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...

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

Science.gov (United States)

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

2018-03-05

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

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

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

1998-09-01

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

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

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

2015-01-01

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

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

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

2008-06-01

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

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

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Nash, Kevin R; Gordon, Marcia N

2016-01-01

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

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

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

1994-09-01

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

Factors influencing recombinant adeno-associated virus production.

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

1998-03-20

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

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

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

2003-10-01

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

PREVALENCE OF BOVINE HERPESVIRUS-1,PARAINFLUENZA-3,BOVINE ROTAVIRUS, BOVINE VIRAL DIARRHEA, BOVINE ADENOVIRUS-7,BOVINE LEUKEMIA VIRUS AND BLUETONGUE VIRUS ANTIBODIES IN CATTLE IN MEXICO

OpenAIRE

SUZAN, Victor M.; ONUMA, Misao; AGUILAR, Romero E.; MURAKAMI, Yosuke

1983-01-01

Sera were collected from dairy and beef cattle in 19 different states of Mexico. These sera were tested for bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PIV-3), bovine rotavirus (BRV), bovine leukemia virus (BLV), bovine adenovirus-7 (BAV-7), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). Seropositive rates for each virus for dairy cattle tested were 158/277(57.0%) for BHV-1,217/286(75.0%) for PIV-3,541/1498(36.1%) for BLV, 134/144(93.1%) for BRV, 39/90(43.3%) for BTV,...

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

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

2012-01-01

Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs) whose transgene expression is activated by Cre (“Cre-On”). Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (“Cre-Off”) and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery. PMID:22866029

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

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Yarong Liu

2014-01-01

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

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

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

1997-06-24

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

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

International Nuclear Information System (INIS)

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

1979-01-01

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

Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

DEFF Research Database (Denmark)

Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari

2010-01-01

delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

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

OpenAIRE

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

2008-01-01

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

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.

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

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

1998-12-01

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

Bovine herpes virus infections in cattle.

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Nandi, S; Kumar, Manoj; Manohar, M; Chauhan, R S

2009-06-01

Bovine herpes virus 1 (BHV-1) is primarily associated with clinical syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis, abortion, infertility, conjunctivitis and encephalitis in bovine species. The main sources of infection are the nasal exudates and the respiratory droplets, genital secretions, semen, fetal fluids and tissues. The BHV-1 virus can become latent following a primary infection with a field isolate or vaccination with an attenuated strain. The viral genomic DNA has been demonstrated in the sensory ganglia of the trigeminal nerve in infectious bovine rhinotracheitis (IBR) and in sacral spinal ganglia in pustular vulvovaginitis and balanoposthitis cases. BHV-1 infections can be diagnosed by detection of virus or virus components and antibody by serological tests or by detection of genomic DNA by polymerase chain reaction (PCR), nucleic acid hybridization and sequencing. Inactivated vaccines and modified live virus vaccines are used for prevention of BHV-1 infections in cattle; subunit vaccines and marker vaccines are under investigation.

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

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Nathalie Alazard-Dany

2009-03-01

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

Monitoring of the antiviral potential of bee venom and wax extracts against Adeno-7 (DNA) and Rift Valley fever virus (RNA) viruses models.

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Hassan, Mostafa I; Mohamed, Aly F; Amer, Moner A; Hammad, Kotb M; Riad, Saber A

2015-04-01

This study monitored the antiviral potential of bee venom and four wax extracts, ethanol white and black beeswax (EWW/EBW) and acetone white and black beeswax (AWW/ABW) extracts. Two different virus models namely Adeno-7 as DNA model and RVFV as RNA virus models. End point calculation assay was used to calculate virus depletion titer. The depletion of viral infectivity titer of ABW to Adeno-7 virus showed strong antiviral activity recorded a depletion of viral infectivity titer (1.66 log (10)/ ml) that gave equal action with bee venom and more than interferon IFN (1 log (10)/ ml). On the other hand, antiviral activity of EBW showed a moderate potential, while AWW showed no antiviral activity. Finally EWW showed synergetic activity against Adeno-7 virus activity. Thus, activity of wax extracts to RVFV was arranged in order of IFN bee venom > AWW & EBW > EWW and ABW recorded 3.34, 0.65, 0.5, 0.34 respectively. It is the first time to study the beeswax effect against DNA and RNA virus' models; acetone black beeswax recorded a depletion titer 1.66 log (10)/ml.

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

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Nance, Michael E; Duan, Dongsheng

2015-12-01

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

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

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

2018-02-01

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

Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

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Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

2010-05-14

The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. Copyright 2010 Elsevier Ltd. All rights reserved.

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

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

2011-05-01

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

Adeno-associated virus rep protein synthesis during productive infection

International Nuclear Information System (INIS)

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

1989-01-01

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

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

Directory of Open Access Journals (Sweden)

Lian-Fang Du

2013-05-01

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

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

Science.gov (United States)

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

2003-06-01

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

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

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

2018-01-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

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

2017-06-13

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

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

Science.gov (United States)

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

2013-01-01

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

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

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Margriet A van Gestel

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

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

Science.gov (United States)

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

2017-06-01

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

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

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Tae Kwann Park

2017-09-01

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

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

NARCIS (Netherlands)

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

2002-01-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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

Science.gov (United States)

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

2013-04-01

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

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

Science.gov (United States)

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

2017-08-01

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

Duplex PCR assay for the detection of avian adeno virus and chicken anemia virus prevalent in Pakistan

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Iqbal Aqib

2011-09-01

Full Text Available Abstract Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.

Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in draught animals in Cambodia.

Science.gov (United States)

Meas, S; Ohashi, K; Tum, S; Chhin, M; Te, K; Miura, K; Sugimoto, C; Onuma, M

2000-07-01

Since bovine immunodeficiency virus (BIV), known as bovine lentivirus, has been detected in dairy and beef cattle in various countries around the world, a prevalence study of antibodies to BIV and bovine leukemia virus (BLV) was conducted in draught animals in five provinces in Cambodia, where protozoan parasite infections were suspected in some animals. To clarify the status of draught animals including Haryana, Brahman, mixed-breed, local breed cattle and muscle water buffaloes, a total of 544 cattle and 42 buffaloes were tested, and 26.3 and 16.7%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting. There were 5.3% positive for anti-BLV antibodies detected by immunodiffusion test among the cattle, but no reactors among buffaloes and no dual infection for both BIV and BLV was determined in this study. Peripheral blood mononuclear cells from BIV-seropositive cattle were found to have BIV-provirus DNA, as detected by polymerase chain reaction and subsequent Southern blot hybridization. This is the first evidence for the presence of BIV and BLV infections in draught animals in tropical countries such as Cambodia. This wide distribution of BIV suggests its association with problems in animal health as reported worldwide, and that a primary BIV infection can predispose death of affected animals by other aggressive pathogens or stresses.

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

Directory of Open Access Journals (Sweden)

Zheng Liu

2012-04-01

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

9 CFR 113.216 - Bovine Rhinotracheitis Vaccine, Killed Virus.

Science.gov (United States)

2010-01-01

... Virus. 113.216 Section 113.216 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.216 Bovine Rhinotracheitis Vaccine, Killed Virus. Infectious Bovine...

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

Science.gov (United States)

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

1995-05-05

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

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

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Qiang Wang

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

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

Science.gov (United States)

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

2018-03-01

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

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

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Charles N de Leeuw

2014-01-01

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

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

Science.gov (United States)

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

2006-07-01

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

associated virus (AAV)-mediated expression of small interfering RNA

African Journals Online (AJOL)

user

2011-04-11

Apr 11, 2011 ... disadvantages. In this study, a siRNA expression recombinant adeno-associated virus (AAV) was .... cleotides were designed, which contained a sense strand of p53 or ..... During MJ, Kaplitt MG, Stem MB, Eidelberg D (2001).

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-09-17

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

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...

Human and bovine viruses and bacteria at three Great Lakes beaches: Environmental variable associations and health risk

Science.gov (United States)

Corsi, Steven R.; Borchardt, Mark A.; Carvin, Rebecca B.; Burch, Tucker R; Spencer, Susan K.; Lutz, Michelle A.; McDermott, Colleen M.; Busse, Kimberly M.; Kleinheinz, Gregory; Feng, Xiaoping; Zhu, Jun

2016-01-01

Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65–87% for pathogenic bacteria, and 13–35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 3 × 10–5, 7 × 10–9, and 3 × 10–7 for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

Science.gov (United States)

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

2005-01-01

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

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

Science.gov (United States)

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

1999-07-01

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

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...

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bovine Virus Diarrhea Vaccine, Killed Virus. 113.215 Section 113.215 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD...

Seroepidemiological study of parainfluenza 3 virus in bovines with reproductive failure, from monteria-colombia

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César Betancur Hurtado

2010-12-01

Full Text Available The virus of the bovine Para influenza 3 is known to be a part of the bovine respiratory complex, along with another infectious agent as the bovine sincitialrespiratory virus, which has not as yet been diagnosed at the geographical area of this study. This work was carried out at Monteria, Colombia, in bovines from 28 farms, with the aim of finding the serological prevalence of the PI-3 virus. Blood samples were collected from 137 females, with a history of reproductive failure, and from 26 bulls from the same farms. The serological test used was the ELISA test. A descriptive analysis was carried out, recording data from positives and from negatives sera. A Chi-square test was used to test for association between the variables: sex, age, reproductive condition and type of production system, with serological reactivity to the PI-3virus. Concerning the results of the study, the point prevalence for the PI-3 virus found was 13, 5%, and under statistical bases, statistical significance was found between age groups and association was not found for the others variables taken in account for the study. According to the results, it was concluded that the PI-3 virus is present in bovines of Monteria, and that a part of the reproductive failure in females of the region, mostly the return to estrus and abortions, is due to the effect of that pathological entity. Finally, the authors recommend more extensive studies on PI-3 Infection, at the different cattle raising areas of Colombia, a country of 24 million heads.

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

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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.

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

Science.gov (United States)

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

1999-06-01

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

Genome-wide association study for host response to bovine leukemia virus in Holstein cows.

Science.gov (United States)

Brym, P; Bojarojć-Nosowicz, B; Oleński, K; Hering, D M; Ruść, A; Kaczmarczyk, E; Kamiński, S

2016-07-01

The mechanisms of leukemogenesis induced by bovine leukemia virus (BLV) and the processes underlying the phenomenon of differential host response to BLV infection still remain poorly understood. The aim of the study was to screen the entire cattle genome to identify markers and candidate genes that might be involved in host response to bovine leukemia virus infection. A genome-wide association study was performed using Holstein cows naturally infected by BLV. A data set included 43 cows (BLV positive) and 30 cows (BLV negative) genotyped for 54,609 SNP markers (Illumina Bovine SNP50 BeadChip). The BLV status of cows was determined by serum ELISA, nested-PCR and hematological counts. Linear Regression Analysis with a False Discovery Rate and kinship matrix (computed on the autosomal SNPs) was calculated to find out which SNP markers significantly differentiate BLV-positive and BLV-negative cows. Nine markers reached genome-wide significance. The most significant SNPs were located on chromosomes 23 (rs41583098), 3 (rs109405425, rs110785500) and 8 (rs43564499) in close vicinity of a patatin-like phospholipase domain containing 1 (PNPLA1); adaptor-related protein complex 4, beta 1 subunit (AP4B1); tripartite motif-containing 45 (TRIM45) and cell division cycle associated 2 (CDCA2) genes, respectively. Furthermore, a list of 41 candidate genes was composed based on their proximity to significant markers (within a distance of ca. 1 Mb) and functional involvement in processes potentially underlying BLV-induced pathogenesis. In conclusion, it was demonstrated that host response to BLV infection involves nine sub-regions of the cattle genome (represented by 9 SNP markers), containing many genes which, based on the literature, could be involved to enzootic bovine leukemia progression. New group of promising candidate genes associated with the host response to BLV infection were identified and could therefore be a target for future studies. The functions of candidate genes

HoBi-like viruses – the typical 'atypical bovine pestivirus'

Science.gov (United States)

HoBi-like viruses, also referred to as bovine viral diarrhea virus 3 (BVDV-3) and atypical pestivirus, have been proposed as a new putative bovine pestivirus species. These viruses were first identified in the last decade and are currently distributed in at least three continents. Published findings...

