potency, in a previous experiment serological response to serotypes 5 and 9 was assayed in guinea-pigs and horses. A durable and comparable serological response was observed in the two animal species. In the present study antibody response in horses and guinea-pigs, immunised with the inactivated-adjuvanted vaccine formulated with serotype 9, was tested over a period of 12 months. When immunity was challenged, horses were protected from infection and disease. Antibody response in horses and guinea-pigs compared favourably.

  15. Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates.

    Science.gov (United States)

    Gaudreault, Natasha N; Jasperson, Dane C; Dubovi, Edward J; Johnson, Donna J; Ostlund, Eileen N; Wilson, William C

    2015-07-01

    Bluetongue virus (BTV) is a vector-transmitted pathogen that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented through serological surveys, and natural infection by the Culicoides vector has been suggested. The report of isolation of BTV serotype 11 (BTV-11) from 2 separate domestic canine abortion cases in the states of Texas in 2011 and Kansas in 2012, were apparently unrelated to BTV-contaminated vaccination or consumption of BTV-contaminated raw meat as had been previously speculated. To elucidate the origin and relationship of these 2 domestic canine BTV-11 isolates, whole genome sequencing was performed. Six additional BTV-11 field isolates from Texas, Florida, and Washington, submitted for diagnostic investigation during 2011 and 2013, were also fully sequenced and analyzed. The phylogenetic analysis indicates that the BTV-11 domestic canine isolates are virtually identical, and both share high identity with 2 BTV-11 isolates identified from white-tailed deer in Texas in 2011. The results of the current study further support the hypothesis that a BTV-11 strain circulating in the Midwestern states could have been transmitted to the dogs by the infected Culicoides vector. Our study also expands the short list of available BTV-11 sequences, which may aid BTV surveillance and epidemiology.

  16. Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1.

    Science.gov (United States)

    Chang, Kyung-Soo; Ohashi, Kazuhiko; Lee, Sung-Il; Takagi, Michihiro; Onum, Misao

    2005-08-01

    The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV 1, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV 1, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV 1. Both meq and L-meq were detected in CD4+ and CD8+ T cells infected with strain Md5 or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8+ T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8+ T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant subpopulation of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8+ T cells which play a role in the clearance of pathogenic agents.

  17. Reverse transcription polymerase chain reaction (RT-PCR based detection and serotyping of FMD Virus from field samples of Gazipur, Bangladesh, and adaptation of the virus in BHK-21 cell

    Directory of Open Access Journals (Sweden)

    Mohammad Ashraful Alam

    2015-09-01

    Full Text Available The study aimed for the detection and serotyping of Foot and Mouth Disease virus (FMDV circulating in Kapasia Upazila, Gazipur district of Bangladesh during 2013. Twelve samples comprising of tongue epithelium (n=8 and inter digital tissue (n=4 were collected from suspected cattle, and inocula were prepared. The inocula were inoculated into confluent BHK-21 cell line for virus propagation. After 3 subsequent passages; progressive cytopathic effects (CPE specific for FMDV i.e., rounding and flattening of cells, breaking down of the intercellular bridge and finally cell death (almost 100% were observed; these were indicative of successful virus propagation in the cells. Viral RNA was extracted, and Reverse Transcription Polymerase Chain Reaction (RT-PCR was performed using three sets of primers corresponding to the serotype and lsquo;O', and lsquo;Asia-1' and and lsquo;A', respectively. Out of the 12 samples, 10 (83.33% were found to be positive for FMDV, and all of those were of serotype and lsquo;O'. It is concluded that FMDV serotype and lsquo;O' is circulating among the cattle of Gazipur district, Bangladesh. [J Adv Vet Anim Res 2015; 2(3.000: 291-295

  18. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  19. Hepatitis A Virus Vaccine Escape Variants and Potential New Serotype Emergence

    Science.gov (United States)

    Pérez-Sautu, Unai; Costafreda, M. Isabel; Caylà, Joan; Tortajada, Cecilia; Lite, Josep; Bosch, Albert

    2011-01-01

    Six hepatitis A virus antigenic variants that likely escaped the protective effect of available vaccines were isolated, mostly from men who have sex with men. The need to complete the proper vaccination schedules is critical, particularly in the immunocompromised population, to prevent the emergence of vaccine-escaping variants. PMID:21470474

  20. African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever

    Science.gov (United States)

    African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of African swine fever virus (ASFV) strain diversity and the viral antigens conferring type specific protective im...

  1. Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

    NARCIS (Netherlands)

    Voorham, Julia M. da Silva; Rodenhuis-Zybert, Izabela A.; Nunez, Nilda Vanesa Ayala; Colpitts, Tonya M.; van der Ende-Metselaar, Heidi; Fikrig, Erol; Diamond, Michael S.; Wilschut, Jan; Smit, Jolanda M.

    2012-01-01

    Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM

  2. Hepatitis A Virus Vaccine Escape Variants and Potential New Serotype Emergence

    OpenAIRE

    Pérez-Sautu, Unai; Costafreda, M. Isabel; Caylà, Joan; Tortajada, Cecilia; Lite, Josep; Bosch, Albert; Pintó, Rosa M.

    2011-01-01

    Six hepatitis A virus antigenic variants that likely escaped the protective effect of available vaccines were isolated, mostly from men who have sex with men. The need to complete the proper vaccination schedules is critical, particularly in the immunocompromised population, to prevent the emergence of vaccine-escaping variants.

  3. Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

    Directory of Open Access Journals (Sweden)

    Júlia M da Silva Voorham

    Full Text Available Cross-reactive dengue virus (DENV antibodies directed against the envelope (E and precursor membrane (prM proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

  4. Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

    Science.gov (United States)

    da Silva Voorham, Júlia M.; Rodenhuis-Zybert, Izabela A.; Ayala Nuñez, Nilda Vanesa; Colpitts, Tonya M.; van der Ende-Metselaar, Heidi; Fikrig, Erol; Diamond, Michael S.; Wilschut, Jan; Smit, Jolanda M.

    2012-01-01

    Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles. PMID:22431958

  5. Comparative Evaluation of Permissiveness to Dengue Virus Serotype 2 Infection in Primary Rodent Macrophages

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    Jeanette Prada-Arismendy

    2012-01-01

    Full Text Available Infection with dengue virus presents a broad clinical spectrum, which can range from asymptomatic cases to severe cases that are characterised by haemorrhagic syndrome and/or shock. The reason for such variability remains unknown. This work evaluated the in vitro permissiveness of mouse, rat, hamster and guinea pig macrophages to infection by dengue virus 2 (DENV2. The results established that macrophages derived from the BALB/c mouse strain showed higher permissiveness to DENV2 infection than macrophages from other rodent species, although all rodent species studied had the C820T mutation in the oligoadenylate synthetase 1b gene, indicating no relationship to the different in vitro susceptibilities of mouse cells at this locus. Other molecular mechanisms related to flavivirus susceptibility remain to be explored.

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

    Science.gov (United States)

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

    2008-06-01

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

  7. Short communication. Bluetongue virus serotype-1 in goats in the Pithoragarh area of Uttarakahand, India

    Directory of Open Access Journals (Sweden)

    Bimalendu Mondal

    2013-12-01

    Full Text Available This short communication reports the results of a bluetongue sero-surveillance conducted in the Pithoragarh hills of Uttarakhand in India during the autumn of 2011. Unclotted blood and serum samples were collected from 51 goats for detection of bluetongue virus (BTV antigen and antibodies. Of the 51 collected samples, 18 (35% were positive to an indirect ELISA and 33 (64% resulted positive to a BTV ELISA antigen. From a strong antigen-positive blood sample, a BTV was isolated (named as PTG-13 on cell culture and was subsequently confirmed as BTV‑1 by RT‑PCR and partial sequencing of genome segment-2. The goat serum samples were found to contain high titer of neutralising antibodies against BTV-23, nonetheless the virus could not be isolated. Interestingly, no neutralizing antibodies were detected against PTG-13 or other BTV-1 isolate, which suggests that sampling was probably done before the development of neutralizing antibodies against PTG-13 virus in the host. Isolation of BTV-1 (PTG-13 and presence of BTV-23 neutralizing antibodies in serum samples indicate that goats were probably infected with BTV-1 and 23 in different periods.

  8. Specific genetic markers for detecting subtypes of dengue virus serotype-2 in isolates from the states of Oaxaca and Veracruz, Mexico

    Directory of Open Access Journals (Sweden)

    Camacho-Nuez Minerva

    2008-07-01

    Full Text Available Abstract Background Dengue (DEN is an infectious disease caused by the DEN virus (DENV, which belongs to the Flavivirus genus in the family Flaviviridae. It has a (+ sense RNA genome and is mainly transmitted to humans by the vector mosquito Aedes aegypti. Dengue fever (DF and dengue hemorrhagic fever (DHF are caused by one of four closely related virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4. Epidemiological and evolutionary studies have indicated that host and viral factors are involved in determining disease outcome and have proved the importance of viral genotype in causing severe epidemics. Host immune status and mosquito vectorial capacity are also important influences on the severity of infection. Therefore, an understanding of the relationship between virus variants with altered amino acids and high pathogenicity will provide more information on the molecular epidemiology of DEN. Accordingly, knowledge of the DENV serotypes and genotypes circulating in the latest DEN outbreaks around the world, including Mexico, will contribute to understanding DEN infections. Results 1. We obtained 88 isolates of DENV, 27 from Oaxaca and 61 from Veracruz. 2. Of these 88 isolates, 16 were serotype 1; 62 serotype 2; 7 serotype 3; and 2 serotype 4. One isolate had 2 serotypes (DENV-2 and -1. 3. Partial nucleotide sequences of the genes encoding C- prM (14 sequences, the NS3 helicase domain (7 sequences, the NS5 S-adenosyl methionine transferase domain (7 sequences and the RNA-dependent RNA polymerase (RdRp domain (18 sequences were obtained. Phylogenetic analysis showed that DENV-2 isolates belonged to the Asian/American genotype. In addition, the Asian/American genotype was divided into two clusters, one containing the isolates from 2001 and the other the isolates from 2005–2006 with high bootstrap support of 94%. Conclusion DENV-2 was the predominant serotype in the DF and DHF outbreak from 2005 to 2006 in Oaxaca State as well as in the 2006

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  11. Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells.

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    Marijke M F Alen

    Full Text Available BACKGROUND: Dendritic cells (DC, present in the skin, are the first target cells of dengue virus (DENV. Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs against all four described serotypes of DENV replication in Raji/DC-SIGN(+ cells and in monocyte-derived DC (MDDC. METHODOLOGY/PRINCIPAL FINDINGS: A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA, Galanthus nivalis (GNA and Urtica dioica (UDA, but not actinohivin (AH was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold than in Raji/DC-SIGN(+ cells. Pradimicin-S (PRM-S, a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN(+ cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection. CONCLUSIONS/SIGNIFICANCE: The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN(+ cells and in primary MDDC.

  12. Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

    Science.gov (United States)

    1982-06-18

    encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika. Two Sandfly Fever viruses (213452 and Candiru) were...thaw at room temperature for 5 minutes,. washed with PBS and drained. The cells were first incubated at 35°C wit an appropriate dilution of hybridoma...include IB8, IB10, ICl0, 4G2, 2C4, 1B6, 4BI0, and ID7 which generally reacted with all of the flaviviruses tested and did not recognize the sandfly

  13. Development and laboratory validation of a lateral flow device for the detection of serotype SAT 2 foot-and-mouth disease viruses in clinical samples.

    Science.gov (United States)

    Ferris, Nigel P; Nordengrahn, Ann; Hutchings, Geoffrey H; Paton, David J; Kristersson, Therese; Brocchi, Emiliana; Grazioli, Santina; Merza, Malik

    2010-02-01

    A lateral flow device (LFD) for the detection of foot-and-mouth disease virus (FMDV) of the SAT 2 serotype was developed using a monoclonal antibody (Mab 2H6). The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia: 305 positive for FMDV type SAT 2 from suspected cases of vesicular disease collected from 30 countries and 1002 samples shown to be negative for FMDV type SAT 2 collected from 67 countries between 1968 and 2008. The diagnostic sensitivity of the LFD for FMDV type SAT 2 was higher at 88% compared to 79% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 100% for the ELISA. The device recognized FMDV strains of wide diversity within the FMDV SAT 2 serotype and gave a superior performance for their detection compared to the 1F10 LFD which had been developed previously and shown to perform less well for the detection of FMDVs of this particular serotype. Reactions in the SAT 2 2H6 LFD with the viruses of other FMDV serotypes and swine vesicular disease (which produces a clinically indistinguishable syndrome in pigs), did not occur. These data illustrate the potential for the LFD to be employed to complement the 1F10 device next to the animal in the pen-side diagnosis of FMD, for providing rapid and objective support to veterinarians in their clinical judgment of the disease and for specific confirmation of a FMDV type SAT 2 infection.

  14. Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

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    Anil Babu Korrapati

    Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  16. Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4

    Science.gov (United States)

    Marín-López, Alejandro; Bermúdez, Roberto; Calvo-Pinilla, Eva; Moreno, Sandra; Brun, Alejandro; Ortego, Javier

    2016-01-01

    Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses. PMID:27994510

  17. Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes.

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    Ruchi Sood

    2015-12-01

    Full Text Available Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.

  18. Positive selection sites in the surface genes of dengue virus: phylogenetic analysis of the interserotypic branches of the four serotypes.

    Science.gov (United States)

    Rodpothong, Patsarin; Auewarakul, Prasert

    2012-06-01

    The existence of four dengue serotypes is associated with a phenomenon called "Antibody-Dependent Enhancement" that has been suggested to cause a severe form of dengue hemorrhagic fever and shock syndrome. To study the evolutionary event that drove the serotype separation, we employed the maximum likelihood approach by focusing on the Premembrane (prM) and Envelop (E) genes. We showed that the separation of dengue serotypes had been dominantly under purifying selection. In spite of the strong selective constraint, one codon of prM gene and twelve codons of E gene were detected to be under positive selection. This indicates that the E protein might have been under a stronger positive pressure than the PrM protein. The codons under positive selection were identified along the interserotypic branches, suggesting that changes at these sites were probably associated with the emergence of the four serotypes and/or adaptation to the new transmission environments.

  19. Co-existence of major and minor viral populations from two different origins in patients secondarily infected with dengue virus serotype 2 in Bangkok.