Characterization of thymus-associated lymphoid depletion in bovine calves acutely or persistently infected with bovine viral diarrhea virus 1, bovine viral diarrhea 2 or HoBi-like pestivirus

Science.gov (United States)

Viruses from recognized pestivirus species bovine viral diarrhea 1 (BVDV-1) and BVDV-2 and the proposed pestivirus species HoBi-like virus infect primarily cattle. Exposure of cattle to these viruses can lead to either acute or persistent infections depending on the timing and status of the animal ...

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

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Tijana Knezevic, PhD

2016-12-01

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

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

Science.gov (United States)

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

1996-07-15

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

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.

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

Science.gov (United States)

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

2008-03-01

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

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

Science.gov (United States)

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

2017-10-15

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

Expression of infectious bovine rhinotracheitis virus glycoprotein D ...

African Journals Online (AJOL)

USER

2010-06-14

Jun 14, 2010 ... Bovine Herpesvirus 1 (BHV-1) belongs to the genus of ... through vaccination with recombinant vaccines of thymidine kinase, manufacturing and applying ..... Resistance to bovine respiratory syncytial virus (BRSV) induced in.

Interferon induction in bovine and feline monolayer cultures by four bluetongue virus serotypes.

OpenAIRE

Fulton, R W; Pearson, N J

1982-01-01

The interferon inducing ability of bluetongue viruses was studied in bovine and feline monolayer cultures inoculated with each of four bluetongue virus serotypes. Interferon was assayed by a plaque reduction method in monolayer cultures with vesicular stomatitis virus as challenge virus. Interferon was produced by bovine turbinate, Georgia bovine kidney, and Crandell feline kidney monolayer cultures in response to bluetongue virus serotypes 10, 11, 13 and 17. The antiviral substances produced...

Quantitative trait loci associated with the immune response to a bovine respiratory syncytial virus vaccine.

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Richard J Leach

Full Text Available Infectious disease is an important problem for animal breeders, farmers and governments worldwide. One approach to reducing disease is to breed for resistance. This linkage study used a Charolais-Holstein F2 cattle cross population (n = 501 which was genotyped for 165 microsatellite markers (covering all autosomes to search for associations with phenotypes for Bovine Respiratory Syncytial Virus (BRSV specific total-IgG, IgG1 and IgG2 concentrations at several time-points pre- and post-BRSV vaccination. Regions of the bovine genome which influenced the immune response induced by BRSV vaccination were identified, as well as regions associated with the clearance of maternally derived BRSV specific antibodies. Significant positive correlations were detected within traits across time, with negative correlations between the pre- and post-vaccination time points. The whole genome scan identified 27 Quantitative Trait Loci (QTL on 13 autosomes. Many QTL were associated with the Thymus Helper 1 linked IgG2 response, especially at week 2 following vaccination. However the most significant QTL, which reached 5% genome-wide significance, was on BTA 17 for IgG1, also 2 weeks following vaccination. All animals had declining maternally derived BRSV specific antibodies prior to vaccination and the levels of BRSV specific antibody prior to vaccination were found to be under polygenic control with several QTL detected.Heifers from the same population (n = 195 were subsequently immunised with a 40-mer Foot-and-Mouth Disease Virus peptide (FMDV in a previous publication. Several of these QTL associated with the FMDV traits had overlapping peak positions with QTL in the current study, including the QTL on BTA23 which included the bovine Major Histocompatibility Complex (BoLA, and QTL on BTA9 and BTA24, suggesting that the genes underlying these QTL may control responses to multiple antigens. These results lay the groundwork for future investigations to identify the

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

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Akikazu Ishihara

2012-01-01

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

Bovine Virus Diarrhea (BVD)

OpenAIRE

Hoar, Bruce R.

2004-01-01

Bovine virus diarrhea (BVD) is a complicated disease to discuss as it can result in a wide variety of disease problems from very mild to very severe. BVD can be one of the most devastating diseases cattle encounter and one of the hardest to get rid of when it attacks a herd. The viruses that cause BVD have been grouped into two genotypes, Type I and Type II. The disease syndrome caused by the two genotypes is basically the same, however disease caused by Type II infection is often more severe...

Bovine Lactoferrin Inhibits Toscana Virus Infection by Binding to Heparan Sulphate

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Agostina Pietrantoni

2015-01-01

Full Text Available Toscana virus is an emerging sandfly-borne bunyavirus in Mediterranean Europe responsible for neurological diseases in humans. It accounts for about 80% of paediatric meningitis cases during the summer. Despite the important impact of Toscana virus infection-associated disease on human health, currently approved vaccines or effective antiviral treatments are not available. In this research, we have analyzed the effect of bovine lactoferrin, a bi-globular iron-binding glycoprotein with potent antimicrobial and immunomodulatory activities, on Toscana virus infection in vitro. Our results showed that lactoferrin was capable of inhibiting Toscana virus replication in a dose-dependent manner. Results obtained when lactoferrin was added to the cells during different phases of viral infection showed that lactoferrin was able to prevent viral replication when added during the viral adsorption step or during the entire cycle of virus infection, demonstrating that its action takes place in an early phase of viral infection. In particular, our results demonstrated that the anti-Toscana virus action of lactoferrin took place on virus attachment to the cell membrane, mainly through a competition for common glycosaminoglycan receptors. These findings provide further insights on the antiviral activity of bovine lactoferrin.

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

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Myrmel, M; Lange, H; Rimstad, E

2015-09-01

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

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

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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.

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

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

2015-10-15

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

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

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

2009-11-01

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

Circulating microRNAs in serum from cattle challenged with Bovine Viral Diarrhea Virus

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Bovine viral diarrhea virus (BVDV) is an RNA virus that is often associated with respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. The objective of this study was to identify microRNAs in cattle that had been challenged with a non-cytopat...

Seroprevalence and factors associated with bovine viral diarrhea virus (BVDV) infection in dairy cattle in three milksheds in Ethiopia.

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Aragaw, Kassaye; Sibhat, Berhanu; Ayelet, Gelagay; Skjerve, Eystein; Gebremedhin, Endrias Z; Asmare, Kassahun

2018-05-31

This work was conducted to estimate the seroprevalence, to identify potential factors that influence seroprevalence of bovine viral diarrhea virus (BVDV), and to investigate the association between BVDV serostatus and occurrence of reproductive disorders in dairy cattle in three milksheds in Ethiopia. A total of 1379 serum samples were obtained from cattle randomly selected from 149 herds from three milksheds representing central, southern, and western Ethiopia. Sera samples were examined for bovine viral diarrhea virus (BVDV) antibodies using commercial competitive enzyme-linked immunosorbent assay (ELISA) kit. Logistic regression analysis was employed to investigate associations between risk factors and the risk of BVDV seroprevalence, and BVDV serostatus and reproductive disorders. Seroreaction to BVDV antigens was detected in 32.6% of the 1379 cattle and 69.8% of the 149 herds sampled. Factors associated with BVDV seroplevalence were age, breed, and herd size (P  0.05). Risk of reproductive disorders was not affected by BVDV serostatus, except for repeat breeding (P > 0.05). The present study demonstrated that BVDV has wide distribution in the country being detected in all the 15 conurbations and 69.8% of herds involved in the study.

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

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Hung-Bo Hsiao

2014-01-01

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

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

International Nuclear Information System (INIS)

Schmitt, J.; Schlehofer, J.R.; Mergener, K.; Gissmann, L.; zur Hausen, H.

1989-01-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur

Evaluation of Fast Technology Analysis (FTA) Cards as an improved method for specimen collection and shipment targeting viruses associated with Bovine Respiratory Disease Complex.

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Liang, Xiao; Chigerwe, Munashe; Hietala, Sharon K; Crossley, Beate M

2014-06-01

In order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acid-based diagnosis, a study was undertaken to verify realtime PCR efficiency of specimens collected and stabilized on FTA Cards™, filter paper which is treated chemically. Nucleic acids collected using FTA Cards without the need for a cold-chain or special liquid media handling provided realtime PCR results consistent (96.8% agreement, kappa 0.923 [95% CI=0.89-0.96]) with the same specimens collected using traditional viral transport media and shipped on ice using the U.S. Department of Transportation mandated liquid handling requirements. Nucleic acid stabilization on FTA Cards was evaluated over a temperature range (-27 °C to +46 °C) for up to 14 days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. No significant difference (P≥0.05) was observed in realtime PCR cycle threshold values over the temperature range and time storage conditions for Bovine Viral Diarrhea virus, Bovine Respiratory Syncytial virus, Bovine Coronavirus, and Bovine Herpesvirus I. The four viruses evaluated in the study are associated with Bovine Respiratory Disease Complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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

2012-01-01

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

Perinatal systemic gene delivery using adeno-associated viral vectors

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Rajvinder eKarda

2014-11-01

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

Inactivation of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of bovine-derived grafting materials.

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Lee, Kwang-Il; Lee, Jung-Soo; Jung, Hong-Hee; Lee, Hwa-Yong; Moon, Seong-Hwan; Kang, Kyoung-Tak; Shim, Young-Bock; Jang, Ju-Woong

2012-01-01

Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

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Lung, O; Furukawa-Stoffer, T; Burton Hughes, K; Pasick, J; King, D P; Hodko, D

2017-12-01

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site. © 2016 Her Majesty the Queen in Right of Canada.

Human and bovine viruses in the Milwaukee River Watershed: hydrologically relevant representation and relations with environmental variables

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Corsi, Steven R.; Borchardt, M. A.; Spencer, S. K.; Hughes, Peter E.; Baldwin, Austin K.

2014-01-01

To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

[Antiviral activity of different drugs in vitro against viruses of bovine infectious rhinotracheitis and bovine diarrhea].

Science.gov (United States)

Glotov, A G; Glotova, T I; Sergeev, A A; Belkina, T V; Sergeev, A N

2004-01-01

In vitro experiments studied the antiviral activity of 11 different drugs against viruses of bovine infective rhinotracheitis (BIRT) and bovine viral diarrhea (BVD). The 50% inhibiting concentrations of the test agents were determined in the monolayers of MDBK and KCT cell cultures. Only did phosprenyl show a virucidal activity against BIRT virus. All the tested drugs significantly inhibited the reproduction of BIRT virus in the sensitive MDBK cell cultures. Thus, bromuridin, acyclovir, ribavirin and methisazonum inhibited the virus by > or = 100,000 times; liposomal ribavirin, gossypolum, anandinum, polyprenolum, phosprenyl, by 1000-10,000 times; eracond and argovit, by 100 times. In experiments on BVD virus, the cultured KCT cells displayed the antiviral activity of bromuridin, phosprenil, polyprenolum, methisazonum, acyclovir, gossypolum, argovit, and ribavirin (in two variants), which caused a statistically significant (100-10,000-fold) decrease in the productive activity of this virus. Eracond and anandid proved to be ineffective.