    Science.gov (United States)

    Puiprom, Orapim; Yamashita, Akifumi; Sasayama, Mikiko; Limkittikul, Kriengsak; Boonha, Khwanchit; Jittmitraphap, Akanitt; Leaungwutiwong, Pornsawan; Kurosu, Takeshi; Ramasoota, Pongrama; Ikuta, Kazuyoshi

    2011-09-16

    Generally, RNA viruses exhibit significant genetic diversity that sometimes effect viral fitness in infected hosts and probably also pathogenesis. Dengue viruses (DENVs) consist of four antigenically distinct serotypes. All the serotypes of DENV can cause mild to severe dengue illnesses. In this study, we examined the sequence variation of DENV in plasma obtained from four patients living in Bangkok who had been secondarily infected with serotype 2 (DENV-2) in 2010. The plasma-derived RNA was directly subjected to reverse transcriptase (RT)-polymerase chain reaction (PCR) at a region including most of domain III of the envelope (E) protein gene, and the PCR products obtained were subjected to clonal sequencing. Using 19-20 clones sequenced from each patient (78 total) plus 601 corresponding sequences from a public database, phylogenetic analysis revealed that the nucleic acid sequences fell into two clusters with clearly different origins. Interestingly, all patients gave sequences indicating that they carried viral populations containing 2, 3 or 5 genetic variants that consisted of one major variant plus one or more minor variants. Three patients showed a major variant from one cluster plus one or more minor components from the other while one showed major and minor variants from a single cluster. Thus, it can be concluded that DENV belonging to two different genetic lineages were co-circulated in Bangkok in 2010. For these two genotype clusters there was also a clear difference in H or Y at the deduced amino acid position 346 (i.e. H346Y) that was consistent for our sequences and 601 sequences from the public database. Thus, one among the mixed viral genotypes introduced into human individuals seems to be variably selected as the predominant component of the carried viral population, and it is possible that the dynamics of this process could influence virus evolution and disease severity.

  20. Comparison of sequences of hypervariable region (HVR subunit S-1 gene of field isolate I-37 infectious bronchitis virus with Connecticut serotype

    Directory of Open Access Journals (Sweden)

    N.L.P Indi Dharmayanti

    2003-06-01

    Full Text Available Infectious Bronchitis is a contagious and acute respiratory disease in chickens caused by infectious bronchitis virus (IBV.Antigenic differences in IBV are associated with changes in the sequence of the spike glycoprotein (S. The subunit S1 which demonstrates more sequence variability than S-2 have been identified as hypervariable region (HVR-1 and 2. There were several IB virus field isolates included I-37 have been identified in Indonesia by serum neutralization method. However, gene sequence variation in HVR subunit S-1 had not yet been identified. Isolate I-37 was close to the serotype Connecticut 46 (Conn 46. The aim of this study is to identify sequence variation of HVR subunit S-1 gene of isolate I-37 produced by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR and sequencing. Several procedures were carried out in the study including virus titration, propagation and was concentrated from the allantoic fluid infected with IBV. Then, RNA was extracted for RTPCR. urther the product was sequnced and its homology with IBV references from GenBank was compared by GenMac version 8.0. Result showed that isolate I-37 produced 515 bp of amplification product. Isolate I-37 and Conn 46 are same serotype, yet their HVR subunit S-1 nucleotides and amino acids (protein differ by 6.9% and 15.6% respectively. It might be concluded that isolate I-37 was variant of Conn 46.

  1. Temporal distribution of dengue virus serotypes in Colombian endemic area and dengue incidence: re-introduction of dengue-3 associated to mild febrile illness and primary infection

    Directory of Open Access Journals (Sweden)

    Raquel Elvira Ocazionez

    2006-11-01

    Full Text Available We have investigated the temporal distribution of dengue (DEN virus serotypes in the department (state of Santander, Colombia, in relation to dengue incidence, infection pattern, and severity of disease. Viral isolation was attended on a total of 1452 acute serum samples collected each week from 1998 to 2004. The infection pattern was evaluated in 596 laboratory-positive dengue cases using an IgG ELISA, and PRNT test. The dengue incidence was documented by the local health authority. Predominance of DEN-1 in 1998 and DEN-3 re-introduction and predominance in 2001-2003 coincided with outbreaks. Predominance of DEN-2 in 2000-2001 coincided with more dengue hemorrhagic fever (DHF. DEN-4 was isolated in 2000-2001 and 2004 but was not predominant. There was an annual increase of primary dengue infections (from 13.7 to 81.4% that correlated with frequency of DEN-3 (r = 0.83; P = 0.038. From the total number of primary dengue infections DEN-3 (81.3% was the most frequent serotype. DHF was more frequent in DEN-2 infected patients than in DEN-3 infected patients: 27.5 vs 10.9% (P < 0.05. DEN-3 viruses belonged to subtype C (restriction site-specific-polymerase chain reaction like viruses isolated in Sri-Lanka and other countries in the Americas. Our findings show the importance of continuous virological surveillance to identify the risk factors of dengue epidemics and severity.

  2. Prevalence of Bluetongue virus serotype 4 in cattle in the State of Sao Paulo, Brazil.

    Science.gov (United States)

    Hellmeister de Campos Nogueira, Adriana; De Stefano, Eliana; de Souza Nunes Martins, Maira; Okuda, Liria Hiromi; Dos Santos Lima, Michele; da Silva Garcia, Thais; Heinz Hellwig, Otto; Alves de Lima, José Eduardo; Savini, Giovanni; Pituco, Edviges Maristela

    2016-09-30

    Bluetongue (BT) is considered endemic in several regions of Brazil. The State of Sao Paulo was divided into 7 cattle production regions (circuits) according the different systems of breeding, operational and logistical capacity of the state veterinary service. At least 1 animal from each property (a total of 1,716 farms) was tested by competitive ELISA for the presence of antibodies against BTV. Sero‑positive sera were subsequently also tested by virus neutralization tests (VNT) using serial dilutions from 1:10 (cutoff) up to 1:640 (in MEM). BTV‑4 neutralizing antibodies were detected in 86% (1,483/1,716) of the animals tested. These results show that BTV‑4 is endemic and widespread in the State of San Paulo and indirectly confirm that in the State there are favourable conditions for the multiplication of competent vectors. However, as no clinical signs have ever been reported in cattle in the region, BTV‑4 infection is likely to occur silently in the State of Sao Paulo.

  3. Quantum dots and duplex-specific nuclease enabled ultrasensitive detection and serotyping of Dengue viruses in one step in a single tube.

    Science.gov (United States)

    Shen, Wei; Gao, Zhiqiang

    2015-03-15

    Leveraging on the enzymatic processing of Dengue virus (DV) RNA hybridized quantum dot-capped DNA capture probes (QD-CPs), an ultrasensitive assay for the detection and serotyping of DVs is described in the report. Briefly, DV-specific DNA CPs are first capped by QDs and then conjugated to magnetic beads. In a sample solution, strands of DV RNA form heteroduplexes with the QD-CPs on the magnetic beads. The CPs together with the QDs in the heteroduplexes are subsequently cleaved off the magnetic beads by a duplex-specific nuclease (DSN), releasing the QDs to the solution, freeing the target RNA strands, and availing them for another around of hybridization with the remaining QD-CPs. After removing the magnetic beads along with unreacted (uncleaved) QD-CPs by using a permanent magnet, ultrasensitive fluorescent detection of DV is realized through the cleaved QDs. Serotyping of DV is accomplished by a judicious design of the QD-CPs. The assay combines excellent signal generation by the highly fluorescent QDs and the effortlessness of utilizing magnetic beads in the removal of the unreacted QD-CPs. The highly efficient DSN cleavage in conjunction with its excellent mismatch discrimination ability permits serotyping of DVs in one tube with excellent sensitivity and selectivity.

  4. Spectrum of VP1 region genetic variants in the foot-and-mouth disease virus serotype O populations derived from infected cattle tongue epithelium.

    Science.gov (United States)

    Sarangi, L N; Mohapatra, J K; Subramaniam, S; Pandey, L K; Das, B; Sanyal, A; Misri, J; Pattnaik, B

    2015-09-01

    RNA virus population exists as a complex distribution of non-identical but closely related sequences known as viral quasispecies. Variant strains are selected from this quasispecies population in response to changing environment. The quasispecies dynamics of a virus existing within an infected host differs from that in a cell culture-adapted population. This study was carried out to explore the genetic variations present in the VP1 coding region of the foot-and-mouth disease (FMD) virus serotype O derived directly from infected cattle tongue epithelium. Molecular clonal populations of two serotype O strains belonging to lineages Ind2001 (IND 30/2011) and PanAsia2 (IND 5/2011) were sequenced at VP1 coding region. For IND 30/2011, 19 clones were sequenced and analysis showed variations at 12 nucleotide positions (nt) resulting in 8 amino acid (aa) replacements. Similarly, for IND 5/2011 virus, 18 clones were sequenced, of which six showed nt variations leading to 3 aa replacements. Most of the variable positions mapped to the surface-exposed loops and some of them were found in the neutralizing antigenic sites (position 81, 149, 169, 186 and 202 of IND 30/2011 and 141 of IND 5/2011), which potentially could be beneficial in rapid adaptive evolution of the virus by giving rise to antigenic variants to overcome neutralizing antibodies. These findings encourage further research into the landscape of the viral quasispecies population in vivo and its implication for viral ecology.

  5. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

  6. Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

    Institute of Scientific and Technical Information of China (English)

    Aiguo; Xin; Mingwang; Zhu; Qi; Hu; Haisheng; Miao; Zhenqi; Peng; Yuwen; He; Lin; Gao; Huachun; Li

    2014-01-01

    An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.

  7. Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency, emergency vaccine.

    Science.gov (United States)

    Horsington, Jacquelyn; Zhang, Zhidong; Bittner, Hilary; Hole, Kate; Singanallur, Nagendrakumar B; Alexandersen, Soren; Vosloo, Wilna

    2015-01-09

    In 2009-2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD50) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n=8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24h prior to contact with the vaccinated animals, or unvaccinated controls (n=4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.

  8. Preferred transduction with AAV8 and AAV9 via thalamic administration in the MPS IIIB model: A comparison of four rAAV serotypes

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    J.A. Gilkes

    2016-03-01

    Full Text Available Sanfilippo syndrome type B (MPS IIIB is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery.

  9. Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle

    NARCIS (Netherlands)

    Jamal, S.M.; Bouma, A.; Broek, van den J.; Stegeman, J.A.; Chenard, G.; Dekker, A.

    2008-01-01

    The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, typ

  10. Individual and bivalent vaccines against botulinum neurotoxin serotypes A and B using DNA-based Semliki Forest virus vectors.

    Science.gov (United States)

    Yu, Yunzhou; Yu, Jiyu; Li, Na; Wang, Shuang; Yu, Weiyuan; Sun, Zhiwei

    2009-10-19

    We evaluated individual and bivalent replicon vaccines against Clostridiumbotulinum neurotoxin serotypes A (BoNT/A) or B (BoNT/B). The DNA replicon vaccine (pSCARSBHc) encoding the Hc domain of BoNT/B (BHc) induced better responses and protection against BoNT/B mouse challenge than conventional DNA vaccine. The dual-expressing DNA vaccine (pSCARSA/BHc) protected similarly to a DNA replicon vaccine mixture (pSCARSAHc+pSCARSBHc). Additionally, recombinant SFV particles, VRP-AHc or VRP-BHc, protected mice from high-dose BoNT/A or BoNT/B challenge, respectively. Mice given either dual-expressing VRP-A/BHc or mixture of VRP-AHc and VRP-BHc were protected from challenge with serotype A/B mixtures. These data justify further testing in other animals or humans.

  11. Genomic Changes in an Attenuated ZB Strain of Foot-and-Mouth Disease Virus Serotype Asia1 and Comparison with Its Virulent Parental Strain

    Directory of Open Access Journals (Sweden)

    Aiguo Xin

    2014-01-01

    Full Text Available The molecular basis of attenuation of foot-and-mouth disease virus (FMDV serotype Asia1 ZB strain remains unknown. To understand the genetic changes of attenuation, we compared the entire genomes of three different rabbit-passaged attenuated ZB strains (ZB/CHA/58(att, ZBRF168, and ZBRF188 and their virulent parental strains (ZBCF22 and YNBS/58. The results showed that attenuation may be brought about by 28 common amino acid substitutions in the coding region, with one nucleotide point mutation in the 5′-untranslated region (5′-UTR and another one in the 3′-UTR. In addition, a total of 21 nucleotides silent mutations had been found after attenuation. These substitutions, alone or in combination, may be responsible for the attenuated phenotype of the ZB strain in cattle. This will contribute to elucidation of attenuating molecular basis of the FMDV ZB strain.

  12. Non-neutralizing monoclonal antibodies to a trypsin-sensitive site on the major glycoprotein of rotavirus which discriminate between virus serotypes.

    Science.gov (United States)

    Coulson, B S; Fowler, K J; White, J R; Cotton, R G

    1987-01-01

    Monoclonal antibodies were derived to a human rotavirus purified from stools. Three of the antibodies immunoprecipitated the rotavirus outer capsid glycoprotein gp 34 and were non-neutralizing. These antibodies reacted by enzyme immunoassay with cultivable rotaviruses showing the "long" RNA electropherotype but were inefficient as detectors of "long" RNA pattern rotaviruses in stools. Treatment of SA 11 rotavirus with 7.5 micrograms/ml porcine trypsin for 30 minutes at 37 degrees C irreversibly reduced binding of the antibodies to SA 11 rotavirus in enzyme immunoassays by 50 per cent. Binding was abolished in the presence of rotavirus-negative faecal extracts. These results indicate that non-neutralizing sites on gp 34 of rotaviruses can vary with RNA electropherotype and serotype, and that levels of trypsin currently in use to assist growth of rotaviruses in cell culture may alter the serological profile of the viruses.

  13. Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

    Energy Technology Data Exchange (ETDEWEB)

    Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard

    2015-10-31

    Infection by the four serotypes ofDengue virus(DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all fourDengue virusserotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

  14. Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

    Institute of Scientific and Technical Information of China (English)

    Tetiana; Krupodorova; Svetlana; Rybalko; Victor; Barshteyn

    2014-01-01

    In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

  15. Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture.