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

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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.

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

International Nuclear Information System (INIS)

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

2003-01-01

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

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

Science.gov (United States)

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

2010-01-01

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

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

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Rajiv R Mohan

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

Human and bovine viruses in the Milwaukee River watershed: Hydrologically relevant representation and relations with environmental variables

Energy Technology Data Exchange (ETDEWEB)

Corsi, S.R., E-mail: srcorsi@usgs.gov [U.S. Geological Survey, Wisconsin Water Science Center, Middleton, WI 53562 (United States); Borchardt, M.A.; Spencer, S.K. [U.S. Department of Agriculture, Agricultural Research Service, 2615 Yellowstone Dr., Marshfield, WI 54449 (United States); Hughes, P.E.; Baldwin, A.K. [U.S. Geological Survey, Wisconsin Water Science Center, Middleton, WI 53562 (United States)

2014-08-15

To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

Human and bovine viruses in the Milwaukee River watershed: Hydrologically relevant representation and relations with environmental variables

International Nuclear Information System (INIS)

Corsi, S.R.; Borchardt, M.A.; Spencer, S.K.; Hughes, P.E.; Baldwin, A.K.

2014-01-01

To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

Radioimmunoassay of bovine leukosis virus antibodies

Energy Technology Data Exchange (ETDEWEB)

Franz, J; Hampl, J; Svoboda, I; Granatova, M; Hofirek, B; Skrobak, F

1986-08-01

A RIA method was developed for identifying the presence of serum antibodies to the bovine leukosis virus. The chosen procedure uses the ability of the virus antigen to bind to the solid phase of a polystyrene carrier. The method was compared with the ELISA method and with the pseudoneutralization and immunodiffusion tests. A high level of agreement was achieved between the RIA and the ELISA methods (95%). By its accuracy the RIA method proves superior to the immunodiffusion test.

Radioimmunoassay of bovine leukosis virus antibodies

International Nuclear Information System (INIS)

Franz, J.; Hampl, J.; Svoboda, I.; Granatova, M.; Hofirek, B.; Skrobak, F.

1986-01-01

A RIA method was developed for identifying the presence of serum antibodies to the bovine leukosis virus. The chosen procedure uses the ability of the virus antigen to bind to the solid phase of a polystyrene carrier. The method was compared with the ELISA method and with the pseudoneutralization and immunodiffusion tests. A high level of agreement was achieved between the RIA and the ELISA methods (95%). By its accuracy the RIA method proves superior to the immunodiffusion test. (author)

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.

Estimating transfer of bovine virus-diarrhoea virus in Danish cattle by use of register data

DEFF Research Database (Denmark)

Alban, L.; Stryhn, H.; Kjeldsen, A.M.

2001-01-01

To study how routinely recorded data (also called "register data") might be used in disease monitoring on a regional or national level, a database for bovine virus-diarrhoea virus (BVDV) was made from existing databases, covering the period January 1995-November 1999. This paper includes a general...... in the same herd during gestation. Among the non-MRS herds, most new infections were associated with movement of a persistently infected animal. The monthly number of newly infected herds is presented; it is seen that the incidence declined substantially during the study period. (C) 2001 Elsevier Science B...

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

Science.gov (United States)

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

2011-06-01

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

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

Science.gov (United States)

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

2002-02-05

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

Bovine respiratory syncytial virus (BRSV): A review

DEFF Research Database (Denmark)

Larsen, Lars Erik

2000-01-01

Bovine respiratory syncytial virus (BRSV) infection is the major cause of respiratory disease in calves during the first year of life. The study of the virus has been difficult because of its lability and very poor growth in cell culture. However, during the last decade, the introduction of new...... complex and unpredictable which makes the diagnosis and subsequent therapy very difficult. BRSV is closely related to human respiratory syncytial virus (HRSV) which is an important cause of respiratory disease in young children. In contrast to BRSV, the recent knowledge of HRSV is regularly extensively...

Antibody Tracing, Seroepidemiology and Risk Factors of Bovine Respiratory Syncytial Virus and Bovine Adenovirus-3 in Dairy Holstein Farms

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Mahsa FARZINPOUR

2016-01-01

Full Text Available Antibody tracing, risk factors and seroepidemiology of bovine respiratory syncytial virus and bovine adenovirus-3 were investigated in 22 Industrial and Semi-Industrial dairy Holstein farms. Serum samples (n=736 from various ages of unvaccinated cows were collected from May to September 2012. Risk factors including age, past history of respiratory diseases, amount of milk production, husbandry type and herd size were considered. Data were analyzed by Chi-square and logistic regression. Results indicated that the infection with some of individual viruses was related to past history of respiratory disease and herd size. No specific pattern was seen on the effect of level of milk production on seropositivity of animals. The seroprevalence for BRSV and BAV-3 were 89.1% and 88%, respectively. The present study indicates that infections of bovine respiratory viruses frequently occur in cattle of Fars province and the main viral cause of primary occurrence of respiratory diseases may be due to aforementioned viruses.

Evidence for the replication of bovine leukemia virus in the B lymphocytes

International Nuclear Information System (INIS)

Paul, P.S.; Pomeroy, K.A.; Johnson, D.W.; Muscoplat, C.C.; Handwerger, B.S.; Soper, F.F.; Sorensen, D.K.

1977-01-01

Bovine peripheral blood lymphocytes from a cow with persistent lymphocytosis were separated on nylon wool columns into nylon-adherent and nonadherent populations. Nylon-adherent cells were highly enriched for surface immunoglobulin (SIg) bearing B lymphocytes (95.5%) and nonadherent cells for SIg negative non-B cells, presumably T lymphocytes (96.3%). The B lymphocytes were found to be the major producers for bovine leukemia virus. A total of 39% of the B-enriched cells, surviving after 72 hours in culture, produced bovine leukemia virus as compared with 0.5% of the non-B cells

Bioinformatics and molecular analysis of the evolutionary relationship between bovine rhinitis A viruses and foot-and-mouth disease virus

Science.gov (United States)

Bovine rhinitis viruses (BRV) cause mild respiratory disease of cattle. In this study, a near full length genome sequence of a virus named RS3X, formerly classified as bovine rhinovirus type 1, isolated from infected cattle from the United Kingdom in the 1960s, was obtained and analyzed. Phylogeneti...

Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

Science.gov (United States)

Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2016-07-15

The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of infectious bovine rhinotracheitis virus glycoprotein D ...

African Journals Online (AJOL)

Bovine Herpesvirus 1 (BHV-1) belongs to the genus of Varicellovirus and the family of Herpesviridae which contains three main gB, gC and gD genes. In order to cloning of the coding region of gD gene of IBR virus , PCR product of the open reading frame of the gene from IBR virus isolated in Iran was amplified by PCR.

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

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Justin Judd

2012-01-01

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

Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs.

OpenAIRE

Wechsler, S J; Luedke, A J

1991-01-01

Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

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

Science.gov (United States)

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

2009-12-28

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

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

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Chang Jin

2009-11-01

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

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Overcoming tumor resistance by heterologous adeno-poxvirus combination therapy

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Markus Vähä-Koskela

2014-01-01

Full Text Available Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.

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

Science.gov (United States)

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

1997-01-01

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

Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2

Science.gov (United States)

The objective of this study was to compare reproductive protection in cattle against the impacts of bovine viral diarrhea virus (BVDV) provided by three different multivalent vaccines containing inactivated BVDV. Beef heifers and cows (n=122), seronegative and virus negative for BVDV, were randomly ...

PREVALENCE OF BOVINE HERPES VIRUS - 1 IN ORGANIZED FARMS OF WEST BENGAL, INDIA

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Sumit Chowdhury

2016-06-01

Full Text Available Infectious Bovine Rhinotracheitis, caused by Bovine Herpesvirus-1 (BoHV-1 maintains latency in trigeminal nerve ganglia of bovine. The sero-positive bull infected with BoHV-1 secretes the virus through semen intermittently, when the immune system is compromised. Sera from bulls housed at different bull stations were analyzed using gE protein specific IDEXX Kit, which showed 78.69% positivity. Each batch of semen from sero-positive bull was investigated further for presence of virus in semen by Real Time-PCR technique for validation of presence of virus in the frozen semen doses using gB specific primers and probe, which showed 0.968 % semen batches positive. This study showed that despite high sero prevalence in bull, the semen excretes very negligible amount of the virus indicating the subtypes circulating in farms of West Bengal, India is assumed to be respiratory type.

Bovine viral diarrhea virus 1b fetal infection with extensive hemorrhages

Science.gov (United States)

Bovine viral diarrhea virus (BVDV) subtype 1b was isolated from tissues of a term bovine fetus with hemorrhages in multiple tissues. At autopsy, multiple petechial hemorrhages were observed at gross examination throughout the body and placenta. Lung, kidney, thymus, and liver fresh tissues were exam...

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

Science.gov (United States)

Sreenivasan, Chithra; Thomas, Milton; Sheng, Zizhang; Hause, Ben M; Collin, Emily A; Knudsen, David E B; Pillatzki, Angela; Nelson, Eric; Wang, Dan; Kaushik, Radhey S; Li, Feng

2015-12-01

Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs

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

Science.gov (United States)

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

2015-01-01

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

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

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Bading Hilmar

2007-12-01

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

In vitro neutralization against HoBi-like viruses by antiobodies in serum of cattle immunized with inactivated or modified live vaccines of bovine viral diarrhea virus 1 and 2

Science.gov (United States)

HoBi-like viruses are an emerging species of pestiviruses with genetic and antigenic similarities to bovine viral diarrhea viruses 1 and 2 (BVDV1 and BVDV2). These viruses have been detected associated with respiratory and/or reproductive disease in cattle in Italy and Brazil. Vaccines for HoBi-like...

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

Science.gov (United States)

Saunders, Arpiar; Sabatini, Bernardo L

2015-07-01

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

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

Science.gov (United States)

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

2018-02-05

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

Seroprevalence and risk factors associated with bovine herpesvirus ...

African Journals Online (AJOL)

Bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) are well known etiological agents of cattle that produce important economic losses due to reproductive failures and calf mortality, as well as enteric and respiratory disease. Tamaulipas is located northeast of Mexico, an important cattle production and ...

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

International Nuclear Information System (INIS)

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

2006-01-01

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

A retrospective analysis of the infectious bovine rhinotracheitis (bovine herpes virus-1) surveillance program in Norway using Monte Carlo simulation models

DEFF Research Database (Denmark)

Paisley, Larry; Tharaldsen, J.; Jarp, J.

2001-01-01

Serological surveillance for antibodies against bovine herpes virus type I (BHV-1) which causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis has been carried out since 1992 in Norway. Since 1993 (when a single infected herd was detected) all bulk-milk and pooled...

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

Science.gov (United States)

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

1997-11-01

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

Bovine immunodeficiency-like virus: inactivation in milk by pasteurisation.

Science.gov (United States)

Venables, C; Lysons, R; Horigan, M; Stagg, D; Dawson, M

1997-03-15

Bioassay was used to determine whether bovine immunodeficiency-like virus (BIV) in milk was inactivated by pasteurisation. Three groups of three calves were inoculated with virus (BIV isolate FL112), milk seeded with virus and milk seeded with virus that had been pasteurised before inoculation, respectively. Seroconversion to BIV was monitored for 12 months by an indirect immunofluorescence assay. The presence of BIV proviral DNA in peripheral blood was determined by a nested polymerase chain reaction (PCR). The animals were euthanized and virus isolation and PCR were attempted on peripheral blood mononunclear cells, prescapular lymph node and spleen. Transmission of BIV was confirmed in the groups that were inoculated with the virus and with the virus in milk, but no evidence of its transmission was demonstrated in the group that received the pasteurised inoculum.

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

Science.gov (United States)

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

2008-10-01

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

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

Science.gov (United States)

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

2016-05-01

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

The relationship between antibody status to bovine corona virus and bovine respiratory syncytial virus and disease incidence, reproduction and herd characteristics in dairy herds

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Tråvén Madeleine

2010-06-01

Full Text Available Abstract Background Bovine respiratory syncytial virus (BRSV and bovine corona virus (BCV affects cattle worldwide. Our objective was to evaluate the effects of these infections on general health and reproduction parameters measurable on herd level and to explore the association between antibody status and some herd characteristics. Methods We collected a pooled milk sample from five primiparous cows from 79 Swedish dairy herds in September 2006. The samples were analysed for immunoglobulin G antibodies to BCV and BRSV with indirect enzyme-linked immunosorbent assays. Herd level data from 1 September 2005 to 30 August 2006 were accessed retrospectively. The location of the herds was mapped using a geographical information system. Results Ten herds were antibody negative to both viruses and were compared with 69 herds positive to BCV or BRSV or both. Positive herds had a higher (P = 0.001 bulk tank milk somatic cell count (BMSCC compared with negative herds. The medians for all other analyzed health and reproductive parameters were consistently in favour of the herds negative to both viruses although the differences were not statistically significant. A higher proportion (P = 0.01 of herds used professional technicians for artificial insemination, rather than farm personnel, amongst the 33 herds negative to BCV compared with the 46 positive herds. Conclusions Our result shows that herds that were antibody positive to BCV and/or BRSV had a higher BMSCC compared with herds negative to BCV and BRSV. There was also tendency that negative herds had a better general herd health compared with positive. A higher proportion amongst the BCV negative herds used external technicians for AI instead of farm personnel, indicating that it is possible to avoid infection although having regular visits. Negative herds were located in close proximity to positive herds, indicating that local spread and airborne transmission between herds might not be of great

Bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk - risk factors and spatial analysis.

Science.gov (United States)

Toftaker, Ingrid; Sanchez, Javier; Stokstad, Maria; Nødtvedt, Ane

2016-10-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) are considered widespread among cattle in Norway and worldwide. This cross-sectional study was conducted based on antibody-ELISA of bulk tank milk (BTM) from 1347 herds in two neighboring counties in western Norway. The study aims were to determine the seroprevalence at herd level, to evaluate risk factors for BRSV and BCoV seropositivity, and to assess how these factors were associated with the spatial distribution of positive herds. The overall prevalence of BRSV and BCoV positive herds in the region was 46.2% and 72.2%, respectively. Isopleth maps of the prevalence risk distribution showed large differences in prevalence risk across the study area, with the highest prevalence in the northern region. Common risk factors of importance for both viruses were herd size, geographic location, and proximity to neighbors. Seropositivity for one virus was associated with increased odds of seropositivity for the other virus. Purchase of livestock was an additional risk factor for BCoV seropositivity, included in the model as in-degree, which was defined as the number of incoming movements from individual herds, through animal purchase, over a period of five years. Local dependence and the contribution of risk factors to this effect were assessed using the residuals from two logistic regression models for each virus. One model contained only the x- and y- coordinates as predictors, the other had all significant predictors included. Spatial clusters of high values of residuals were detected using the normal model of the spatial scan statistic and visualized on maps. Adjusting for the risk factors in the final models had different impact on the spatial clusters for the two viruses: For BRSV the number of clusters was reduced from six to four, for BCoV the number of clusters remained the same, however the log-likelihood ratios changed notably. This indicates that geographical differences in proximity to

Perspectives on avian and bovine leukemia virus immunological studies

International Nuclear Information System (INIS)

Higuchi, T.; Souza, J.M.M. de; Nogueira, Z.M.; Ogata, H.

1984-01-01

The avian and bovine RNA virus are studied. The mechanism of replication, the genome, the ultrastructural composition, the immunogens reactivity, the class of determinants and affinity are presented. Purification techniques of viral proteins and immunoassay proceeding are reported. (M.A.C.) [pt

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

DEFF Research Database (Denmark)

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

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

Directory of Open Access Journals (Sweden)

Tomohiro Okagawa

2017-06-01

Full Text Available Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1/PD-ligand 1 (PD-L1, is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2 was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV. Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

Short communication. Microculture syncytia assay for bovine leukemia virus

Energy Technology Data Exchange (ETDEWEB)

Paul, P.S.; Castro, A.E.; Pomeroy, K.A.; Johnson, D.W.; Muscoplat, C.C.

1978-01-01

A microculture syncytia assay for the detection of bovine leukemia virus (BLV) has been described and compared with the conventional macroculture assay. The microculture assay required fewer indicator cells, was as sensitive as the macroculture assay and provided a reproducible test for the detection and titration of BLV.

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

Directory of Open Access Journals (Sweden)

Yingpeng Liu

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

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

Science.gov (United States)

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

2017-03-01

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

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

Science.gov (United States)

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

2008-01-01

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

Glycoprotein-G-gene-based molecular and phylogenetic analysis of rabies viruses associated with a large outbreak of bovine rabies in southern Brazil.

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Cargnelutti, Juliana F; de Quadros, João M; Martins, Mathias; Batista, Helena B C R; Weiblen, Rudi; Flores, Eduardo F

2017-12-01

A large outbreak of hematophagous-bat-associated bovine rabies has been occurring in Rio Grande do Sul (RS), the southernmost Brazilian state, since 2011, with official estimates exceeding 50,000 cattle deaths. The present article describes a genetic characterization of rabies virus (RABV) recovered from 59 affected cattle and two sheep, from 56 herds in 16 municipalities (2012-2016). Molecular analysis was performed using the nucleotide (nt) and predicted amino acid (aa) sequences of RABV glycoprotein G (G). A high level of nt and aa sequence identity was observed among the examined G sequences, ranging from 98.4 to 100%, and from 97.3 to 100%, respectively. Likewise, high levels of nt and aa sequence identity were observed with bovine (nt, 99.8%; aa, 99.8%) and hematophagous bat (nt, 99.5%; aa, 99.4%) RABV sequences from GenBank, and lower levels were observed with carnivore RABV sequences (nt, 92.8%; aa, 88.1%). Some random mutations were observed in the analyzed sequences, and a few consistent mutations were observed in some sequences belonging to cluster 2, subcluster 2b. The clustering of the sequences was observed in a phylogenetic tree, where two distinct clusters were evident. Cluster 1 comprised RABV sequences covering the entire study period (2012 to 2016), but subclusters corresponding to different years could be identified, indicating virus evolution and/or introduction of new viruses into the population. In some cases, viruses from the same location obtained within a short period grouped into different subclusters, suggesting co-circulation of viruses of different origins. Subcluster segregation was also observed in sequences obtained in the same region during different periods, indicating the involvement of different viruses in the cases at different times. In summary, our results indicate that the outbreaks occurring in RS (2012 to 2016) probably involved RABV of different origins, in addition to a possible evolution of RABV isolates within this

Competitive virus assay method for titration of noncytopathogenic bovine viral diarrhea viruses (END⁺ and END⁻ viruses).

Science.gov (United States)

Muhsen, Mahmod; Ohi, Kota; Aoki, Hiroshi; Ikeda, Hidetoshi; Fukusho, Akio

2013-03-01

A new, reliable and secure virus assay method, named the competitive virus assay (CVA) method, has been established for the titration of bovine viral diarrhea viruses (BVDVs) that either show the exaltation of Newcastle disease virus (END) phenomenon or heterologous interference phenomenon (but not the END phenomenon). This method is based on the principle of (1) homologous interference between BVDVs, by using BVDV RK13/E(-) or BVDV RK13/E(+) strains as competitor virus, and (2) END phenomenon and heterologous interference, by using attenuated Newcastle disease virus (NDV) TCND strain as challenge virus. In titration of BVDV END(+) and BVDV END(-) viruses, no significant difference in estimated virus titer was observed between CVA and conventional methods. CVA method demonstrated comparable levels of sensitivity and accuracy as conventional END and interference methods, which require the use of a velogenic Miyadera strain of NDV and vesicular stomatitis virus (VSV), both of which are agents of high-risk diseases. As such, the CVA method is a safer alternative, with increased bio-safety and bio-containment, through avoidance of virulent strains that are commonly employed with conventional methods. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

Shi, Xin; Wang, Jianzhong; Qin, Yan

2014-12-01

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

Comparison of the cross-antibody response induced in sheep by inactivated bovine viral diarrhoea virus 1 and Hobi-like pestivirus.

Science.gov (United States)

Decaro, Nicola; Mari, Viviana; Sciarretta, Rossana; Lucente, Maria Stella; Camero, Michele; Losurdo, Michele; Larocca, Vittorio; Colao, Valeriana; Cavaliere, Nicola; Lovero, Angela; Lorusso, Eleonora; Buonavoglia, Canio

2013-06-01

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species. Copyright © 2012 Elsevier Ltd. All rights reserved.

Improved detection of Bovine Viral Diarrhea Virus in Bovine lymphoid cell lines using PrimeFlow RNA assay

Science.gov (United States)

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains

Science.gov (United States)

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but many other species. Serological evidence suggests that nearly 100% of children in the United States have been infected with PIV-3 by five years of age. Similarly, in cattle PIV-3 is commonly associated with bovine re...

HoBi-like virus challenge of pregnant cows that had previously given birth to calves persistently infected with bovine viral diarrhea virus

Science.gov (United States)

The ability of bovine viral diarrhea viruses (BVDV) to establish persistent infection (PI) following fetal infection is central to keeping these viruses circulating. Similarly, an emerging species of pestivirus, HoBi-like viruses, is also able to establish PIs. Dams that are not PI, but carrying PI ...

Identification and Characterization of Bovine Viral Diarrhea Virus from Indonesian Cattle (IDENTIFIKASI DAN KARAKTERISASI VIRUS BOVINE VIRAL DIARRHEA DARI SAPI INDONESIA

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Muharam Saepulloh

2015-05-01

Full Text Available Bovine viral diarrhea virus (BVDV is an important viral disease, which a ubiquitous pathogen ofcattle with worldwide economic importance and due to its misdiagnose with other viruses. The goal of thecurrent study was to identify and characterize of BVDV by reverse transcriptase polymerase chainreaction (RT-PCR and followed by sequence genome analyses. Blood, feces, and semen samples werecollected from 588 selected cattle from animals suffering from diarrhea and respiratory manifestation. RTPCRresults showed that the 69 (11.74% samples were positive to BVDV. Further molecularcharacterization was conducted only with 17 PCR positive samples. The results indicated the 17 IndonesianBVD virus isolates were belonging to the genotype-1 of BVDV (BVDV-1 based on sequence analysis anda phylogenetic relationship between Indonesian BVDV isolates and BVDV in the world. This finding is thefirst report of BVD-1 circulated in Indonesian cattle.