    Science.gov (United States)

    Krupodorova, Tetiana; Rybalko, Svetlana; Barshteyn, Victor

    2014-10-01

    In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A (serotype H1N1) and herpes simplex virus type 2 (HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47 (H1N1) in MDCK cells reducing the infectious titer by 2.0-6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species-Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes-this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index (324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes (amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

  16. Evaluation of the Protection Efficacy of a Serotype 1 Marek's Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek's Disease.

    Science.gov (United States)

    Gimeno, Isabel M; Cortes, Aneg L; Faiz, Nik M; Hernandez-Ortiz, Byron A; Guy, James S; Hunt, Henry D; Silva, Robert F

    2015-06-01

    Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.

  17. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells.

    Science.gov (United States)

    Hung, Jan-Jong; Hsieh, Meng-Ti; Young, Ming-Jer; Kao, Chuan-Liang; King, Chwan-Chuen; Chang, Wen

    2004-01-01

    Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

  18. Divergence of the dengue virus type 2 Cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in Surabaya, Indonesia, 2008-2014.

    Science.gov (United States)

    Kotaki, Tomohiro; Yamanaka, Atsushi; Mulyatno, Kris Cahyo; Churrotin, Siti; Sucipto, Teguh Hari; Labiqah, Amaliah; Ahwanah, Nur Laila Fitriati; Soegijanto, Soegeng; Kameoka, Masanori; Konishi, Eiji

    2016-01-01

    Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1-3, dominated by Surabaya strains, were defined as the "Surabaya lineage". Clusters 4-6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the "South East Asian lineage". The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013-14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013-14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries.

  19. Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle.

    Science.gov (United States)

    Jamal, Syed M; Bouma, Annemarie; van den Broek, Jan; Stegeman, Arjan; Chénard, Gilles; Dekker, Aldo

    2008-11-25

    The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, type of adjuvant, valency and type of virus culture on the dose-response curve. Data from 297 potency tests of FMD vaccines, comprising 4004 vaccinated cattle, performed at the FMD vaccine production facility in the Netherlands, were used for the present study. A generalised linear mixed effect model was used to analyse the results. Our study showed that the relation between FMD vaccine potency and fraction protected was also affected by the serotype and type of adjuvant. No common level of protection could be assigned to all FMD vaccines with the same amount of PD50 per dose, this information is essential when designing a new standard FMD vaccines control.

  20. Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis.

    Science.gov (United States)

    Lin, Xian; Huang, Canhui; Shi, Jian; Wang, Ruifang; Sun, Xin; Liu, Xiaokun; Zhao, Lianzhong; Jin, Meilin

    2015-01-01

    Swine influenza virus and Streptococcus suis are two important contributors to the porcine respiratory disease complex, and both have significant economic impacts. Clinically, influenza virus and Streptococcus suis co-infections in pigs are very common, which often contribute to severe pneumonia and can increase the mortality. However, the co-infection pathogenesis in pigs is unclear. In the present study, co-infection experiments were performed using swine H1N1 influenza virus and Streptococcus suis serotype 2 (SS2). The H1N1-SS2 co-infected pigs exhibited more severe clinical symptoms, serious pathological changes, and robust apoptosis of lungs at 6 days post-infection compared with separate H1N1 and SS2 infections. A comprehensive gene expression profiling using a microarray approach was performed to investigate the global host responses of swine lungs against the swine H1N1 infection, SS2 infection, co-infection, and phosphate-buffered saline control. Results showed 457, 411, and 844 differentially expressed genes in the H1N1, SS2, and H1N1-SS2 groups, respectively, compared with the control. Noticeably, genes associated with the immune, inflammatory, and apoptosis responses were highly overexpressed in the co-infected group. Pathway analysis indicated that the cytokine-cytokine receptor interactions, MAPK, toll-like receptor, complement and coagulation cascades, antigen processing and presentation, and apoptosis pathway were significantly regulated in the co-infected group. However, the genes related to these were less regulated in the separate H1N1 and SS2 infection groups. This observation suggested that a certain level of synergy was induced by H1N1 and SS2 co-infection with significantly stronger inflammatory and apoptosis responses, which may lead to more serious respiratory disease syndrome and pulmonary pathological lesion.

  1. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    Science.gov (United States)

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine.

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

    Science.gov (United States)

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

    2002-03-01

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

  3. Secondary infection with Streptococcus suis serotype 7 increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs

    Directory of Open Access Journals (Sweden)

    Xu Min

    2010-08-01

    Full Text Available Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV and Streptococcus suis are common pathogens in pigs. In samples collected during the porcine high fever syndrome (PHFS outbreak in many parts of China, PRRSV and S. suis serotype 7 (SS7 have always been isolated together. To determine whether PRRSV-SS7 coinfection was the cause of the PHFS outbreak, we evaluated the pathogenicity of PRRSV and/or SS7 in a pig model of single and mixed infection. Results Respiratory disease, diarrhea, and anorexia were observed in all infected pigs. Signs of central nervous system (CNS disease were observed in the highly pathogenic PRRSV (HP-PRRSV-infected pigs (4/12 and the coinfected pigs (8/10; however, the symptoms of the coinfected pigs were clearly more severe than those of the HP-PRRSV-infected pigs. The mortality rate was significantly higher in the coinfected pigs (8/10 than in the HP-PRRSV- (2/12 and SS7-infected pigs (0/10. The deceased pigs of the coinfected group had symptoms typical of PHFS, such as high fever, anorexia, and red coloration of the ears and the body. The isolation rates of HP-PRRSV and SS7 were higher and the lesion severity was greater in the coinfected pigs than in monoinfected pigs. Conclusion HP-PRRSV infection increased susceptibility to SS7 infection, and coinfection of HP-PRRSV with SS7 significantly increased the pathogenicity of SS7 to pigs.

  4. Questionnaire survey about the motives of commercial livestock farmers and hobby holders to vaccinate their animals against Bluetongue virus serotype 8 in 2008-2009 in the Netherlands.

    Science.gov (United States)

    Elbers, A R W; de Koeijer, A A; Scolamacchia, F; van Rijn, P A

    2010-03-16

    After a massive epidemic of Bluetongue virus serotype 8 (BTV-8) among ruminants in 2006-2007 in the European Union (EU), the Netherlands started a voluntary emergency vaccination campaign in May 2008, subsidized by the EU. At the start of a new campaign in 2009, without subsidized vaccination, we investigated by mail survey the motives of farmers and hobby holders to vaccinate against BTV-8 in 2008 and 2009. Mean vaccine uptake in 2008 was: 73% in sheep, 71% in cattle, 43% in goat farms and 67% in hobby holdings. Top-5 motives pro-vaccination were: prevention of production loss; subsidized vaccination; recommendation by practitioner; welfare reasons; contribution to the eradication campaign. Top-5 motives against vaccination were: vaccination costs; absence of clinical BT-problems; presumed low infection risk; balance between vaccination costs and loss without vaccination; bad experience with earlier vaccination campaigns. Willingness to vaccinate was significantly lower in 2009: 42% in sheep, 58% in cattle, 19% in goat farms and 49% in hobby holdings. Measures to stimulate vaccination among those that did not want to vaccinate in 2009 were: subsidized vaccination; possibility to vaccinate their own animals; more information on efficacy/safety of vaccine and why animals had to be vaccinated again; availability of a BT vaccine combined with vaccine(s) against other diseases.

  5. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available BACKGROUND: Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS. METHODS AND FINDINGS: This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP. CONCLUSION: This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  6. Foot-and-Mouth Disease Virus Serotype O Phylodynamics: Genetic Variability Associated with Epidemiological Factors in Pakistan

    DEFF Research Database (Denmark)

    Brito, B. P.; Perez, A. M.; Jamal, S. M.;

    2013-01-01

    One of the most challenging aspects of foot-and-mouth disease (FMD) control is the high genetic variability of the FMD virus (FMDV). In endemic settings such as the Indian subcontinent, this variability has resulted in the emergence of pandemic strains that have spread widely and caused devastati...... into Europe (Bulgaria) and Africa (Libya)....

  7. Dromedaries (Camelus dromedarius) are of Low Susceptibility to Inoculation with Foot-and-Mouth Disease Virus Serotype O

    DEFF Research Database (Denmark)

    Alexandersen, Søren; Wernery, U.; Nagy, P.;

    2008-01-01

    Two sheep and five dromedaries were inoculated with a highdose of a cattle-passaged type O strain of foot-and-mouth disease virus (FMDV). The sheep developed typical FMD. The inoculated camels, which were placed in contact with five further dromedaries and four sheep, showed no visible sign...

  8. Serotype Specificity of Antibodies against Foot-and-Mouth Disease Virus in Cattle in Selected Districts in Uganda

    DEFF Research Database (Denmark)

    Mwiine, F.N.; Ayebazibwe, C.; Olaho-Mukani, W.;

    2010-01-01

    Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non...

  9. Phylogenetic study reveals co-circulation of Asian II and Cosmopolitan genotypes of Dengue virus serotype 2 in Nepal during 2013.

    Science.gov (United States)

    Singh, Sneha; Gupta, Birendra P; Manakkadan, Anoop; Das Manandhar, Krishna; Sreekumar, Easwaran

    2015-08-01

    The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come.

  10. Co-circulation of two extremely divergent serotype SAT 2 lineages in Kenya highlights challenges to foot-and-mouth disease control

    DEFF Research Database (Denmark)

    Sangula, Abraham; Belsham, Graham; Muwanika, Vincent;

    2010-01-01

    Amongst the SAT serotypes of foot-and-mouth disease virus (FMDV), the SAT 2 serotype is the most widely distributed throughout sub-Saharan Africa. Kenyan serotype SAT 2 viruses have been reported to display the highest genetic diversity for the serotype globally. This complicates diagnosis and co...

  11. A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

    Science.gov (United States)

    Yang, Huiqiang; Li, Zhushi; Lin, Hua; Wang, Wei; Yang, Jian; Liu, Lina; Zeng, Xianwu; Wu, Yonglin; Yu, Yongxin; Li, Yuhua

    2016-06-01

    To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

  12. Multifunctional Virus-Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer

    Science.gov (United States)

    2012-06-01

    cancer cells in synergy with gene therapy. We proposed to develop virus- nanoshell assemblies by attaching adeno-associated virus (AAV) to gold... nanoshells (Au NS) through chemical bonds. We have successfully completed majority of tasks 1 and 2 of our Statement of Work. Specifically, we have...therapy, virus, Au nanoshell Multifunctional Virus- Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer Dr. Fang Wei

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

    Directory of Open Access Journals (Sweden)

    Hélène Hall

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

  14. Infecciones concurrentes por dos serotipos del virus dengue durante un brote en el noroeste de Perú, 2008 Concurrent infections by two dengue virus serotypes during an outbreak in northwestern Peru, 2008

    Directory of Open Access Journals (Sweden)

    Enrique Mamani

    2010-03-01

    Full Text Available Objetivo. Describir la existencia de infecciones concurrentes por diferentes serotipos del virus dengue (DENV en un brote ocurrido en el noroeste de Perú durante el 2008. Materiales y métodos. Se analizó 73 muestras séricas de pacientes con dengue en un brote en el noroeste de Perú entre mayo y junio de 2008. Para la serotipificación del DENV se utilizó técnicas de biología molecular; así, primero se realizó la extracción del ARN con el kit QIAamp viral RNA Mini, luego se realizó la transcripción inversa y amplificación de los fragmentos de ADNc viral mediante las técnicas de reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR multiplex y de RT-Anidada PCR (RT-Nested PCR, y finalmente de realizó el secuenciamiento genético de los fragmentos de ADNc viral utilizando el kit Big Dye Terminator v.3,1. Resultados. Los 73 casos de dengue presentaron infecciones por diferentes serotipos: 34 (46,6% por DENV-3, 29 (39,7% por DENV-1, 4 (5,5% por DENV-4 y 6 casos (8,2% por DENV-1 y DENV-3. Las manifestaciones clínicas más frecuentes fueron fiebre y cefalea (100%, mialgia (94,5%, dolor ocular (83,6%, artralgia (78,1%, escalofríos (63,0%, nauseas/vómitos (38,4%, prueba de lazo positiva (30,1% y erupción cutánea (20,5%. Los pacientes con infecciones concurrentes presentaron cuadros leves, excepto una paciente que presentó prueba de lazo positivo y sangrado genital. Conclusión. Es el primer reporte de pacientes peruanos con infecciones concurrentes por dos serotipos del DENV sin formas graves de la enfermedad.Objetives. To establish the existence of concurrent infections by different dengue virus (DENV serotypes in an outbreak in the Northwestern in Peru during 2008. Material and methods. 73 serum samples from patients with dengue were analyzed during an outbreak that occurred in Northwestern in Peru between May and June 2008. Molecular biology techniques were used to serotype the DENV, thus, firstly the viral RNA

  15. Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines.

    Science.gov (United States)

    Chang, Kyung-Soo; Ohashi, Kazuhiko; Onuma, Misao

    2002-12-01

    The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.

  16. Bluetongue virus serotype 1 outbreak in the Basque Country (Northern Spain 2007-2008. Data support a primary vector windborne transport.

    Directory of Open Access Journals (Sweden)

    Rodrigo García-Lastra

    Full Text Available BACKGROUND: Bluetongue (BT is a vector-borne disease of ruminants that has expanded its traditional global distribution in the last decade. Recently, BTV-1 emerged in Southern Spain and caused several outbreaks in livestock reaching the north of the country. The aim of this paper was to review the emergence of BTV-1 in the Basque Country (Northern Spain during 2007 and 2008 analyzing the possibility that infected Culicoides were introduced into Basque Country by winds from the infected areas of Southern Spain. METHODOLOGY/PRINCIPAL FINDINGS: We use a complex HYSPLIT (Hybrid Single-Particle Lagrangian Integrated Trajectory model to draw wind roses and backward wind trajectories. The analysis of winds showed September 28 to October 2 as the only period for the introduction of infected midges in the Basque Country. These wind trajectories crossed through the areas affected by serotype 1 on those dates in the South of the Iberian Peninsula. Additionally meteorological data, including wind speed and humidity, and altitude along the trajectories showed suitable conditions for Culicoides survival and dispersion. CONCLUSIONS/SIGNIFICANCE: An active infection in medium-long distance regions, wind with suitable speed, altitude and trajectory, and appropriate weather can lead to outbreaks of BTV-1 by transport of Culicoides imicola, not only over the sea (as reported previously but also over the land. This shows that an additional factor has to be taken into account for the control of the disease which is currently essentially based on the assumption that midges will only spread the virus in a series of short hops. Moreover, the epidemiological and serological data cannot rule out the involvement of other Culicoides species in the spread of the infection, especially at a local level.