Sero prevalence and risk factors associated with bovine herpes virus type 1 (BHV1) infection in non-vaccinated cattle herds in Andalusia (South of Spain)

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Gonzalez-Garcia, M. A.; Arenas-Casas, A.; Carbonero-Martinez, A.; Borge-Rodriguez, C.; Garcia-Bocanegra, I.; Maldonado, J. L.; Gomez-Pacheco, J. M.; Perea-Remujo, J. A.

2009-07-01

An epidemiological and serological survey of bovine herpes virus 1 (BHV1) infection was conducted in Andalusia from January to April of 2000. A total of 4,035 blood samples were collected from 164 herds. A questionnaire, which included variables potentially associated with infection, was filled out for each herd. Serum samples were obtained to identify specific BHV1 antibodies and were tested using a blocking ELISA test. The observed crude odds ratio (OR) (estimate of the chance of a particular event occurring in an exposed group in relation to its rate of occurrence in a nonexposed group) for vaccination is 9.8 (95 % confidence interval: 8.3-11.7). The vaccinated group comprised large dairy farms. This study can only be considered as representative of unvaccinated, small to medium size dairy farms and beef farms in Andalusia. True sero prevalence of the BHV1 virus in non vaccinated bovine populations in Andalusia reached 45.7% of individuals and 70.4% of herds. Risk factors for BHV1 infection in bovine Andalusian non vaccinated herds are nonexistence of specific cattle infrastructure (OR: 3.07), beef crossbreeding (OR: 7.90), affiliation with Livestock Health Defence Associations (OR: 2.57), a history of reproductive disorders (OR: 8.39), external replacement (OR: 2.74), proximity to an urban area (OR: 6.11) and herd size (41.98). To control for confounding effects, a binomial logistic regression model was developed. From this regression, BHV1 infections are concentrated in large herds, with external replacement, located close to urban areas. This is the first published report on BHV1 prevalence in the South of Spain. (Author) 14 refs.

A survey of free-ranging deer in Ireland for serological evidence of exposure to bovine viral diarrhoea virus, bovine herpes virus-1, bluetongue virus and Schmallenberg virus.

Science.gov (United States)

Graham, David A; Gallagher, Clare; Carden, Ruth F; Lozano, Jose-Maria; Moriarty, John; O'Neill, Ronan

2017-01-01

Deer are an important wildlife species in both the Republic of Ireland and Northern Ireland having colonised most regions across the island of Ireland. In comparison to cattle and sheep which represent the main farmed ruminant species on the island, there is a lack of data concerning their exposure, as measured by the presence of antibodies, to important viral pathogens of ruminants. A study was therefore undertaken to investigate the seroprevalence of wild deer to four viruses, namely bovine viral diarrhoea virus (BVDV), bovine herpesvirus-1 (BoHV-1), Schmallenberg virus (SBV) and bluetongue virus (BTV). Two panels of sera were assembled; Panel 1 comprised 259 samples (202 collected in the Republic of Ireland and 57 in Northern Ireland) between 2013 and 2015, while Panel 2 comprised 131 samples collected in the Republic of Ireland between 2014 and 2015. Overall sika deer ( Cervus nippon ) were sampled most commonly (54.8%), followed by fallow deer ( Dama dama ) (35.3%), with red deer ( Cervus elaphus ) (4.3%) and hybrid species (0.3%) sampled less frequently, with the species not being recorded for the remaining 5.3% of deer sampled. Age was not recorded for 96 of the 390 deer sampled. 196 of the remainder were adults, while 68 and 30 were yearlings and calves, respectively. Using commercially available enzyme-linked immunosorbent assays, true prevalence and 95% confidence intervals were calculated as 9.9%, (6.8-13.0% CI), SBV; 1.5% (0.1-3.0% CI), BoHV-1; 0.0%, 0-1.7% CI), BVDV; and 0.0%, (0.01-0.10% CI), BTV. The results indicate a very low seroprevalence for both BVDV and BoHV-1 in the wild deer tested within the study and, are consistent with a very low prevalence in Ireland. While serological cross-reaction with cervid herpesviruses cannot be excluded, the results in both cases suggest that the presence of these viruses in deer is not a significant risk to their control and eradication from the cattle population. This is important given the ongoing programme

Viral infections and bovine mastitis: a review

NARCIS (Netherlands)

Wellenberg, G.J.; Poel, van der W.H.M.; Oirschot, van J.T.

2002-01-01

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or

Bovine virus diarrhea virus in free-living deer from Denmark.

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Nielsen, S S; Roensholt, L; Bitsch, V

2000-07-01

Free-living deer are suggested as a possible source of infection of cattle with bovine virus diarrhea (BVD) virus. To examine this hypothesis blood samples from 476 free-living deer were collected during two different periods and tested for BVD virus and antibody in Denmark. In 1995-96, 207 animals were tested. These included 149 roe deer (Capreolus capreolus), 29 fallow deer (Dama dama), 20 red deer (Cervus elaphus) and one sika deer (Cervus sika). For the remaining eight animals no species information was available. In 1998-99, 269 animals were tested including 212 roe deer and 57 red deer. The animals were selected from areas with a relatively high prevalence of cattle herds with a BVD persistent infection status in 1997 and 1998. All 207 samples from 1995-96 were found antibody-negative except two samples from red deer. Only 158 of the 207 samples were tested for virus and were all found negative. Of the 269 samples from 1998-99 all but one were antibody negative. The positive sample was from a red deer. All samples were virus-negative. It appears that BVD infection does not occur in roe deer in Denmark. The presence of antibody in a few red deer from various districts in Jutland probably results from cattle to deer transmission, rather than spread among deer. Hence, the possibility of free-living deer as a source of infection for cattle in Denmark seems to be remote.

Vertical transmission of bovine viral diarrhoea virus (BVDV) in mousedeer (Tragulus javanicus) and spread to domestic cattle

DEFF Research Database (Denmark)

Uttenthal, Åse; Høyer, M.J.; Grøndahl, C.

2006-01-01

This study investigates the transmission of bovine viral diarrhoea virus (BVDV) 1f from a persistently infected (PI) lesser Malayan mousedeer to two bovine calves. Different contact routes to two calves were analysed: 1) aerosol contact between two adjacent pens without physical contact; 2) indir...... mousedeer BVD virus in the E2 region (420 nucleotides) through 4 generations showed only 7 mutations, which were maintained from mother to offspring....

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

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

2012-01-01

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

Molecular detection of Bluetongue Virus (BTV and Bovine Leukemia Virus (BLV in uterine biopsies of dairy cows with or without reproductive problems

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Juliana Marques Bicalho

2016-10-01

Full Text Available Reproductive performance of dairy cows has a direct impact on herd productivity. Infectious agents, such as Bluetongue Virus (BTV and Bovine Leukemia Virus (BLV, are associated with reproductive failure. However, it remains unknown if these viruses are present in the uterus and cause gestational loss. This study used molecular methods to assess if BTV and BLV can be detected in the uterus of serologically positive dairy cows with a record of abortions, stillbirths and repeat breeding (n=23 and without a record of reproductive problems (n =23. The cows came from three dairy herds of the state of Minas Gerais, Brazil. BTV was not detected in any of the uterine biopsies. Proviral DNA of BLV was detected in 54.5 % of the seropositive cows, but positivity for BLV in the uterus was not associated with the existence of reproductive problems. In conclusion, this study shows that BLV, but not BTV, is present in the uterus of seropositive cows, regardless of reproductive performance.

Characterization of a chimeric foot-and-mouth disease virus bearing bovine rhinitis B virus leader proteinase

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Our recent study has shown that bovine rhinovirus type 2 (BRV2), a new member of the Aphthovirus genus, shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). Despite low sequence conservation (36percent amino acid identity) and N- and C-terminus folding differences,...

Case Report: Emergence of bovine viral diarrhea virus persistently infected calves in a closed herd

Science.gov (United States)

Bovine viral diarrhea virus (BVDV) continues to have significant economic impact on the cattle industry worldwide. The virus is primarily maintained in the cattle population due to persistently infected animals. Herd surveillance along with good vaccination programs and biosecurity practices are the...

Comparison of Detection of Bovine Virus Diarrhea Virus Antigen in Various Types of Tissue and Fluid Samples Collected from Persistently Infected Cattle

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Bovine viral diarrhea viruses are economically important pathogens of cattle. Most new infections are acquired from animals persistently infected with the virus. Surveillance programs rely on skin biopsies for detection of persistently infected cattle. The purpose of this study was to compare ant...

Bovine respiratory syncytial virus ISCOMs - protection in the presence of maternal antibodies

DEFF Research Database (Denmark)

Hägglund, Sara; Hu, Ke-Fei; Larsen, Lars Erik

2004-01-01

The protection induced by immunostimulating complexes (ISCOMs) against bovine respiratory syncytial virus (BRSV) was evaluated and compared to that of a commercial inactivated vaccine (CV) in calves with BRSV-specific maternal antibodies. Following experimental challenge, controls (n = 4...

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.

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection.

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Suzuki, Saori; Konnai, Satoru; Okagawa, Tomohiro; Ikebuchi, Ryoyo; Nishimori, Asami; Kohara, Junko; Mingala, Claro N; Murata, Shiro; Ohashi, Kazuhiko

2015-02-15

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4(+) T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. Copyright © 2014 Elsevier B.V. All rights

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

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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…

Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats

Science.gov (United States)

2013-01-01

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable. PMID:23675947

Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats.

Science.gov (United States)

Bachofen, Claudia; Vogt, Hans-Rudolf; Stalder, Hanspeter; Mathys, Tanja; Zanoni, Reto; Hilbe, Monika; Schweizer, Matthias; Peterhans, Ernst

2013-05-15

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable.

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

NARCIS (Netherlands)

Poel, van der W.H.M.; Langedijk, J.P.M.; Kramps, J.A.; Middel, W.G.J.; Brand, A.; Oirschot, van J.T.

1997-01-01

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV

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.

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

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

1997-03-01

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

Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

Energy Technology Data Exchange (ETDEWEB)

Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

2007-08-06

Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

Comparison of Bovine coronavirus-specific and Bovine respiratory syncytial virus-specific antibodies in serum versus milk samples detected by enzyme-linked immunosorbent assay.

Science.gov (United States)

Ohlson, Anna; Blanco-Penedo, Isabel; Fall, Nils

2014-01-01

Bovine coronavirus (BCV; Betacoronavirus 1) and Bovine respiratory syncytial virus (BRSV) are significant causes of enteric and respiratory disease in beef and dairy cattle throughout the world. Indirect enzyme-linked immunosorbent assays are widely used to detect serum antibodies for herd monitoring and prevalence studies. In dairy herds, milk is more readily collected than serum. Hence, in order to investigate the test agreement between serum and milk, both serum and milk samples from 105 cows in 27 dairy herds were analyzed in parallel for presence of immunoglobulin G antibodies to BCV and BRSV. The Bland-Altman analyses of data demonstrated good agreement between serum and milk antibody titers for both viruses. The results indicate milk samples are sufficient for surveillance of antibodies to BCV and BRSV.

Development and Characterization of a Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out Supplemental Materials

Energy Technology Data Exchange (ETDEWEB)

Smith, S; Danganan, L; Tammero, L; Lenhoff, R; Naraghi-arani, P; Hindson, B

2007-08-06

Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed advanced rapid diagnostics that may be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the potential to improve our nation's ability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect animal populations of high economic importance in the United States. Under 2005 DHS funding we have developed multiplexed (MUX) nucleic-acid-based PCR assays that combine foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease (SVD) and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1 or Infectious Bovine Rhinotracheitus IBR), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus BPSV, Orf of sheep, and Pseudocowpox). Under 2006 funding we have developed a Multiplexed PCR [MUX] porcine assay for detection of FMDV with rule out tests for VESV and SVD foreign animal diseases in addition to one other domestic vesicular animal disease vesicular stomatitis virus (VSV) and one domestic animal disease of swine porcine reproductive and respiratory syndrome (PRRS). We have also developed a MUX bovine assay for detection of FMDV with rule out tests for the two bovine foreign animal diseases malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis virus (VSV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitus virus (BHV-1), bluetongue virus (BTV), and the Parapox

Replication and clearance of respiratory syncytial virus - Apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus

DEFF Research Database (Denmark)

Viuff, B.; Tjørnehøj, Kirsten; Larsen, Lars Erik

2002-01-01

and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared...... and the infections with human respiratory syncytial. virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication......Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus...

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.

Comparison of the prevalence and incidence of infection with bovine virus diarrhoea virus (BVDV) in Denmark and Michigan and association with possible risk factors

DEFF Research Database (Denmark)

Houe, H.; Baker, J.C.; Maes, R.K.

1995-01-01

Based on 2 previous surveys on the occurrence of infection with bovine virus diarrhoea virus (BVDV) in Danish and Michigan dairy herds, the prevalence and incidence of the infection were compared. The presence of certain possible risk factors for the occurrence of infection in the 2 areas were...... summarized and it was investigated if any of these risk factors had significant effect on the presence of animals persistently infected (PI) with BVDV in the dairy herds. Information on the cattle population density in the 2 areas was obtained from statistical yearbooks. Further information...... for the individual farms on age distribution, housing of animals, herd size, pasturing and purchasing policy was gathered. The prevalence of PI animals was more than 10 times higher in Denmark as compared to Michigan. In herds without PI animals, the annual incidence of seroconversion as calculated from the age...

In Vivo Characterisation of Five Strains of Bovine Viral Diarrhoea Virus 1 (Subgenotype 1c

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Rebecca K. Ambrose

2018-01-01

Full Text Available Bovine viral diarrhoea virus 1 (BVDV-1 is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.

Virus survival in slurry: Analysis of the stability of foot-and-mouth disease, classical swine fever, bovine viral diarrhoea and swine influenza viruses

DEFF Research Database (Denmark)

Bøtner, Anette; Belsham, Graham

2012-01-01

of an outbreak of disease before it has been recognized. The survival of foot-and-mouth disease virus, classical swine fever virus, bovine viral diarrhoea virus and swine influenza virus, which belong to three different RNA virus families plus porcine parvovirus (a DNA virus) was examined under controlled...... conditions. For each RNA virus, the virus survival in farm slurry under anaerobic conditions was short (generally ≤1h) when heated (to 55°C) but each of these viruses could retain infectivity at cool temperatures (5°C) for many weeks. The porcine parvovirus survived considerably longer than each of the RNA...... viruses under all conditions tested. The implications for disease spread are discussed....

Bovine Necrotic Vulvovaginitis Associated with Porphyromonas levii

Science.gov (United States)

Friedgut, Orly; Alpert, Nir; Stram, Yehuda; Lahav, Dan; Tiomkin, Doron; Avramson, Miriam; Grinberg, Kalia; Bernstein, Michael

2004-01-01

An outbreak of bovine necrotic vulvovaginitis associated with Porphyromonas levii, an emerging animal and human pathogen, affected 32 cows on a dairy farm in the northeast of Israel. Five animals had to be culled. This report appears to be the first that associates P. levii with bovine necrotic vulvovagnitis. PMID:15109423

Studies on genetic diversity of bovine viral diarrhea viruses in Danish cattle herds

DEFF Research Database (Denmark)

Nagy, Abdou; Fahnøe, Ulrik; Rasmussen, Thomas Bruun

2014-01-01

Scandinavian countries have successfully pursued bovine viral diarrhea virus (BVDV) eradication without the use of vaccines. In Denmark, control and eradication of BVDV were achieved during the last two decades, but occasionally new BVDV infections are detected in some Danish cattle herds. The aim...

Eradication of bovine viral diarrhea virus in Germany-Diversity of subtypes and detection of live-vaccine viruses.

Science.gov (United States)

Wernike, Kerstin; Schirrmeier, Horst; Strebelow, Heinz-Günter; Beer, Martin

2017-09-01

Bovine viral diarrhea (BVD) causes high economic losses in the cattle population worldwide. In Germany, an obligatory control program with detection and removal of persistently infected animals is in force since 2011. For molecular tracing of virus transmission, a comprehensive sequence data base of the currently circulating BVD viruses was established. Partial sequences of 1007 samples collected between 2008 and 2016 were generated. As dominant viruses, subtypes 1b (47.0%) and 1d (26.5%) could be identified with no marked geographic or sampling year effect, a much higher amount of BVDV-2c was detected in 2013 compared to other years, predominantly in Western Germany. In addition, subtypes 1a, 1e, 1f, 1h, 1g, 1k, and 2a were found. Interestingly, besides field-viruses, two different live-vaccine viruses were detected in tissue samples of newborn calves (n=37) whose mothers were immunized during pregnancy. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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Barbara Bogner

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

Effects of interferon-tau on cattle persistently infected with bovine viral diarrhea virus.

Science.gov (United States)

Kohara, Junko; Nishikura, Yumiko; Konnai, Satoru; Tajima, Motoshi; Onuma, Misao

2012-08-01

In this study, the antiviral effects of bovine interferon-tau (boIFN-tau) on bovine viral diarrhea virus (BVDV) were examined in vitro and in vivo. In the in vitro experiments, the replication of cytopathic and non-cytopathic BVDV was inhibited in the bovine cells treated with boIFN-tau. The replication of BVDV was completely suppressed by boIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the effect of boIFN-tau on virus propagation in cattle persistently infected (PI) with non-cytopathic BVDV, boIFN-tau was subcutaneously administered to PI cattle at 10(5) U/kg or 10(6) U/kg body weight 5 times per week for 2 weeks. No physical abnormality such as depression was observed in the cattle during the experiment. The mean BVDV titers in the serum of the PI cattle decreased slightly during the boIFN-tau administration period with the dose of 10(6) U/kg. However, the BVDV titers in the serum returned to the pre-administration level after the final boIFN-tau administration. These results suggest that boIFN-tau demonstrates an anti-BVDV effect, reducing the BVDV level in serum transiently when injected into PI cattle.

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

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

2014-09-01

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

Proteins of bovine viral diarrhea virus: characterization, biotype-specific differences, and immunological properties

International Nuclear Information System (INIS)

Donis, R.O.

1987-01-01

Virus-specific polypeptides in bovine viral diarrhea-mucosal disease (BVD) virus-infected bovine cells were studied by radiolabeling. A total of 12 polypeptides with apparent Mr of 165, 135, 118, 80, 75, 62, 56-58, 48, 37, 32, 25 and 19 kilodaltons (k) were identified in infected cells. Five glycoproteins were detected in infected cells. Two abundant species had apparent Mr of 48 k and 56-58 k while the minor species had masses of 118, 75 and 65 k. When cells were radiolabeled with L-[ 35 S]-methionine in the presence of tunicamycin the 56-58 k migrated with apparent masses of 54 k and 48-50 K in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4-6 K reduction in the apparent molecular mass of the 56-58 k yielding a 52 k digested product. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a vital role in the infection cycle of BVD virus. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be consistently differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of unique polypeptide profiles they induce in the infected cell: the most abundant polypeptide in CB-BVD infected cells is the 80 kD polypeptide while NCB-BVD lack this polypeptide and induce a predominant 118 k polypeptide. A panel of 25 murine monoclonal antibodies (Mabs) against the two major glycoproteins of BVD virus was produced. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity the Mabs in the panel were divided into 3 classes: Class 1 Mabs reacted with the 56-58 k glycoprotein and neutralized the virus, Class 2 Mabs recognized the 56-58 k glycoprotein but were not neutralizing and Class 3 Mabs reacted with the 48 k glycoprotein and did not neutralize the virus. These results identify the 56-58 k as one of the envelope glycoproteins of BVD virus

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

NARCIS (Netherlands)

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

2003-01-01

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

Bovine lymphosarcoma: development of a radioimmunologic techniques for detection of the etiologic agent

International Nuclear Information System (INIS)

Devare, S.G.; Stephenson, J.R.; Sarma, P.S.; Aaronson, S.A.

1976-01-01

A highly sensitive and specific radioimmunoassay has been developed for the major structural protein of an oncornavirus etiologically associated with bovine lymphosarcoma. This test can be used to identify cattle which have been exposed to the bovine leukemia virus and may thus develop or transmit the disease. Analysis of randomly obtained serums indicates that infection with this virus is widespread among cattle

Bovine leukemia virus seroprevalence among cattle presented for slaughter in the United States

Science.gov (United States)

Infection with bovine leukemia virus (BLV) results in economic loss due reduced productivity, especially the reduction of milk production and early culling. In the USA.,USA, previous studies in 1996, 1999 and 2007 showed BLV infections widespread, especially in the dairy herds. The goal of this stud...

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV, Bovine Leukemia Virus (BLV, Human Papilloma Virus (HPV, and Epstein–Barr Virus (EBV

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James S. Lawson

2018-01-01

Full Text Available BackgroundAlthough the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV, bovine leukemia virus (BLV, human papilloma viruses (HPVs, and Epstein–Barr virus (EBV-also known as human herpes virus type 4. Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence.The evidenceMMTV and human breast cancer—the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer—the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer—the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer—the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal.ConclusionThe influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer.

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV), Bovine Leukemia Virus (BLV), Human Papilloma Virus (HPV), and Epstein-Barr Virus (EBV).

Science.gov (United States)

Lawson, James S; Salmons, Brian; Glenn, Wendy K

2018-01-01

Although the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV), bovine leukemia virus (BLV), human papilloma viruses (HPVs), and Epstein-Barr virus (EBV-also known as human herpes virus type 4). Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence. MMTV and human breast cancer-the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer-the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer-the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer-the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal. The influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer.

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

NARCIS (Netherlands)

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

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

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.

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

OpenAIRE

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

2000-01-01

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

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

Cis-drivers and trans-drivers of bovine leukemia virus oncogenesis.