  17. A novel dengue virus serotype-2 nanovaccine induces robust humoral and cell-mediated immunity in mice.

    Science.gov (United States)

    Hunsawong, Taweewun; Sunintaboon, Panya; Warit, Saradee; Thaisomboonsuk, Butsaya; Jarman, Richard G; Yoon, In-Kyu; Ubol, Sukathida; Fernandez, Stefan

    2015-03-30

    Dengue virus (DENV), a member of the Flaviviridae family, can be transmitted to humans through the bite of infected Aedes mosquitoes. The incidence of dengue has increased worldwide over the past few decades. Inadequate vector control, changing global ecology, increased urbanization, and faster global travel are factors enhancing the rapid spread of the virus and its vector. In the absence of specific antiviral treatments, the search for a safe and effective vaccine grows more imperative. Many strategies have been utilized to develop dengue vaccines. Here, we demonstrate the immunogenic properties of a novel dengue nanovaccine (DNV), composed of ultraviolet radiation (UV)-inactivated DENV-2, which has been loaded into the nanoparticles containing chitosan/Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (CS/BCG-NPs). We investigated the immunogenicity of DNV in a Swiss albino mouse model. Inoculation with various concentrations of vaccine (0.3, 1, 3 and 10μg/dose) with three doses, 15-day apart, induced strong anti-dengue IgM and IgG antibodies in the mouse serum along with neutralizing antibody against DENV-2 reference strain (16681), a clinical-isolate strain (00745/10) and the mouse-adapted New Guinea-C (NGC) strain. Cytokine and chemokine secretion in the serum of DNV-immunized mice showed elevated levels of IFN-γ, IL-2, IL-5, IL-12p40, IL-12p70, IL-17, eotaxin and RANTES, all of which have varying immune functions. Furthermore, we observed a DNV dose-dependent increase in the frequencies of IFN-γ-producing CD4(+) and CD8(+) T cells after in vitro stimulation of nucleated cells. Based on these findings, DNV has the potential to become a candidate dengue vaccine.

  18. Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

    Directory of Open Access Journals (Sweden)

    Peter Charbel Issa

    Full Text Available Adeno-associated viral vectors (AAV have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/- mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1 mouse in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/- mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

  19. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    . Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection......Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV...

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  1. O型口蹄疫病毒间接免疫荧光检测方法的建立%Establishment of Indirect Immunofluorescence Assay for Detection of Foot-and-Mouth Disease Virus Serotype O

    Institute of Scientific and Technical Information of China (English)

    蔡扩军; 乔军; 孟庆玲; 陈创夫; 马铈委; 黄炯; 魏婕

    2012-01-01

    To establish an indirect immunofluorescence assay (IFA) test to diagnose swine foot-and-mouth disease virus(FMDV) serotype O, the porcine anti-FMDV serotype O serum was served as the primary antibody and the fluorescein isothiocyanated (FITC) labeled Protein A protein (a surface protein of S.aures) as the secondary antibody. This detection method was developed by the optimization of reaction conditions. The optimum conditions of IFA were as follows: the optimum working dilutions of anti-FMDV Serotype O antibody and FITC-labeled protein A were 1: 50 and 1 : 800 respectively, the optimum incubating time was 1 hour for both. IFA can specifically detect FMDV serotype O in the BHK-21 cells, while the results were negative in BHK-21 cells infected with FMDV serotype A, Asia I and SVDV. The results showed that the IFA test is specific, sensitive, simple and rapid to detect FMDV serotype O, which may be applied in diagnosis and studies of the localization and dynamic distribution of the FMDV in infected organisms.%为建立猪O型口蹄疫病毒(FMDV)间接免疫荧光(IFA)检测方法,以猪抗O型口蹄疫病毒阳性血清为一抗、异硫氰酸荧光素(FITC)标记的SPA蛋白(葡萄球菌蛋白A)为二抗,通过反应条件的优化,建立检测方法.结果表明,IFA最佳工作条件为,一抗最适稀释度为1∶50,FITC标记的二抗的最适稀释度为1 ∶ 800,最适孵育时间均为1h.特异性试验表明,用建立的IFA检测方法只能检测O型FMDV,而A型、AsiaIFMDV型和猪水疱病病毒(SVDV)检测结果均为阴性.建立的检测O型FMDV抗原的IFA检测方法具有特异、敏感、简便、快速等优点,可用于FMDV感染的实验室诊断及其在感染机体中的定位和动态分布研究.

  2. Identification of multiple novel viruses, including a parvovirus and a hepevirus, in feces of red foxes.

    Science.gov (United States)

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

    2013-07-01

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

  3. Injection parameters and virus dependent choice of promoters to improve neuron targeting in the nonhuman primate brain.

    Science.gov (United States)

    Lerchner, W; Corgiat, B; Der Minassian, V; Saunders, R C; Richmond, B J

    2014-03-01

    We, like many others, wish to use modern molecular methods to alter neuronal functionality in primates. For us, this requires expression in a large proportion of the targeted cell population. Long generation times make germline modification of limited use. The size and intricate primate brain anatomy poses additional challenges. We surved methods using lentiviruses and serotypes of adeno-associated viruses (AAVs) to introduce active molecular material into cortical and subcortical regions of old-world monkey brains. Slow injections of AAV2 give well-defined expression of neurons in the cortex surrounding the injection site. Somewhat surprisingly we find that in the monkey the use of cytomegalovirus promoter in lentivirus primarily targets glial cells but few neurons. In contrast, with a synapsin promoter fragment the lentivirus expression is neuron specific at high transduction levels in all cortical layers. We also achieve specific targeting of tyrosine hydroxlase (TH)- rich neurons in the locus coeruleus and substantia nigra with a lentvirus carrying a fragment of the TH promoter. Lentiviruses carrying neuron specific promoters are suitable for both cortical and subcortical injections even when injected quickly.

  4. Domain Ⅲ of Dengue Virus Serotype 2 Envelope:Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Obejective The domainⅢof dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the inhibited effects of recombinant protein on virus was detected. Methods In this study, the domainⅢ(DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was puriifed using enzymatic cleavage and afifnity puriifcation. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢprotein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a speciifc soluble 29 kD protein was obtained, and the expression product accounted for 68.87%of the total protein of the cell lysate. Western blot demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was puriifed using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢprotein. At a 1︰16 dilution, the antibodies produced at least 90%neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢantibody titer reached 1︰1 024. Conclusions DENV-2 EDⅢwas cloned and expressed successfully. DENV-2 EDⅢprotein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

  5. 口服重组腺相关病毒基因药物%Oral recombinant adeno-associated virus gene medicine

    Institute of Scientific and Technical Information of China (English)

    刁勇; 许瑞安

    2009-01-01

    重组腺相关病毒(rAAV)载体介导的口服基冈药物引起业界广泛的重视.尽管经口服给药后转基因的有效表达面临许多障碍,但该技术的有效性已得到大量实验证实.本文总结了口服rAAV基冈药物的临床前研究结果,重点阐述了该类型药物的传递、吸收、分布和基冈转导等药动学特点.已证实rAAV基因药物对人体的安全性高,但口服rAAV基因药物的临床应用仍需对其作用机制和生物约剂学特征进行深入和广泛的研究.

  6. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    DEFF Research Database (Denmark)

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari;

    2010-01-01

    Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation...

  7. Definition of the region on NS3 which contains multiple epitopes recognized by dengue virus serotype-cross-reactive and flavivirus-cross-reactive, HLA-DPw2-restricted CD4+ T cell clones.

    Science.gov (United States)

    Okamoto, Y; Kurane, I; Leporati, A M; Ennis, F A

    1998-04-01

    The epitopes recognized by six CD4+ CD8- cytotoxic T lymphocyte (CTL) clones established from a dengue-3 virus-immune donor were defined. (i) Three CTL clones, JK10, JK34 and JK39, were cross-reactive for dengue virus types 1-4. (ii) One clone, JK28, was cross-reactive for dengue virus types 1-4 and West Nile virus. (iii) Two clones, JK26 and JK49, were cross-reactive for dengue virus types 1-4, West Nile virus and yellow fever virus. The clones, except for JK49, recognized the same epitope on NS3 in an HLA-DPw2-restricted fashion. The smallest synthetic peptide recognized by the five CTL clones was a 10 aa peptide which comprises aa 255-264 on dengue virus NS3. JK49 recognized the overlapping epitope which comprises aa 257-266 in an HLA-DPw2-restricted fashion. Analysis of T cell receptor (TCR) usage by these T cell clones revealed that (i) JK10 and JK34 use V alpha11, and JK34 and JK28 use V beta23, and (ii) the amino acid sequences of the V(D)J junctional region of the TCR were different among these five CTL clones. There were, however, single amino acid conservations among TCRs of some of these T cell clones. These results indicate that the region on NS3 which comprises aa 255-266 contains multiple epitopes recognized by dengue serotype-cross-reactive and flavivirus-cross-reactive CD4+ CTL in an HLA-DPw2-restricted fashion and that a single epitope can be recognized by T cells which have heterogeneous virus specificities.

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

    Institute of Scientific and Technical Information of China (English)

    阮喜云; 魏敏; 毕建忠; 杨广笑; 王全颍

    2011-01-01

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

  9. Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis.

    Science.gov (United States)

    Renz, K G; Cheetham, B F; Walkden-Brown, S W

    2013-01-01

    Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.

  10. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen.

    Science.gov (United States)

    Wu, Han-Chung; Jung, Mei-Ying; Chiu, Chien-Yu; Chao, Ting-Ting; Lai, Szu-Chia; Jan, Jia-Tsrong; Shaio, Men-Fang

    2003-10-01

    In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.

  11. Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions▿

    OpenAIRE

    Wang, Hongjie; Li, ZongYi; Yumul, Roma; Lara, Stephanie; Hemminki, Akseli; Fender, Pascal; Lieber, André

    2011-01-01

    Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96–104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob...

  12. Prevalencia de anticuerpos neutralizantes contra los serotipos del virus dengue en universitarios de Tabasco, México Prevalence of neutralizing antibodies to dengue virus serotypes in university students from Tabasco, Mexico

    Directory of Open Access Journals (Sweden)

    Gilma Guadalupe Sánchez-Burgos

    2008-10-01

    Full Text Available OBJETIVO: Determinar la seroprevalencia de anticuerpos neutralizantes de los serotipos del virus dengue en estudiantes universitarios de Tabasco, México, durante los meses de septiembre a noviembre del año 2005. MATERIAL Y MÉTODOS: Se determinó la presencia de IgG contra el virus en el suero de estudiantes que acudieron al centro clínico de la universidad; en los sueros positivos se determinaron los anticuerpos neutralizantes mediante el ensayo de reducción de placa lítica. RESULTADOS: La prevalencia de IgG contra el dengue fue de 9.1%; de esta proporción, los anticuerpos neutralizantes fueron DENV-1 (20%, DENV-2 (100%, DENV-3 (4% y DENV-4 (68%. CONCLUSIONES: Este estudio muestra que el serotipo transmitido con mayor frecuencia en el estado de Tabasco es el DENV-2, aunque no ha sido el aislado con más frecuencia. La elevada prevalencia de anticuerpos neutralizantes contra el DENV-4, al parecer de reacción cruzada, podría explicar la baja circulación de este serotipo en Tabasco.OBJECTIVE: Determine the seroprevalence of neutralizing antibodies to dengue virus in students from the state university of Tabasco, Mexico. MATERIAL AND METHODS: A transversal study was conducted of serum collected from students between September and November, 2005. The sera were screened for anti-dengue IgG and those that had evidence of dengue antibodies were analyzed by a plaque reduction neutralization test. RESULTS: Prevalence of anti-dengue IgG was 9.1%. The frequency of neutralizing antibodies was 100% for DENV-2, 68% for DENV-4, 20% for DENV-1, and 4 % for DENV-3. CONCLUSIONS: We found that in this population, DENV-2 circulates more than DENV-3 despite the fact that DENV-3 is more frequently isolated. Unexpectedly, neutralizing antibodies against DENV-4 were frequently found even though this serotype is almost extinct; thus, it is probable that cross-immunity could suppress DEN-4 transmission, as has been suggested.

  13. Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones.

    OpenAIRE

    Gagnon, S J; Zeng, W.; Kurane, I; Ennis, F A

    1996-01-01

    We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we ...

  14. Pneumonia due to pandemic (H1N1) 2009 influenza virus and Klebsiella pneumoniae capsular serotype K16 in a patient with nasopharyngeal cancer.

    Science.gov (United States)

    Lai, Chih-Cheng; Lee, Pei-Lin; Tan, Che-Kim; Huang, Yu-Tsung; Kao, Chiang-Lian; Wang, Jin-Town; Hsueh, Po-Ren

    2012-10-01

    Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and group A Streptoccocus, but no Klebsiella pneumoniae were responsible for bacterial coinfections during the 2009 and previous influenza pandemics. We hereby report a case with concurrent bacteremic pneumonia due to an unusual capsular serotype K16 K. pneumoniae and pandemic (H1N1) 2009 influenza in a patient with nasopharyngeal cancer. Such a coinfection has not previously been described.

  15. Serotype specificity of B-haplotype influence on the relative efficacy of Marek's disease vaccines.

    Science.gov (United States)

    Bacon, L D; Witter, R L

    1994-01-01

    B-haplotype genes in the chicken were previously shown to differentially influence vaccine efficacy against challenge with very virulent Marek's disease virus according to the type of Marek's disease (MD) vaccine used. To determine whether MD vaccines of the same serotype gave comparable levels of protection against MD in chickens of the same haplotype challenged with MD virus strain Md5, two serotype 1 and two serotype 2 vaccines were compared with one serotype 3 vaccine using chickens of 15-B-congenic lines. There was a strong correlation in development of MD lesions among chickens of the different lines receiving the two serotype 2 vaccines (r = 0.94) as well as among chickens receiving the two serotype 1 vaccines (r = 0.76). The serotype 1 vaccines were preferable for B2, B13, B15, and B21, but serotype 2 vaccines were more protective for B5 chickens. The two serotype 2 vaccines gave equivalent protection; however, of the serotype 1 vaccines, CVI988/Rispens provided more protection than Md11/75c/R2/23. We conclude that the B-haplotype influence on MD vaccine efficacy is dependent on the serotype of the vaccine.