Science.gov (United States)

Safari, Roghaiyeh; Hamaidia, Malik; de Brogniez, Alix; Gillet, Nicolas; Willems, Luc

2017-10-01

The bovine leukemia virus (BLV) is a retrovirus inducing an asymptomatic and persistent infection in ruminants and leading in a minority of cases to the accumulation of B-lymphocytes (lymphocytosis, leukemia or lymphoma). Although the mechanisms of oncogenesis are still largely unknown, there is clear experimental evidence showing that BLV infection drastically modifies the pattern of gene expression of the host cell. This alteration of the transcriptome in infected B-lymphocytes results first, from a direct activity of viral proteins (i.e. transactivation of gene promoters, protein-protein interactions), second, from insertional mutagenesis by proviral integration (cis-activation) and third, from gene silencing by microRNAs. Expression of viral proteins stimulates a vigorous immune response that indirectly modifies gene transcription in other cell types (e.g. cytotoxic T-cells, auxiliary T-cells, macrophages). In principle, insertional mutagenesis and microRNA-associated RNA interference can modify the cell fate without inducing an antiviral immunity. Despite a tight control by the immune response, the permanent attempts of the virus to replicate ultimately induce mutations in the infected cell. Accumulation of these genomic lesions and Darwinian selection of tumor clones are predicted to lead to cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Herd-level prevalence and associated risk factors for Toxoplasma gondii, Neospora caninum, Chlamydia abortus and bovine viral diarrhoea virus in commercial dairy and beef cattle in eastern, northern and northeastern China.

Science.gov (United States)

Sun, Wu-Wen; Meng, Qing-Feng; Cong, Wei; Shan, Xiao-Feng; Wang, Chun-Feng; Qian, Ai-Dong

2015-11-01

Although the seroprevalence of Toxoplasma gondii, Neospora caninum, Chlamydia abortus and bovine viral diarrhea virus infection in cattle have been reported in some areas in China, most of them were conducted with small number of cattle samples and very limited districts and neglected the assessment of herd management factors associated with herd-level prevalence of these pathogen infections. Thus, from September 2013 to December 2014, a large-scale seroprevalence study was conducted to determine the animal-level and herd-level seroprevalence and identify herd-level risk factors associated with these pathogen infections in 4487 cattle from 134 herds in five provinces (Heilongjiang, Jilin, Liaoning, Shandong, Hebei) and Inner Mongolia Autonomous Region of China. At animal level, the true prevalence of antibodies against T. gondii, N. caninum, C. abortus and bovine viral diarrhoea virus (BVDV) was 10.48, 17.14, 11.92 and 50.10%, respectively. At herd level, the true prevalence of antibodies against T. gondii, N. caninum, C. abortus and BVDV was 27.16, 29.10, 37.31 and 40.30%, respectively. Multivariate analysis of these characteristics showed that source of water and presence of felids were significantly associated with T. gondii infection in the studied cattle herds. Source of water was significantly associated with N. caninum infection in the studied cattle herds. While herd size and management system were significantly associated with BVDV infection in the studied cattle herds, this is the first report of herd-level prevalence and associated risk factors of T. gondii, N. caninum, C. abortus and BVDV infection in cattle in China.

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of bovine viral diarrhea virus type 1 and 2

NARCIS (Netherlands)

Jelsma, H.; Loeffen, W.L.A.; Beuningen, van A.R.; Rijn, van P.A.

2013-01-01

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent

Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade

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Ikebuchi Ryoyo

2011-09-01

Full Text Available Abstract The inhibitory receptor programmed death-1 (PD-1 and its ligand, programmed death-ligand 1 (PD-L1 are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection.

Comparison of some storage and isolation methods to recover bluetongue virus from bovine blood.

OpenAIRE

Thomas, F C

1984-01-01

Bovine blood containing bluetongue virus was stored as whole blood with EDTA (4 degrees C), as washed cellular components (4 degrees C), and as washed cellular components with 10% DMSO (-70 degrees C). Periodic isolation attempts were made over a period of 330 days in four cell lines and embryonating chicken eggs (intravenous inoculation). Bluetongue virus was successfully isolated in all systems from most samples throughout the test period. There appeared to be more variation amongst days of...

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

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Vandaele Leen

2011-01-01

Full Text Available Abstract Bluetongue virus serotype 8 (BTV-8, which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

Experimental infection of pregnant goats with bovine viral diarrhea virus (BVDV)1 or 2

Science.gov (United States)

Infections with bovine viral diarrhea virus (BVDV) of the genus pestivirus, family Flaviviridae, are not limited to cattle but occur in various artiodactyls. Persistently infected (PI) cattle are the main source of BVDV. Persistent infections also occur in heterologous hosts such as sheep and deer. ...

Differential expression of miRNA-423-5p in serum from cattle challenged with bovine viral diarrhea virus

Science.gov (United States)

Bovine viral diarrhea virus (BVDV) is an RNA virus that causes respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. However, microRNA profiles in cattle exposed to BVDV are currently nonexistent and few studies have been reported; therefore,...

Le virus de la leucémie bovine et l’homéostasie du compartiment lymphocytaire périphérique

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Luc Willems

2007-01-01

Full Text Available Bovine leukaemia virus and peripheral blood lymphocytes homeostasis. Bovine leukaemia virus (BLV is the etiological agent of a lymphoproliferative disease in cattle. This retrovirus can also be transmitted experimentally to the ovine species, in which pathology is more rapid and more frequent. In this model, infection leads to an increased cell turnover. This accelerated lymphocyte dynamics might be related to viral expression which induces cellular proliferation and host cell destruction by the immune system.

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

Science.gov (United States)

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

2015-08-01

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

BOVINE RESPIRATORY SYNCYTIAL VIRUS EPIDEMIOLOGY AND RISK FACTORS ON CATTLE HERDS OF CAMPECHE STATE, MEXICO

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Lisandro Alberto Encalada Mena

2016-12-01

Full Text Available High seroprevalence in Yucatan and proximity to the state of Campeche make it necessary to determine the seroprevalence and risk factors of bovine respiratory syncytial virus (VRSB in the state of Campeche, Mexico. Thus the objective of the present work was to determine the seroprevalence and risk factors bovine respiratory syncytial virus (BRSV of the state of Campeche, Mexico. The sampled of 36 cattle herds (842 sera were analyzed by indirect ELISA kit, in the 11 municipalities of Campeche. A survey to obtain risk factors (sex, age of animals, number of animals grazing density, management system, presence of sheep on the farm and access to the roadside was applied and calculated X2 for each variable considered. Of the total number of samples analyzed (842, 273 were positive (32.47%. The prevalence ranges found ranged from 0% to 84%, so in 9 of the herds there were no positive samples, indicating a 75% (27/36 of dispersion of this virus. X2 analysis indicated that all variables were significant and are risk factors regarding with respect to the variable seroprevalence of BRSV. The results indicate a wide circulation of BRSV and we suggest implement recommendations that will enable a lower spread of this virus in the cattle population.

Detection of bovine viral diarrhoea virus in specimens from cattle in South Africa and possible association with clinical disease

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N. Kabongo

2004-06-01

Full Text Available Studies covering all aspects of bovine viral diarrhoea virus (BVDV have been conducted in several countries in Europe, Asia and America. In southern Africa, more information is required about the nature of BVDV infection, the prevalence of different strains and the economic importance of the disease. The presence of BVDV in southern Africa has been known since the early 1970s through serological surveys but few reports confirming its presence by virus isolation and correlation with clinical disease are available. Specimens (n = 312 collected in 1998/99, from live and dead cattle from different farming systems, were obtained from private practitioners, feedlot consultants and abattoirs throughout the country. Specimens (n=37 from African buffaloes (Syncerus caffer in the Kruger National Park were also included. All specimens were processed for virus isolation in cell culture with confirmation by means of immunofluorescent antibody tests and some also by means of an antigen capture ELISA. BVDV was isolated from 15 (4.7 % cattle and were all noncytopathic biotypes. BVDV was not detected in 37 lymph nodes obtained from buffaloes in the Kruger National Park. Of the clinical signs in cattle from which virus were isolated, respiratory signs was the most frequent (10/15, followed by diarrhoea (5/15. Abortion, congenital malformations, haemorrhagic diarrhoea and poor growth were also included as criteria for selection of animals for specimen collection, but no BVD viruses were isolated from cattle manifesting these clinical signs.

Seroprevalence of some bovine viral respiratory diseases among non vaccinated cattle in Saudi Arabia

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Mohamed Abd El Fatah Mahmoud

2013-02-01

Full Text Available Aim: Four viral pathogens, bovine viral diarrhea virus (BVDV, and bovine herpes virus type 1 (BHV-1, bovine parainfluenza type 3 virus (PI-3V, bovine respiratory syncytial virus (BRSV are mainly associated with bovine respiratory diseases that cause major economic losses in the dairy cattle industry. This study aimed to document exposure of cattle in Saudi Arabia to infectious BVDV, BHV-1, PI-3V and BRSV viruses in non vaccinated cattle in order to obtain epidemiological and immunological information. Materials and Methods: In the present study, 460 random serum samples obtained from non vaccinated cattle in five districts (Riyadh, Eastern Province, Jizan, Najran, Asir of Saudi Arabia between January to March 2011. These samples were tested for presence of antibodies against BVDV, BHV-1, BRSV and PIV-3 by commercial indirect ELISA kits. Results: Our findings displayed that Seropositivity rates were 26 % for BVD, 17.4 % for BHV-1, 69.1 % for PI-3V and 75.6 % for BRSV in the sampled population. In addition, coinfections with more than one virus were considerably common among non-vaccinated dairy cattle. Conclusion: These results indicate that exposure to these agents is common within the study areas. Preventive and control measures against these infectious agents should therefore be adopted. [Vet World 2013; 6(1.000: 1-4

Evaluation of bovine viral diarrhea virus in New World camelids.

Science.gov (United States)

Wentz, Philip A; Belknap, Ellen B; Brock, Kenneth V; Collins, James K; Pugh, David G

2003-07-15

To determine the effect of experimental infection with bovine viral diarrhea virus (BVDV) on llamas and their fetuses, evaluate seroprevalence of BVDV in llamas and alpacas, and genetically characterize BVDV isolates from llamas. Prospective study. 4 pregnant llamas for the experimental infection study and 223 llamas and alpacas for the seroprevalence study. Llamas (seronegative to BVDV) were experimentally infected with a llama isolate of BVDV via nasal aerosolization. After inoculation, blood samples were collected every other day for 2 weeks; blood samples were obtained from crias at birth and monthly thereafter. For the seroprevalence study, blood was collected from a convenience sample of 223 camelids. Isolates of BVDV were characterized by reverse transcription-polymerase chain reaction assay. Viremia and BVDV-specific antibody response were detected in the experimentally infected llamas, but no signs of disease were observed. No virus was detected in the crias or aborted fetus, although antibodies were evident in crias after colostrum consumption. Seroprevalence to BVDV was 0.9% in llamas and alpacas. Sequences of the llama BVDV isolates were comparable to known bovine isolates. Findings suggest that llamas may be infected with BVDV but have few or no clinical signs. Inoculation of llamas during gestation did not result in fetal infection or persistent BVDV infection of crias. Seroprevalence to BVDV in llamas and alpacas is apparently low. The most likely source for BVDV infection in camelids may be cattle.

Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

International Nuclear Information System (INIS)

Brock, K.V.