  16. Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

    Indian Academy of Sciences (India)

    S B Nagendrakumar; M Madhanmohan; P N Rangarajan; V A Srinivasan

    2009-03-01

    The leader protease (Lpro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968–2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups – Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (< 5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or convergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the Lpro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the Lpro ( < 0.05; 0.046*) and at aa 171 in the capsid protein VP1 ( < 0.01; 0.003**).

  17. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.;

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  18. Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.

    Science.gov (United States)

    Neeraja, M; Lakshmi, V; Lavanya, Vanjari; Priyanka, E N; Parida, M M; Dash, P K; Sharma, Shashi; Rao, P V Lakshmana; Reddy, Gopal

    2015-01-01

    Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.

  19. 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....

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  1. Strains of Lentinula edodes suppress growth of phytopathogenic fungi and inhibit Alagoas serotype of vesicular stomatitis virus Linhagens de Lentinula edodes inibem fungos fitopatogênicos e o vírus da estomatite vesicular, sorotipo Alagoas

    Directory of Open Access Journals (Sweden)

    Selma H. Sasaki

    2001-03-01

    Full Text Available Four Lentinula edodes strains (Le10, 46, K2, Assai were assessed for their antagonistic effect on four filamentous fungus species of agricultural importance (Helminthosporium euphorbiae, Helminthosporium sp, Fusarium solani and Phomopsis sojae and on Alagoas serotype of Vesicular Stomatitis Virus (VSA. The L. edodes strains studied had variable effects on the filamentous fungi and on VSA. The K2 and Le10 strains were antagonistic on the fungi assessed and the 46 and K2 strains were efficient on the Vesicular Stomatitis Virus. The results widened the list of beneficial effects of L. edodes on the control and prevention of animal pathogenic virus and filamentous fungi.Quatro linhagens de Lentinula edodes (Le10, 46, K2, ASSAI foram avaliadas quanto ao seu efeito inibitório sobre quatro espécies de fungos filamentosos de importância agrícola (Helminthosporium euphorbiae, Helminthosporium sp., Fusarium solani, Phomopsis sojae e sobre o sorotipo Alagoas vírus da estomatite vesicular (VSA. Foi observado que as linhagens de L. edodes estudadas apresentaram variabilidade quanto ao seu efeito, tanto sobre os fungos filamentosos quanto sobre o vírus VSA. As linhagens K2 e Le10 apresentaram-se antagônicas sobre os fungos e as linhagens 46 e K2 foram eficientes na inibição do vírus VSA. Os resultados obtidos permitem ampliar a lista de efeitos benéficos de algumas linhagens de L. edodes no controle e prevenção de vírus patogênicos animais e de fungos filamentosos.

  2. Construction and preliminary investigation of a novel dengue serotype 4 chimeric virus using Japanese encephalitis vaccine strain SA14-14-2 as the backbone.

    Science.gov (United States)

    Li, Zhushi; Yang, Huiqiang; Yang, Jian; Lin, Hua; Wang, Wei; Liu, Lina; Zhao, Yu; Liu, Li; Zeng, Xianwu; Yu, Yongxin; Li, Yuhua

    2014-10-13

    For the purpose of developing a novel dengue vaccine candidate, recombinant plasmids were constructed which contained the full length cDNA clone of Japanese encephalitis (JE) vaccine strain SA14-14-2 with its premembrane (PreM) and envelope (E) genes replaced by the counterparts of dengue virus type 4 (DENV4). By transfecting the in vitro transcription products of the recombinant plasmids into BHK-21 cells, a chimeric virus JEV/DENV4 was successfully recovered. The chimeric virus was identified by complete genome sequencing, Western blot and immunofluorescent staining. Growth characteristics revealed it was well adapted to primary hamster kidney (PHK) cells. Its genetic stability was investigated and only one unintentional mutation in 5'-untranslated region (5'-UTR) was found after 20 passages in PHK cells. Neurotropism, neurovirulence and immunogenicity of the chimeric virus were tested in mice. Besides, the influence of JE vaccine pre-immunization on the neutralizing antibody level induced by the chimeric virus was illuminated. To our knowledge, this is the first chimeric virus incorporating the JE vaccine stain SA14-14-2 and DENV4. It is probably a potential candidate to compose a tetravalent dengue chimeric vaccine.

  3. EFSA Panel on Animal Health and Welfare (AHAW); Scientific Opinion on bluetongue serotype 8

    DEFF Research Database (Denmark)

    Bøtner, Anette; Oura, Chris; Saegerman, Claude

    To answer a question from the European Commission on the potential special characteristics of bluetongue virus (BTV) serotype 8 (BTV-8) compared to other serotypes and their possible impact on the epidemiology of the disease, a systematic literature review was carried out by a working group...

  4. 登革病毒EDⅢ特异性单抗的体内外中和活性观察%A murine monoclone antibody against envelope protein domain Ⅲ neutralizes dengue virus four serotypes in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    黄艳芬; 邓永强; 丘立文; 刘利东; 秦鄂德; 车小燕; 秦成峰

    2013-01-01

    目的 鉴定1株能同时识别登革病毒Ⅰ~Ⅳ型(dengue virus serotypesⅠ-Ⅳ,DENV 1~4)的EDⅢ特异性单抗2D73的体内外中和活性.方法 采用间接酶联免疫吸附实验(ELISA)和间接免疫荧光法(IFA)对单抗2D73的特性进行鉴定,并用基于酶联免疫斑点法的微中和实验(ELISPOT-MNT)和乳鼠保护实验观察单抗2D73对DENV 1~4的体内外中和活性.结果 单抗2D73对4型DENV均具有较强的中和活性,其50%抑制浓度(IC50)分别为0.28、0.16、0.18和18.82 μg/ml.乳鼠保护实验显示,该抗体对DENV 1~4同样具有较好的体内保护效果,与对照组相比,实验组小鼠发病时间明显延迟,生存率显著提高(P<0.05).结论 获得1株同时针对DENV 1~4且具有体内外中和活性的单抗,该单抗可进一步用于DENV致病和免疫机制的研究以及抗病毒药物的研制.%Objective To evaluate the neutralizing activities of a murine monoclonal antibody ( mAb) 2D73 against dengue virus serotypes Ⅰ-Ⅳ( DENV1-4) in vitro and in vivo. Methods Indirect ELISA and immuno fluorescence assay (IFA) were applied to identify specificity of mAb 2D73. Enzyme-linked immunospot micro -neutralizing test (ELISPOT-MNT) was performed to identify its neutralizing activities in vitro, and protective capacity was evaluated in a stringent in -tracranial challenge suckling mice model. Results All the four DENV serotypes were strongly neutralized by mAb 2D73 with 50% inhibitory concentrations (IC50) of 0. 28 , 0. 16 , 0. 18 and 18. 82 μg/ml to DENV1-4 , respectively. The onset of pathogenesis was delayed and the survival rate was improved obviously (P < 0. 05 ) in experimental group compared with control group. Conclusion MAb 2D73 against all the four DENV serotypes has strong neutralizing activities in vitro and protective efficacy in vivo, and might be used for the study of the pathogenic and immunologic mechanism of DENV and for the development of antiviral therapy.

  5. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    Directory of Open Access Journals (Sweden)

    Xia Feng

    Full Text Available The efficacy of an inactivated foot-and-mouth disease (FMD vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01. In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

  6. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    Science.gov (United States)

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

  7. Form, symmetry and packing of biomacromolecules. II. Serotypes of human rhinovirus

    Science.gov (United States)

    Janner, A.

    2010-05-01

    The differentiation of the human rhinovirus into serotypes, all having very similar structures and the same architecture, is shown to be related to the packing of the viruses in the crystal and to its space-group symmetry.

  8. Seroepidemiological investigation of foot-and-mouth disease virus serotypes in cattle around Lake Mburo National Park in South-Western Uganda

    DEFF Research Database (Denmark)

    Mwiine, Frank Norbert; Ayebazibwe, Chrisostom; Alexandersen, Søren

    2010-01-01

    Foot-and-mouth disease (FMD) outbreaks in cattle occur annually in Uganda. In this study the authors investigated antibodies against FMD virus (FMDV) in cattle in surrounding areas of Lake Mburo National Park in South-western Uganda. Two hundred and eleven serum samples from 23 cattle herds were...

  9. Ns1 is a key protein in the vaccine composition to protect Ifnar(-/- mice against infection with multiple serotypes of African horse sickness virus.

    Directory of Open Access Journals (Sweden)

    Francisco de la Poza

    Full Text Available African horse sickness virus (AHSV belongs to the genus Orbivirus. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA expressing VP2 and NS1 proteins from AHSV-4. IFNAR((-/- mice inoculated with DNA/rMVA-VP2,-NS1 from AHSV-4 in an heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies specific of AHSV-4. In addition, vaccination stimulated specific T cell responses against the virus. The vaccine elicited partial protection against an homologous AHSV-4 infection and induced cross-protection against the heterologous AHSV-9. Similarly, IFNAR((-/- mice vaccinated with an homologous prime-boost strategy with rMVA-VP2-NS1 from AHSV-4 developed neutralizing antibodies and protective immunity against AHSV-4. Furthermore, the levels of immunity were very high since none of vaccinated animals presented viraemia when they were challenged against the homologous AHSV-4 and very low levels when they were challenged against the heterologous virus AHSV-9. These data suggest that the immunization with rMVA/rMVA was more efficient in protection against a virulent challenge with AHSV-4 and both strategies, DNA/rMVA and rMVA/rMVA, protected against the infection with AHSV-9. The inclusion of the protein NS1 in the vaccine formulations targeting AHSV generates promising multiserotype vaccines.

  10. Morphologic and phenotypic characteristics of myocarditis in two pigs infected by foot-and mouth disease virus strains of serotypes O or A

    Science.gov (United States)

    Myocarditis is often cited as the cause of fatalities associated with foot-and-mouth disease virus (FMDV) infection; however the pathogenesis of FMDV-associated myocarditis has not been described in detail. The current report describes substantial quantities of FMDV in association with a marked mono...

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

    Science.gov (United States)

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

    2008-12-19

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

  12. Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi, India.

    Science.gov (United States)

    Afreen, Nazia; Naqvi, Irshad H; Broor, Shobha; Ahmed, Anwar; Parveen, Shama

    2015-01-01

    Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.

  13. Optimizing celgosivir therapy in mouse models of dengue virus infection of serotypes 1 and 2: The search for a window for potential therapeutic efficacy.

    Science.gov (United States)

    Watanabe, Satoru; Chan, Kitti Wing-Ki; Dow, Geoffrey; Ooi, Eng Eong; Low, Jenny G; Vasudevan, Subhash G

    2016-03-01

    Although the antiviral drug celgosivir, an α-glucosidase I inhibitor, is highly protective when given twice daily to AG129 mice infected with dengue virus, a similar regimen of twice daily dosing did not significantly reduce serum viral loads in patients in a recent clinical trial. This failure presumably might reflect the initiation of treatment when patients were already viremic. To better mimic the clinical setting, we used viruses isolated from patients to develop new mouse models of DENV1 and DENV2 infection and employed the models to test the twice daily treatment, begun either on the day of infection or on the third day post-infection, when the mice had peak of viremia. We found that, although the treatment started on day 0 was effective on viral load reduction, it provided no benefit when begun on day 3, indicating that in vivo antiviral efficacy becomes less prominent once viremia reaches the peak level. To determine if the therapeutic regimen in humans could be improved, we tested regimen of four-times daily treatment and found that the treatment significantly reduced viremia, suggesting that a similar regimen may be effective in a human clinical trial. A new clinical trial to investigate an altered dosing regimen has been approved (NCT02569827).

  14. 4种血清型登革病毒NS1蛋白序列及B细胞抗原表位特异性分析%Analysis on genome and amino acids sequence and B cell epitopes for 4 serotypes of dengue virus NS1 protein

    Institute of Scientific and Technical Information of China (English)

    陈艳佳; 熊建英; 朱利; 曹虹; 赵卫

    2013-01-01

    目的 比较4种血清型登革病毒NS1蛋白型特异性抗原表位基因序列及氨基酸序列之间的差异,为利用基因差异进行血清学分型及疫苗研究提供新的线索.方法 利用DNAstar数据包中的Editseq程序,从20株登革病毒分离株的全基因组序列中将NS1基因型特异性抗原表位序列截取出来,再用Clustal X软件进行多序列比对,进行同源性分析,找出型内最为保守的抗原表位序列.并将比对结果在120株登革病毒序列中进一步验证.结果 NS1蛋白36~45和71~85位氨基酸为型特异性抗原表位,高度保守,型内完全相同,型间不同.结论 NS1蛋白36~45位氨基酸可以作为登革病毒血清分型和研制亚单位疫苗的靶标.%OBJECTIVE To compare genome and amino acids sequences and possible B cell epitopes of 4 serotypes of dengue virus NS1 protein, to explore a new method of gene typing and provide new clues to vaccine research. METHODS Cut off the corresponding gene sequences of NS1 protein from the complete genome of 20 dengue virus isolates, then multisequencing was carried out to find the gene which was conservative within the same serotype and variant in the other serotypes. RESULTS Specific antigens of NS1 protein (36-45 and 71-85 amino acids) were conservative within the same serotype and variant in other serotypes. CONCLUSION Specific antigens of NS1 protein (36-45 amino acids) are the bases of dengue virus typing and targets of subunit vaccine development.

  15. Evaluation of a DNA vaccine candidate expressing prM-E-NS1 antigens of dengue virus serotype 1 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) in immunogenicity and protection.

    Science.gov (United States)

    Zheng, Qun; Fan, Dongying; Gao, Na; Chen, Hui; Wang, Juan; Ming, Ying; Li, Jieqiong; An, Jing

    2011-01-17

    Dengue is one of the most important mosquito-borne viral diseases. In past years, although considerable effort has been put into the development of a vaccine, there is currently no licensed dengue vaccine. In this study, we constructed DNA vaccines that carried the prM-E-NS1 genes of dengue virus serotype 1 (DV1) with or without the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, an attractive DNA vaccine adjuvant. Immunization with the plasmid pCAG-DV1/E/NS1, which expresses viral prM-E-NS1, or the bicistronic plasmid pCAG-DV1-GM, which co-expresses viral prM-E-NS1 and GM-CSF, resulted in long-term IgG response, high levels of splenocyte-secreted interferon-γ and interleukin-2, strong cytotoxic T lymphocyte activity and sufficient protection in the DV1-challenged mice. This suggested that both humoral and cellular immune responses were induced by the immunizations and that they played important roles in protection against the DV1 challenge. Interestingly, the magnitude, quality and protective capacity of the immune responses induced by immunization with pCAG-DV1/E/NS1 or pCAG-DV1-GM seemed stronger than those induced by pCAG-DV1/E (expressing viral prM-E alone). Taken together, we demonstrated that prM/E plus NS1 would be a suitable solution for the development of a DNA vaccine against DV.

  16. The distinct distribution and phylogenetic characteristics of dengue virus serotypes/genotypes during the 2013 outbreak in Yunnan, China: Phylogenetic characteristics of 2013 dengue outbreak in Yunnan, China.

    Science.gov (United States)

    Wang, Binghui; Yang, Henglin; Feng, Yue; Zhou, Hongning; Dai, Jiejie; Hu, Yunzhang; Zhang, Li; Wang, Yajuan; Baloch, Zulqarnain; Xia, Xueshan

    2016-01-01

    Since 2000, sporadic imported cases of dengue fever were documented almost every year in Yunnan Province, China. Unexpectedly, a large-scale outbreak of dengue virus (DENV) infection occurred from August to December 2013, with 1538 documented cases. In the current study, 81 dengue-positive patient samples were collected from Xishuangbanna, the southernmost prefecture of the Yunnan province, and 23 from Dehong, the westernmost prefecture of the Yunnan province. The full-length envelope genes were amplified and sequenced. Phylogenetic analysis revealed that nine strains (39.1%) and 14 strains (60.9%) from the Dehong prefecture were classified as genotype I of DENV-1 and Asian I genotype of DENV-2, respectively. All strains from Xishuangbanna were identified as genotype II of DENV-3. Bayesian coalescent analysis indicates that the outbreak originated from bordering southeastern Asian countries. These three epidemic genotypes were predicted to originate in Thailand and then migrate into Yunnan through different routes.

  17. Analysis of the acute phase responses of Serum Amyloid A, Haptoglobin and Type 1 Interferon in cattle experimentally infected with foot-and-mouth disease virus serotype O

    DEFF Research Database (Denmark)

    Stenfeldt, Carolina; Heegaard, Peter M. H.; Stockmarr, Anders;

    2011-01-01

    periods exceeding 28 days in order to determine the carrier-status of individual animals. The systemic host response to FMDV in infected animals was evaluated in comparison to similar measurements in sera from 6 mock-inoculated control animals.There was a significant increase in serum concentrations...... of both APPs and type 1 IFN in infected animals coinciding with the onset of viremia and clinical disease. The measured parameters declined to baseline levels within 21 days after inoculation, indicating that there was no systemically measurable inflammatory reaction related to the carrier state of FMD......A series of challenge experiments were performed in order to investigate the acute phase responses to foot-and-mouth disease virus (FMDV) infection in cattle and possible implications for the development of persistently infected "carriers". The host response to infection was investigated through...

  18. Experimental infection of mice with avian paramyxovirus serotypes 1 to 9.

    Directory of Open Access Journals (Sweden)

    Sunil K Khattar

    Full Text Available The nine serotypes of avian paramyxoviruses (APMVs are frequently isolated from domestic and wild birds worldwide. APMV-1, also called Newcastle disease virus, was shown to be attenuated in non-avian species and is being developed as a potential vector for human vaccines. In the present study, we extended this evaluation to the other eight serotypes by evaluating infection in BALB/c mice. Mice were inoculated intranasally with a prototype strain of each of the nine serotypes and monitored for clinical disease, gross pathology, histopathology, virus replication and viral antigen distribution, and seroconversion. On the basis of multiple criteria, each of the APMV serotypes except serotype 5 was found to replicate in mice. Five of the serotypes produced clinical disease and significant weight loss in the following order of severity: 1, 2>6, 9>7. However, disease was short-lived. The other serotypes produced no evident clinical disease. Replication of all of the APMVs except APMV-5 in the nasal turbinates and lungs was confirmed by the recovery of infectious virus and by substantial expression of viral antigen in the epithelial lining detected by immunohistochemistry. Trace levels of infectious APMV-4 and -9 were detected in the brain of some animals; otherwise, no virus was detected in the brain, small intestine, kidney, or spleen. Histologically, infection with the APMVs resulted in lung lesions consistent with broncho-interstitial pneumonia of varying severity that were completely resolved at 14 days post infection. All of the mice infected with the APMVs except APMV-5 produced serotype-specific HI serum antibodies, confirming a lack of replication of APMV-5. Taken together, these results demonstrate that all APMV serotypes except APMV-5 are capable of replicating in mice with minimal disease and pathology.

  19. Analysis of the acute phase responses of Serum Amyloid A, Haptoglobin and Type 1 Interferon in cattle experimentally infected with foot-and-mouth disease virus serotype O

    Directory of Open Access Journals (Sweden)

    Stenfeldt Carolina

    2011-05-01

    Full Text Available Abstract A series of challenge experiments were performed in order to investigate the acute phase responses to foot-and-mouth disease virus (FMDV infection in cattle and possible implications for the development of persistently infected "carriers". The host response to infection was investigated through measurements of the concentrations of the acute phase proteins (APPs serum amyloid A (SAA and haptoglobin (HP, as well as the bioactivity of type 1 interferon (IFN in serum of infected animals. Results were based on measurements from a total of 36 infected animals of which 24 were kept for observational periods exceeding 28 days in order to determine the carrier-status of individual animals. The systemic host response to FMDV in infected animals was evaluated in comparison to similar measurements in sera from 6 mock-inoculated control animals. There was a significant increase in serum concentrations of both APPs and type 1 IFN in infected animals coinciding with the onset of viremia and clinical disease. The measured parameters declined to baseline levels within 21 days after inoculation, indicating that there was no systemically measurable inflammatory reaction related to the carrier state of FMD. There was a statistically significant difference in the HP response between carriers and non-carriers with a lower response in the animals that subsequently developed into FMDV carriers. It was concluded that the induction of SAA, HP and type 1 IFN in serum can be used as markers of acute infection by FMDV in cattle.

  20. Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo.

    Science.gov (United States)

    Williams, Katherine L; Wahala, Wahala M P B; Orozco, Susana; de Silva, Aravinda M; Harris, Eva

    2012-07-20

    The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.

  1. A Tetravalent Dengue Vaccine Based on a Complex Adenovirus Vector Provides Significant Protection in Rhesus Monkeys against All Four Serotypes of Dengue Virus▿

    OpenAIRE

    Raviprakash, Kanakatte; Wang, Danher; Ewing, Dan; Holman, David H.; Block, Karla; Woraratanadharm, Jan; Chen, Lan; Hayes, Curtis; Dong, John Y.; Porter, Kevin

    2008-01-01

    Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vect...

  2. Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes.

    OpenAIRE

    Bukowski, J F; Kurane, I; Lai, C J; Bray, M.; Falgout, B.; Ennis, F A

    1989-01-01

    Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type...

  3. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    Directory of Open Access Journals (Sweden)

    Jiandong Shi

    Full Text Available Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB, CD8+ T-cell epitopes of the dengue virus (DENV NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21,-B (53, and-C (42 had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01, 515NS5525 (98.71%, A*24:02, 225NS5232 (99.35%, A*33:03, 516NS5523 (98.71%, A*33:03, and 284NS5291 (98.06%, A*33:03, were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01, 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01, 262NS5269 (92.90%, B*38:02, and 538NS5547 (90.97%, B*51:01, were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02, 514NS5524 (98.71%, C*01:02 and C*14:02, 92NS599 (98.06%, C*03:02 and C*15:02, 362NS5369 (44.84%, C*03:04 and C*08:01, 225NS5232 (99.35%, C*04:01, 234NS5241(96.77%, C*04:01, 361NS5369 (94.84%, C*04:01, 515NS5522 (98.71%, C*14:02, 515NS5524 (98.71%, C*14:02, were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02, 200NS5210 (A*11:01, 362NS5369 (C*03:04, C*08:01, and 514NS5524 (C*01:02, C*14:02 could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  4. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    Science.gov (United States)

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Li, Jianfan; Li, Yanhan; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  5. Cross-protective efficacy of engineering serotype A foot-and-mouth disease virus vaccine against the two pandemic strains in swine.

    Science.gov (United States)

    Zheng, Haixue; Lian, Kaiqi; Yang, Fan; Jin, Ye; Zhu, Zixiang; Guo, Jianhong; Cao, Weijun; Liu, Huanan; He, Jijun; Zhang, Keshan; Li, Dan; Liu, Xiangtao

    2015-10-26

    Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that affects domestic and wild cloven-hoofed animals worldwide. Recently, a series of outbreaks of type A FMDV occurred in Southeast Asian countries, China, the Russia Federation, Mongolia, Kazakhstan and South Korea. The FMD virus (A/GDMM/CHA/2013) from China's Guangdong province (2013) is representative of those responsible for the latest epidemic, and has low amino acid identity (93.9%) in VP1 protein with the epidemic strain A/WH/CHA/09 from Wuhan, China in 2009. Both of isolates belong to the Sea-97 genotype of ASIA topotype. Therefore, the application of a new vaccine strain with cross-protective efficacy is of fundamental importance to control the spread of the two described pandemic strains. A chimeric strain rA/P1-FMDV constructed by our lab previously through replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09, has been demonstrated to exhibit good growth characteristics in culture, and the rA/P1-FMDV inactivated vaccine can provide protection against epidemic strain A/WH/CHA/09 in cattle. However, it is still unclear whether the vaccine produces efficient protection against the new pandemic strain (A/GDMM/CHA/2013). Here, vaccine matching and pig 50% protective dose (PD50) tests were performed to assess the vaccine potency. The vaccine matching test showed cross-reactivity of sera from full dose vaccine vaccinated pigs with A/WH/CHA/09 and A/GDMM/CHA/2013 isolates, with average r1 values of 0.94±0.12 and 0.68±0.06 (r1≥0.3), which indicates that the rA/P1-FMDV vaccine is likely to confer good cross-protection against the two isolates. When challenged with two pandemic isolates A/WH/CHA/09 and A/GDMM/CHA/2013 strain, the vaccine achieved 12.51 PD50 and 10.05 PD50 per dose (2.8μg), respectively. The results indicated that the rA/P1-FMDV inactivated vaccine could protect pigs against both A/WH/CHA/09 and A/GDMM/CHA/2013 pandemic isolates.

  6. Dengue virus type 3, South Pacific Islands, 2013.

    Science.gov (United States)

    Cao-Lormeau, Van-Mai; Roche, Claudine; Musso, Didier; Mallet, Henri-Pierre; Dalipanda, Tenneth; Dofai, Alfred; Nogareda, Francisco; Nilles, Eric J; Aaskov, John

    2014-06-01

    After an 18-year absence, dengue virus serotype 3 reemerged in the South Pacific Islands in 2013. Outbreaks in western (Solomon Islands) and eastern (French Polynesia) regions were caused by different genotypes. This finding suggested that immunity against dengue virus serotype, rather than virus genotype, was the principal determinant of reemergence.

  7. Simultaneous circulation of all four dengue serotypes in Manaus, State of Amazonas, Brazil in 2011

    Directory of Open Access Journals (Sweden)

    Michele de Souza Bastos

    2012-06-01

    Full Text Available INTRODUCTION: Manaus, the capital city of the state of Amazon with nearly 2 million inhabitants, is located in the middle of the Amazon rain forest and has suffered dengue outbreaks since 1998. METHODS: In this study, blood samples were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR, aimed at identifying dengue virus serotypes. RESULTS: Acute phase sera from 432 patients were tested for the presence of dengue virus. Out of the 432 patients, 137 (31.3% were found to be positive. All the four dengue virus serotypes were observed. CONCLUSIONS: The simultaneous circulation of the four dengue serotypes is described for the first time in Manaus and in Brazil.

  8. Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated nucleus pulposus (NP) cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.

  9. 重组腺相关病毒质量控制的qPCR技术研究进展%Advances in Real-Time Quantitative PCR Technology for Quality Control of Recombinant Adeno-Associated Virus

    Institute of Scientific and Technical Information of China (English)

    肖桂清; 杨会勇; 刁勇

    2014-01-01

    综述实时荧光定量PCR(qPCR)技术在重组腺相关病毒(rAAV)的基因组滴度测定中的应用成果,以及qPCR技术在rAAV质量控制过程中的应用,如感染滴度及rcAAV污染率的测定等.分析qPCR技术存在的适用范围窄、易产生系统性误差和易受rAAV杂质干扰等问题的原因,并探讨相关的解决策略.

  10. Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum.

    Science.gov (United States)

    Wang, Hsi-Kai; Tsai, Cheng-Han; Chen, Kuan-Hung; Tang, Chung-Tao; Leou, Jiun-Shyang; Li, Pi-Chun; Tang, Yin-Liang; Hsieh, Hsyue-Jen; Wu, Han-Chung; Cheng, Chao-Min

    2014-02-01

    Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

  11. Infecciones concurrentes por dos serotipos del virus dengue durante un brote en el noroeste de Perú, 2008 Concurrent infections by two dengue virus serotypes during an outbreak in northwestern Peru, 2008

    OpenAIRE

    Enrique Mamani; Dana Figueroa; María Paquita García; María del Carmen Garaycochea; Pozo, Edwar J.

    2010-01-01

    Objetivo. Describir la existencia de infecciones concurrentes por diferentes serotipos del virus dengue (DENV) en un brote ocurrido en el noroeste de Perú durante el 2008. Materiales y métodos. Se analizó 73 muestras séricas de pacientes con dengue en un brote en el noroeste de Perú entre mayo y junio de 2008. Para la serotipificación del DENV se utilizó técnicas de biología molecular; así, primero se realizó la extracción del ARN con el kit QIAamp viral RNA Mini, luego se realizó la transcri...

  12. Co-circulation of two extremely divergent serotype SAT 2 lineages in Kenya highlights challenges to foot-and-mouth disease control.

    Science.gov (United States)

    Sangula, A K; Belsham, G J; Muwanika, V B; Heller, R; Balinda, S N; Siegismund, H R

    2010-10-01

    Amongst the SAT serotypes of foot-and-mouth disease virus (FMDV), the SAT 2 serotype is the most widely distributed throughout sub-Saharan Africa. Kenyan serotype SAT 2 viruses have been reported to display the highest genetic diversity for the serotype globally. This complicates diagnosis and control, and it is essential that patterns of virus circulation are known in order to overcome these difficulties. This study was undertaken to establish patterns of evolution of FMDV serotype SAT 2 in Kenya using complete VP1 coding sequences in a dataset of 65 sequences from Africa, collected over a period of 50 years. Two highly divergent lineages were observed to co-circulate, and occasional trans-boundary spread was inferred, emphasizing the value of constant monitoring and characterization of field strains for improved diagnosis and appropriate vaccine application as well as the need for regional approaches to control.

  13. Geo-spatial distribution of serologically detected bovine Foot and Mouth Disease (FMD serotype outbreaks in Ilesha Baruba, Kwara State-Nigeria

    Directory of Open Access Journals (Sweden)

    Hamza Olatunde Olabode

    2014-09-01

    Full Text Available The study was aimed at assessing the prevalence and distribution of bovine Foot and Mouth Disease (FMD serotypes in Ilesha Baruba, Kwara state-Nigeria. To identify the source of epidemics, geo-spatial analysis was done on the FMD outbreak locations (n=15 using Global Positioning Service (GPS device (EtrexR. Randomly sampled bovine sera (n=64 from herd representatives were subjected to FMD 3ABC enzyme-linked immunosorbent assay (FMD 3ABC ELISA and solid-phase competitive ELISA (SP-cELISA, for the screening and serotyping of FMD virus, respectively. Through ELISA, the FMD serotypes detected in this study were- serotype O (83%; n=53/64, serotype A (7.8%; n=5/64, serotype vaccine O (1.6%; n=1/64, and serotype vaccine SAT2 (1.6%; n=1/64. Multiple serotypes were observed in two different combinations; these were O and A (4.7%; n=3/64, and O and SAT2 (1.6%; n=1/64. FMD multiple serotype infections were associated with absence of cross-immunity between serotypes and cross reactivity enhanced by clustered herds, highland study area topography, road and river interconnectivity, possible human settlements, activities and traffic. This study provides baseline information on geo-spatial distribution, and identification of prevalent FMD serotypes in Ilesha Baruba, Kwara state-Nigeria.

  14. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

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    Akseli Hemminki

    2010-09-01

    Full Text Available Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

  15. Evaluation of Infectivity, Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea.

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    Juan M Pacheco

    Full Text Available Since the early 2000s outbreaks of foot-and-mouth disease (FMD have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea. One outbreak with FMD virus (FDMV serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.

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

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    Hildegund CJ Ertl

    2011-07-01

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

  17. Identification of Avian Paramyxovirus Serotype-1 in Wild Birds in the USA

    Science.gov (United States)

    In the US, sampling for avian paramyxovirus serotype-1 (APMV-1) is generally conducted when morbidity or mortality events occur involving certain families of wild birds known to be affected by the virus, such as cormorants (Family Phalacrocoracidae), pigeons, doves (Family Columbidae), or pelicans (...

  18. Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds

    Science.gov (United States)

    Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we se...

  19. Establishment and Application of Real-time Quantitative PCR to Detect Duck Hepatitis A Virus Serotype 1%血清1型鸭甲型肝炎病毒实时定量P CR检测方法的建立与初步应用

    Institute of Scientific and Technical Information of China (English)

    孙庆歌; 谢红玲; 李晶梅; 朱薇; 肖爱芳; 赖志; 薛霜; 高俊锋; 马慧慧; 祝春花

    2014-01-01

    In order to develop a real-time quantitative PCR to detect duck hepatitis A virus serotype 1 ( DHAV-1) , a pair of primers and one TaqMan probe were designed and synthesized, according to the 5 ’ untranslated sequences of DHAV-1 published on GenBank, The recombinant plasmid was built as a standard control for the method, the specificity, sensitivity, and repeatability of the method were determined, and also preliminarily applicated in the detection of clinical samples. The results showed that the detection assay was specific for DHAV-1, and there was no cross reaction between duck hepatitis A virus serotype 3( DHAV-3) and the other viruses including duck plague virus, Newcastle disease virus, avian influenza virus, duck reovirus, avian infectious bronchitis virus, etc. The method also showed high sensitivity, as low as 10 copies virus could be detected for each reaction, and good reproducibility, there was a coefficient of variations less than 3% for both inter-assay and intra-assay. The detection results of clinical samples were consistent with the sequencing. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and repeatability;it was useful for the rapid diagnosis and quantification analysis of DHAV-1.%为建立一种可检测血清1型鸭甲型肝炎病毒( DHAV-1)的实时定量PCR方法,根据GenBank中DHAV-15’非编码区的保守区,设计合成1对引物和1条TaqMan探针,以构建的重组质粒作为标准品,绘制标准曲线,并对所建立方法进行了特异性、敏感性和可重复性试验以及临床病料检测初步应用。结果,该方法与血清3型鸭甲型肝炎病毒、鸭瘟病毒、新城疫病毒、禽流感病毒、呼肠孤病毒、传染性支气管炎病毒等无交叉反应性;最低可以检测到10 copies/μL;组内和组间变异系数均小于3%;临床病料的检测结果与测序检测结果一致。

  20. New Salmonella serotype: Salmonella enteritidis serotype Grandhaven (30(1):r:1,2).

    OpenAIRE

    McDougal, D L; Treleaven, B E; Renshaw, E C

    1982-01-01

    A new Salmonella serotype, Salmonella enteritidis serotype Grandhaven (30(1):r:1,2), was isolated from the stool of a 35-year-old man with mild gastroenteritis. He had just returned from Sudan, Africa.

  1. Pneumococcal capsular polysaccharide structure predicts serotype prevalence.

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    Daniel M Weinberger

    2009-06-01

    Full Text Available There are 91 known capsular serotypes of Streptococcus pneumoniae. The nasopharyngeal carriage prevalence of particular serotypes is relatively stable worldwide, but the host and bacterial factors that maintain these patterns are poorly understood. Given the possibility of serotype replacement following vaccination against seven clinically important serotypes, it is increasingly important to understand these factors. We hypothesized that the biochemical structure of the capsular polysaccharides could influence the degree of encapsulation of different serotypes, their susceptibility to killing by neutrophils, and ultimately their success during nasopharyngeal carriage. We sought to measure biological differences among capsular serotypes that may account for epidemiological patterns. Using an in vitro assay with both isogenic capsule-switch variants and clinical carriage isolates, we found an association between increased carriage prevalence and resistance to non-opsonic neutrophil-mediated killing, and serotypes that were resistant to neutrophil-mediated killing tended to be more heavily encapsulated, as determined by FITC-dextran exclusion. Next, we identified a link between polysaccharide structure and carriage prevalence. Significantly, non-vaccine serotypes that have become common in vaccinated populations tend to be those with fewer carbons per repeat unit and low energy expended per repeat unit, suggesting a novel biological principle to explain patterns of serotype replacement. More prevalent serotypes are more heavily encapsulated and more resistant to neutrophil-mediated killing, and these phenotypes are associated with the structure of the capsular polysaccharide, suggesting a direct relationship between polysaccharide biochemistry and the success of a serotype during nasopharyngeal carriage and potentially providing a method for predicting serotype replacement.

  2. Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments

    NARCIS (Netherlands)

    Gennip, van H.G.P.; Veldman, D.; Water, van de S.G.P.; Rijn, van P.A.

    2010-01-01

    Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was repor

  3. Circulación de un linaje diferente del virus dengue 2 genotipo América / Asia en la región amazónica de Perú, 2010 Circulation of a different lineage of dengue virus serotype 2 American / Asian genotype in the Peruvian amazon, 2010

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    Enrique Mamani

    2011-03-01

    Full Text Available El objetivo del estudio fue determinar el genotipo del virus dengue tipo 2 (DENV-2 que circuló en la región Amazónica de Perú entre noviembre de 2010 y enero de 2011. Se analizaron ocho muestras de pacientes captados durante la vigilancia para dengue en las ciudades de Iquitos, Yurimaguas, Trujillo, Tarapoto y Lima entre noviembre de 2010 y enero de 2011 que fueron remitidas al Instituto Nacional de Salud. Se realizó el aislamiento viral en la línea C6/36 HT y la extracción del ARN viral. Se aplicaron técnicas de biología molecular para establecer el serotipo (RT - PCR múltiple y genotipo (RT-Nested PCR de la región E/NS1 seguidas de secuenciación y análisis filogenético. El análisis filogenético reveló la introducción de un linaje diferente que ingresó a Perú a finales del 2010. Estos aislamientos encontrados en Iquitos y otras ciudades de Perú están muy relacionados con aislamientos de DENV-2 que circularon en Brasil durante el 2007 y 2008 asociados con casos de dengue grave y muertes. En conclusión se detectó la introducción de un linaje diferente del DENV-2 genotipo América/Asia en Perú que podría estar asociado con la presencia de casos más graves de dengue.Our objective was to determine the genotype of the dengue virus type 2 (DENV-2 that circulated in the Amazon region of Peru between November 2010 and January 2011. We analyzed eight samples collected during dengue surveillance activities in the cities of Iquitos, Yurimaguas, Trujillo, Tarapoto and Lima between November 2010 and January 2011 that were sent to Insitituto Nacional de Salud. The viruses were isolated in C6/36 HT cell line. Viral RNA was extracted and the serotype (RT - PCR multiplex and genotype (RT-Nested PCR of the region E/NS1 were determined. Finally, the E/ NS1 amplicons were sequenced and analyzed by phylogeny. The phylogenetic analysis revealed the introduction of a different lineage which entered in Peru by the end of 2010. These isolates

  4. Serotype assignment by sero-agglutination, ELISA, and PCR

    Science.gov (United States)

    For assessing isolates of Listeria monocytogenes serotype designation is the foremost subtyping method used. Traditionally serotyping has been done with agglutination reactions. In the last decade alternative serotyping methods were described using Enzyme Linked Immunosorbent Assay(ELISA)and Polymer...

  5. PCR specific for Actinobacillus pleuropneumoniae serotype 3

    DEFF Research Database (Denmark)

    Zhou, L.; Jones, S.C.P.; Angen, Øystein

    2008-01-01

    , but the method has liminations, for example, cross-reactions between serotypes 3, 6, and 8. This study describes the development of a serotype 3-specific PCR, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The PCR test was evaluated on 266 strains...

  6. Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

    Directory of Open Access Journals (Sweden)

    Lucile Tran

    Full Text Available Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT imaging of gene expression to determine whether local virus administration (direct injection in the kidney could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting.

  7. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    Science.gov (United States)

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  8. Complete genome sequence of avian paramyxovirus (APMV serotype 5 completes the analysis of nine APMV serotypes and reveals the longest APMV genome.

    Directory of Open Access Journals (Sweden)

    Arthur S Samuel

    Full Text Available BACKGROUND: Avian paramyxoviruses (APMV consist of nine known serotypes. The genomes of representatives of all APMV serotypes except APMV type 5 have recently been fully sequenced. Here, we report the complete genome sequence of the APMV-5 prototype strain budgerigar/Kunitachi/74. METHODOLOGY/PRINCIPAL FINDINGS: APMV-5 Kunitachi virus is unusual in that it lacks a virion hemagglutinin and does not grow in the allantoic cavity of embryonated chicken eggs. However, the virus grew in the amniotic cavity of embryonated chicken eggs and in twelve different established cell lines and two primary cell cultures. The genome is 17,262 nucleotides (nt long, which is the longest among members of genus Avulavirus, and encodes six non-overlapping genes in the order of 3'N-P/V/W-M-F-HN-L-5' with intergenic regions of 4-57 nt. The genome length follows the 'rule of six' and contains a 55-nt leader sequence at the 3'end and a 552 nt trailer sequence at the 5' end. The phosphoprotein (P gene contains a conserved RNA editing site and is predicted to encode P, V, and W proteins. The cleavage site of the F protein (G-K-R-K-K-R downward arrowF conforms to the cleavage site motif of the ubiquitous cellular protease furin. Consistent with this, exogenous protease was not required for virus replication in vitro. However, the intracerebral pathogenicity index of APMV-5 strain Kunitachi in one-day-old chicks was found to be zero, indicating that the virus is avirulent for chickens despite the presence of a polybasic F cleavage site. CONCLUSIONS/SIGNIFICANCE: Phylogenetic analysis of the sequences of the APVM-5 genome and proteins versus those of the other APMV serotypes showed that APMV-5 is more closely related to APMV-6 than to the other APMVs. Furthermore, these comparisons provided evidence of extensive genome-wide divergence that supports the classification of the APMVs into nine separate serotypes. The structure of the F cleavage site does not appear to be a

  9. Dengue serotype cross-reactive, anti-E protein antibodies confound specific immune memory for one year after infection

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    Ying Xiu eToh

    2014-08-01

    Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

  10. Serotype Distribution and Demographics of Dengue Patients in a Tertiary Hospital of Lahore, Pakistan during the 2011 Epidemic

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    Muhammad MUNIR

    2013-06-01

    Full Text Available The dengue outbreak in Lahore, Pakistan during 2011 was unprecedented in terms of its severity and magnitude. This research aims to determine the serotype distribution of the dengue virus during this outbreak and classify the patients demographically. 5ml of venous blood was drawn aseptically from 166 patients with dengue like signs to test for the virus between the months of August to November 2011. The samples were sent to CDC, Atlanta, Georgia for the purpose of molecular assays to determine their serotype. RT-PCR protocol was performed targeting the 4 dengue serotypes. Out of 166 cases, dengue infection was detected with RT-PCR in 95 cases, all infected with the same serotype DEN-2. 75 % of positive cases were males while 25 % were females. Most positive patients were in the age range of 16 - 30 years. 33 % positive cases had accompanying bleeding. This is first study during the 2011 dengue epidemic in Lahore that reports DEN-2 as the only prevalent serotype. It also indicates that more infected patients were males, adults, within the age range of 16 - 30 years, the epidemic peaked in the month of November, Dengue hemorrhagic fever (DHF is manifested more in females, and that Ravi town was heavily hit by the dengue virus infection.

  11. Predicting antigenic sites on the foot-and-mouth disease virus capsid of the South African Territories (SAT) types using virus neutralization data

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) outer capsid proteins 1B, 1C and 1D contribute to the virus serotype distribution and antigenic variants that exist within each of the seven serotypes. This study presents a phylogenetic, genetic and antigenic analysis of the South African Territories (SAT) seroty...

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

    Science.gov (United States)

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

    2014-12-01

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

  13. Estimation of the time of seroconversion to the New Jersey serotype of vesicular stomatitis virus in sentinel cattle of dairy herds located at high and low elevations in southern Mexico

    Science.gov (United States)

    To estimate time to seroconversion to vesicular stomatitis 1 New Jersey virus (VSNJV) in sentinel cattle in southern Mexico, ninety-two sentinel cattle in four free-ranging dairy herds at high- (=500 mts) and low-elevation (<500 mts) locations in southern Mexico were studied. A prospective cohort s...

  14. Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9

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    Samuel Arthur S

    2011-02-01

    Full Text Available Abstract Avian paramyxoviruses (APMVs are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9. Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi. All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.

  15. Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

    Science.gov (United States)

    Apt, Doris; Raviprakash, Kanakatte; Brinkman, Alice; Semyonov, Andrey; Yang, Shumin; Skinner, Craig; Diehl, Lori; Lyons, Richard; Porter, Kevin; Punnonen, Juha

    2006-01-16

    We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.

  16. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

    Directory of Open Access Journals (Sweden)

    Monica Poggianella

    Full Text Available Dengue virus (DENV infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

  17. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

    Science.gov (United States)

    Poggianella, Monica; Slon Campos, José L; Chan, Kuan Rong; Tan, Hwee Cheng; Bestagno, Marco; Ooi, Eng Eong; Burrone, Oscar R

    2015-01-01

    Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

  18. Isolation of avian serotype 3 paramyxoviruses from imported caged birds in Israel.

    Science.gov (United States)

    Shihmanter, E; Weisman, Y; Lublin, A; Mahani, S; Panshin, A; Lipkind, M

    1998-01-01

    Ten avian serotype 3 paramyxoviruses were isolated for the first time in Israel from passerine and psittacine imported caged birds. The birds were submitted for investigation of an illness characterized by nonspecific signs of weakness, anorexia, vomiting, and sneezing. In addition, only the parakeets developed specific neurologic signs. In bacteriologic and pathologic investigation, cachexia and diarrhea were observed in both groups of birds. In psittacines, considerable alterations were observed in lungs, liver, and spleen. Some nonviral pathogens were occasionally isolated. The isolates appeared to belong to serotype 3b avian paramyxovirus (APMV), the prototype strain of which is APMV-3b/parakeet/Netherlands/449/75. The isolation of APMV-3 viruses from imported caged birds may represent a way of introduction of these viruses into the country.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-07-15

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

  20. Multi-serotype pneumococcal nasopharyngeal carriage prevalence in vaccine naive Nepalese children, assessed using molecular serotyping.

    Directory of Open Access Journals (Sweden)

    Rama Kandasamy

    Full Text Available Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49.5% of samples with more than one serotype being found in 67/297 (20.2% of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44.4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.

  1. The cps locus of Streptococcus suis serotype 16: Development of a serotype-specific PCR assay

    NARCIS (Netherlands)

    Wang, K.; Weixing, Fan; Wisselink, H.J.; Chengping, Lu

    2011-01-01

    Streptococcus suis serotype 16 can infect pigs and humans. We describe the identification and the characterization of the capsular polysaccharides synthesis locus of S. suis serotype 16. Using PCR primers flanking the capsular polysaccharides synthesis locus, a 30,101-bp fragment was amplified. Twen

  2. In silico antigenic site evaluation and antiviral therapy against dengue serotypes

    Directory of Open Access Journals (Sweden)

    Pratap Parida

    2014-03-01

    Full Text Available Non Structural protein 3 (NS3 constitute protease, helicase and polymerase that are essential for dengue virus replication. The aim of the present study is to block the replication of the virus by targeting the NS3 Protein. The retrieved sequences of NS3 protein from National Centre for Biotechnology information (NCBI shows that the antigenic sites of the protein are highly variable in all the four serotypes of dengue virus (DENV i.e. DENV I, DENV II, DENV III and DENV IV. DENV III found to be most distantly related serotype among all the serotypes studied using UPGMA method. The 3D structure of NS3 protein was modeled using homology modeling by MODELLER 9v8. Evaluation of the constructed NS3 protein models were done by PROCHECK, WhatIf using Exome Horizon. The derived compounds of mycophenolic acid and ribavirin were docked as ligands to the constructed models of NS3 protein using Autodock 4.2 for Protein-ligand interaction study.

  3. Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1-9 in chickens and ducks.

    Directory of Open Access Journals (Sweden)

    Shin-Hee Kim

    Full Text Available Avian paramyxovirus (APMV serotypes 1-9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2-9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT assay in chicken eggs and intracerebral pathogenicity index (ICPI test in 1-day-old SPF chicks demonstrated that APMV types 2-9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2-9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2-9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1 and exhibited restricted viral replication of the APMVs (including APMV-1 to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1-9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.

  4. Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test

    DEFF Research Database (Denmark)

    Dubreuil, J.D.; Letellier, A.; Stenbæk, Eva;

    1996-01-01

    A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS......-PAGE and Western blotting, A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A, pleuropneumoniae, were tested by mixing 25 mu L of polystyrene reagent with the same volume of a dense...... suspension of bacterial cells grown for 18 h. All A, pleuropneumoniae strains had been previously serotyped using standard procedures, The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found...

  5. Serotypes in Saccharomyces telluris: Their relation to source of isolation

    Science.gov (United States)

    Hasenclever, H.F.; Kocan, R.M.

    1973-01-01

    Three serotypes have been characterized with three reference strains of Saccharomyces telluris and designated as A, B, and C. One reference strain of Torpulopsis bovina, the imperfect form of S. telluris, belonged to serotype B. Strains of S. telluris isolated from four columbid species were serotyped. All 98 strains of this yeast isolated from Columba livia belonged to serotype B. Three other columbid species, C. leucocephala, C. fasciata, and Zenaidura macroura harbored strains of serotype C only. Serotype A was not isolated from any of the avian species.

  6. The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers.

    Science.gov (United States)

    Shigekane, H; Kawaguchi, Y; Shirakata, M; Sakaguchi, M; Hirai, K

    1999-01-01

    In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

  7. Vesicular stomatitis virus (indiana 2 serotype as experimental model to study acute encephalitis – morphological features Vírus da estomatite vesicular (sorotipo indiana 2 como modelo experimental para o estudo de encefalite aguda – aspectos morfológicos

    Directory of Open Access Journals (Sweden)

    Florêncio Figueiredo Cavalcanti Neto

    2003-10-01

    Full Text Available The Vesicular Stomatitis Virus (VSV is a Vesiculovirus of the Rhabdoviridae family that infects mammals and causes vesicular lesions similar to those of foot-and-mouth disease. VSV experimental encephalitis can be induced in rodents and the symptoms are similar to those observed in rabies. However, the lesions observed in the animals´ encephalon are different. Inclusion bodies are not observed. There is necrosis, particularly in the region of the olfactory bulb, and, in some cases, ventriculitis. It was observed that the time pattern of VSV dissemination and the morphological aspects of the lesions are similar to those described in literature. The virus seems to be disseminated through the brain ventricles, being multiplied in the ependyma cells and in the neurons, besides using retrograde and anterograde transport. It was noticed that, due to the facility of virus manipulation, this experimental model has been used in innumerable research studies in several fields. If, on the one hand there are plenty of reports on the infection pathogenesis, on the other hand there are many gaps involving, for instance, aspects about virus transmission, recovery of infected animals and participation of glial cells in the acute as well as in the recovery phases.   O vírus da estomatite vesicular (VEV é um Vesiculovírus da família Rhabdoviridae que infecta mamíferos e causa lesões vesiculares semelhantes às observadas na febre aftosa. A encefalite experimental pode ser induzida em roedores e os sintomas são semelhantes aos observados na raiva; entretanto, as lesões observadas no encéfalo dos animais são diferentes. Corpúsculos de inclusão não são observados, há necrose especialmente da região do bulbo olfatório e em alguns casos, ventriculite. Observamos que o padrão temporal de disseminação do VEV e os aspectos morfológicos das lesões são similares aos descritos na literatura. O vírus parece se disseminar através dos ventr

  8. Experimental avian paramyxovirus serotype-3 infection in chickens and turkeys.

    Science.gov (United States)

    Kumar, Sachin; Militino Dias, Flavia; Nayak, Baibaswata; Collins, Peter L; Samal, Siba K

    2010-01-01

    Avian paramyxoviruses (APMV) are divided into nine serotypes. Newcastle disease virus (APMV-1) is the most extensively characterized, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two divergent strains of APMV-3, Netherlands and Wisconsin, in (i) 9-day-old embryonated chicken eggs, (ii) 1-day-old specific pathogen free (SPF) chicks and turkeys, and (iii) 2-week-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was 112 h for APMV-3 strain Netherlands and > 168 h for strain Wisconsin. The intracerebral pathogenicity index in 1-day-old chicks for strain Netherlands was 0.39 and for strain Wisconsin was zero. Thus, both strains are lentogenic. Both the strains replicated well in brain tissue when inoculated intracerebrally in 1-day-old SPF chicks, but without causing death. Mild respiratory disease signs were observed in 1-day-old chickens and turkeys when inoculated through oculonasal route with either strain. There were no overt signs of illness in 2-weeks-old chickens and turkeys by either strain, although all the birds seroconverted after infection. The viruses were isolated predominantly from brain, lungs, spleens, trachea, pancreas and kidney. Immunohistochemistry studies also showed the presence of large amount of viral antigens in both epithelial and sub-epithelial lining of respiratory and alimentary tracts. Our result suggests systemic spread of APMV-3 even though the viral fusion glycoprotein does not contain the canonical furin proteases cleavage site. Furthermore, there was little or no disease despite systemic viral spread and abundant viral replication in all the tissues tested.

  9. Viruses isolated from Panamanian sloths.

    Science.gov (United States)

    Seymour, C; Peralta, P H; Montgomery, G G

    1983-11-01

    Seven virus strains were isolated in Vero cells from whole blood samples from 80 wild-caught sloths, Bradypus variegatus and Choloepus hoffmanni, from Central Panamá. Four strains of at least two different serotypes are related to Changuinola virus; two of these were associated with prolonged or recrudescent viremias. One strain is an antigenic subtype of Punta Toro virus, and another, described here as Bradypus-4 virus, is a new, antigenically ungrouped virus. A second new virus from sloths, Utive virus, forms an antigenic complex within the Simbu serogroup with Utinga and Pintupo viruses. Tests on sequential plasma samples from radio-marked free-ranging sloths and from recently captured animals maintained in captivity showed that both species develop neutralizing antibodies following naturally acquired virus infections. Antibodies against the Changuinola and Simbu serogroup viruses are widespread in both sloth species and are especially prevalent in Choloepus, but are virtually absent in all other wild vertebrate species tested.

  10. Dengue serotypes 1–4 exhibit unique host specificity in vitro

    Directory of Open Access Journals (Sweden)

    Barr KL

    2012-10-01

    Full Text Available Kelli L Barr, Benjamin D Anderson, Gary L Heil, John A Friary, Gregory C Gray, Dana A FocksDepartment of Environmental and Global Health, College of Public Health and Health Professions and the Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USABackground: Over 3000 cell lines from over 150 species are commercially available today from the American Type Culture Collection. These cell lines offer alternative approaches to investigating the interactions between arboviruses and other vertebrates at the cellular level. The various cell origins, types, and morphologies can be valuable resources for studying viral ecology and examining hypotheses regarding viral reservoirs. Dengue viruses (DENV are major re-emerging pathogens that have been studied classically in only a few cell lines.Methods: We evaluated the susceptibility of 19 distinct mammalian, avian, and reptilian cell lines to DENV infection. Cell lines were infected with DENV serotypes 1–4 and evaluated for susceptibility via focus-forming unit assays and quantitative reverse-transcription polymerase chain reaction.Results: Both methods demonstrated the ability of DENV to replicate in 14 cell lines derived from various vertebrates with viral titers ranging from 1 × 103 to 1 × 107 infectious units per milliliter. Cell line susceptibility to DENV infection was serotype specific, with DENV-1 and DENV-4 infecting more cell lines than either DENV-2 or DENV-3. Cellular type also seemed to affect the infectivity of DENV. Human endothelial cells were only susceptible to DENV-4. Of six fibroblast lines, 100% were susceptible to at least one DENV serotype whereas only 62% of 13 epithelial lines were susceptible to DENV serotypes 1–4.Conclusion: These data indicate that a variety of cell lines from human and animal species can be used to culture DENV. The serotype-specific susceptibility for certain cell lines may provide a tool to help characterize specific DENV

  11. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes.

    Science.gov (United States)

    Kang, W; Wang, L; Harrell, H; Liu, J; Thomas, D L; Mayfield, T L; Scotti, M M; Ye, G J; Veres, G; Knop, D R

    2009-02-01

    Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

  12. Field Evaluation of a Deployable RT-PCR Assay System for Real-Time Identification of Dengue Virus

    Science.gov (United States)

    2004-06-01

    strains of dengue serotypes 1-4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses as well as dengue virus infected...JA, Pyke A, Smith GA. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes . J Med Virol. 2002 April; 66(4

  13. Luminex(®) multiplex bead suspension arrays for the detection and serotyping of Salmonella spp.

    Science.gov (United States)

    Dunbar, Sherry A; Ritchie, Vivette Brown; Hoffmeyer, Michaela R; Rana, Gunjot S; Zhang, Hongwei

    2015-01-01

    In this chapter we describe two commercially available bead-based molecular assays for detection, identification and serotyping of Salmonella. The xTAG(®) Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplex test for the simultaneous detection of nucleic acids from Salmonella plus 14 other gastroenteritis-causing bacteria, viruses, and parasites from stool specimens. xTAG GPP uses the Luminex(®) xTAG universal array technology for the identification of specific target sequences combined with the xMAP(®) bead multiplexing platform for detection of the targets that were present in the starting sample. The xMAP Salmonella Serotyping Assay (SSA) is a multiplex nucleic acid-based direct hybridization assay for molecular identification of the serotype of Salmonella isolates. In xMAP SSA, target sequences amplified from cultured Salmonella isolates are captured by hybridization to sequence-specific capture probes which have been coupled to the multiplexed bead sets. Herein we provide detailed protocols for each of these assays and present data which describe their performance characteristics for detection and serotyping Salmonella.

  14. Serotype distribution in non-bacteremic pneumococcal pneumonia

    DEFF Research Database (Denmark)

    Benfield, Thomas Lars Vibe; Skovgaard, Marlene; Schønheyder, Henrik Carl

    2013-01-01

    There is limited knowledge of serotypes that cause non-bacteremic pneumoco