1987-01-01

Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

Evaluation of envelope glycoprotein E(rns) of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus.

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Vijayaraghavan, Balaje; Xia, Hongyan; Harimoorthy, Rajiv; Liu, Lihong; Belák, Sándor

2012-11-01

Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way. Copyright © 2012 Elsevier B.V. All rights reserved.

Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine αV Integrin Utilization by Type A and O Viruses

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Duque, Hernando; Baxt, Barry

2003-01-01

Three members of the αV integrin family of cellular receptors, αVβ1, αVβ3, and αVβ6, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other αV integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the αV integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β1, β5, and β6 integrin subunits. Using these subunits, along with previously cloned bovine αV and β3 subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by αVβ1, αVβ3, αVβ5, and αVβ6 among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used αVβ3 and αVβ6 with relatively high efficiency, while only one virus utilized αVβ1 with moderate efficiency. In contrast, both type O viruses utilized αVβ6 and αVβ1 with higher efficiency than αVβ3. Only low levels of viral replication were detected in αVβ5-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host. PMID:12551988

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.

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

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

2013-01-01

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

Genetic and antigenic relatedness of bovine herpesvirus-1 and pseudorabies virus

International Nuclear Information System (INIS)

Bush, C.E.

1985-01-01

The DNA sequence homology between the genomes of bovine herpesvirus-1 (BHV-1) and pseudorabies virus (PRV) was examined. Reciprocal cross hybridization of viral DNA labeled by nick translation to Southern blots of Kpnl, BamH1, EcoR1, and HindIII restriction endonuclease digested DNA, detected homologous sequences dispersed throughout the genomes of the two viruses. The DNA-DNA hybrids were found to be stable under high stringency wash conditions. Sequences of a 32 P-labeled PRV DNA A fragment probe were found to hybridize only to the BHV-1 HindIII G fragment. This indicated that the sequence homology detected between these two viruses was not simply due to fortuitous hybridization of guanine plus cytosine rich sequences. The homology between BHV-1 and PRV was determined by liquid reassociation. It was found that the hybridization rates between 32 P-labeled PRV DNA and unlabeled BHV-1 DNA and 32 P-labeled BHV-1 DNA and unlabeled PRV DNA corresponded to approximately 8% reassociation. The antigenic relatedness between BHV-1 and PRV was also examined. Eighty percent plaque reduction serum neutralization tests showed that BHV-1 rabbit hyperimmune antiserum neutralized BHV-1 virus at a serum neutralization titer (SNT) of 1:256 and PRV virus at an (SNT) of 1:8. PRV rabbit hyperimmune antiserum neutralized PRV virus at an SNT of 1:4 and BHV-1 virus at an SNT of 1:2

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis

DEFF Research Database (Denmark)

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria

2018-01-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study...... was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR® BCV-Ab and SVANOVIR® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high...

Tunable protease-activatable virus nanonodes.

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

2014-05-27

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

Extensive sequence divergence among bovine respiratory syncytial viruses isolated during recurrent outbreaks in closed herds

DEFF Research Database (Denmark)

Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

2000-01-01

and veal calf production units) in different years and from all confirmed outbreaks in Denmark within a short period. The results showed that identical viruses were isolated within a herd during outbreaks and that viruses from recurrent infections varied by up to 11% in sequence even in closed herds......The nucleotides coding for the extracellular part of the G glycoprotein and the full SH protein of bovine respiratory syncytial virus (BRSV) were sequenced from viruses isolated from numerous outbreaks of BRSV infection. The isolates included viruses isolated from the same herd (closed dairy farms....... It is possible that a quasispecies variant swarm of BRSV persisted in some of the calves in each herd and that a new and different highly fit virus type (master and consensus sequence) became dominant and spread from a single animal in connection with each new outbreak. Based on the high level of diversity...

Lack of evidence for the presence of emerging HoBi-like viruses in North American fetal bovine serum lots.

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The detection of HoBi-like virus in fetal bovine serum (FBS) labeled as United States of America (USA) origin, but packaged in Europe, raised concerns that HoBi-like virus may have entered the USA. In this study, 90 lots of FBS originating in North America (NA) were screened for pestivirus antigen ...

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

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Verhaagen Joost

2010-02-01

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

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

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Nagesh Pulicherla

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

Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus

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Marina Aiello Padilla

2015-01-01

Full Text Available Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV. Two bacterial strains were identified as active, with percentages of inhibition (IP equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection.

Sequences outside that of residues 93-102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells.

Science.gov (United States)

Ma, Xueqing; Li, Pinghua; Bai, Xingwen; Sun, Pu; Bao, Huifang; Lu, Zengjun; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2014-10-13

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. During 2010 and 2011, there was an epidemic of the Mya-98 lineage of the Southeast Asia (SEA) topotype in East Asia, including China. Changes in the FMDV 3A protein have been previously reported to be associated with the inability of FMDV to grow in bovine cells and cause disease in cattle. In this paper, we report the generation of a full-length infectious cDNA clone of FMDV O/SEA/Mya-98 strain O/GZSB/2011 for the first time along with two genetically modified viruses with deletion at positions 93-102 and 133-143 in 3A based on the established infectious clone. All the recombinant viruses grew well and displayed growth properties and plaque phenotypes similar to those of the parental virus in baby hamster kidney (BHK-21) cells, porcine kidney (PK-15) cells, and primary fetal porcine kidney (FPK) cells. While the recombinant viruses rvGZSB and rvSBΔ133-143 exhibited similar growth properties and plaque phenotypes with the parental virus in primary fetal bovine kidney (FBK) cells, the recombinant virus rvSBΔ93-102, containing deletion at positions 93-102 in 3A, grew at a slower rate and had a smaller plaque size phenotype in FBK cells than that of the parental virus. Therefore, the results suggest that the deletion at positions 93-102 of 3A protein does not affect FMDV replication efficiency in BHK-21, PK-15 and FPK cells, but affects virus replication efficiency in FBK cells, although, cannot alone account for the inability to replicate in bovine cells. Copyright © 2014 Elsevier B.V. All rights reserved.

High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

Science.gov (United States)

Erster, Oran; Stram, Rotem; Menasherow, Shopia; Rubistein-Giuni, Marisol; Sharir, Binyamin; Kchinich, Evgeni; Stram, Yehuda

2017-02-02

In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses' geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms. Copyright © 2016. Published by Elsevier B.V.

Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains

DEFF Research Database (Denmark)

Elvander, M.; Vilcek, S.; Baule, C.

1998-01-01

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were...... of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88-100% among BRSV...

Knowledge Gaps Impacting the Development of Bovine Viral Diarrhea Virus Control Programs in the United States

Science.gov (United States)

This paper identifies knowledge gaps that impact on the design of programs to control and or eradicate bovine viral diarrhea viruses (BVDV) in the United States. Currently there are several voluntary regional BVDV control programs in place. These control programs are aimed at the removal of animals ...

Antiviral activity of Petiveria alliacea against the bovine viral diarrhea virus.

Science.gov (United States)

Ruffa, M J; Perusina, M; Alfonso, V; Wagner, M L; Suriano, M; Vicente, C; Campos, R; Cavallaro, L

2002-07-01

Natural products are a relevant source of antiviral drugs. Five medicinal plants used in Argentina have been assayed to detect inhibition of viral growth. Antiviral activity of the infusions and methanolic extracts of Aristolochia macroura, Celtis spinosa, Plantago major, Schinus areira, Petiveria alliacea and four extracts obtained from the leaves and stems of the last plant were evaluated by the plaque assay. P. alliacea, unlike A. macroura, C. spinosa, P. major and S. areira, inhibited bovine viral diarrhea virus (BVDV) replication. Neither P. alliacea nor the assays of the other plants were active against herpes simplex virus type 1, poliovirus type 1, adenovirus serotype 7 and vesicular stomatitis virus type 1. Four extracts of P. alliacea were assayed to detect anti-BVDV activity. Ethyl acetate (EC(50) of 25 microg/ml) and dichloromethane (EC(50) of 43 microg/ml) extracts were active; moreover, promising SI (IC(50)/EC(50)) values were obtained. BVDV is highly prevalent in the cattle population, there are no antiviral compounds available; additionally, it is a viral model of the hepatitis C virus. For these reasons and in view of the results obtained, the isolation and characterization of the antiviral components present in the P. alliacea extracts is worth carrying out in the future. Copyright 2002 S. Karger AG, Basel

Bovine parvovirus uses clathrin-mediated endocytosis for cell entry.

Science.gov (United States)

Dudleenamjil, Enkhmart; Lin, Chin-Yo; Dredge, Devin; Murray, Byron K; Robison, Richard A; Johnson, F Brent

2010-12-01

Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.

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

Science.gov (United States)

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

2016-01-01

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

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

Science.gov (United States)

Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

2009-01-01

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

Bovine respiratory syncytial virus: first serological evidence in Uruguay.

Science.gov (United States)

Costa, M; García, L; Yunus, A S; Rockemann, D D; Samal, S K; Cristina, J

2000-01-01

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves resulting in a substantial economic loss for the cattle industry worldwide. In order to determine the presence of BRSV in Uruguay, an immunoenzymatic test was set up, using a recombinant BRSV nucleocapsid (N) protein as the antigen. The N protein was produced in Sf9 insect cells by a recombinant baculovirus expressing the N protein. Serum samples collected from one hundred cattle from four different geographic regions of Uruguay were analyzed. Antibodies against the N protein of BRSV were detected in 95% of the serum samples analyzed. These results show for the first time the presence of BRSV antibodies and suggest a widespread BRSV infection in the cattle population of Uruguay.

Cytopathic bovine viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction.

Science.gov (United States)

Peterhans, Ernst; Bachofen, Claudia; Stalder, Hanspeter; Schweizer, Matthias

2010-01-01

Bovine viral diarrhea virus (BVDV), a Flaviviridae pestivirus, is arguably one of the most widespread cattle pathogens worldwide. Each of its two genotypes has two biotypes, non-cytopathic (ncp) and cytopathic (cp). Only the ncp biotype of BVDV may establish persistent infection in the fetus when infecting a dam early in gestation, a time point which predates maturity of the adaptive immune system. Such fetuses may develop and be born healthy but remain infected for life. Due to this early initiation of fetal infection and to the expression of interferon antagonistic proteins, persistently infected (PI) animals remain immunotolerant to the infecting viral strain. Although only accounting for some 1% of all animals in regions where BVDV is endemic, PI animals ensure the viral persistence in the host population. These animals may, however, develop the fatal mucosal disease, which is characterized by widespread lesions in the gastrointestinal tract. Cp BVD virus, in addition to the persisting ncp biotype, can be isolated from such animals. The cp viruses are characterized by unrestrained genome replication, and their emergence from the persisting ncp ones is due to mutations that are unique in each virus analyzed. They include recombinations with host cell mRNA, gene translocations and duplications, and point mutations. Cytopathic BVD viruses fail to establish chains of infection and are unable to cause persistent infection. Hence, these viruses illustrate a case of "viral emergence to extinction" - irrelevant for BVDV evolution, but fatal for the PI host. © INRA, EDP Sciences, 2010.

First Results in the Use of Bovine Ear Notch Tag for Bovine Viral Diarrhoea Virus Detection and Genetic Analysis.

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Christian Quinet

Full Text Available Infection due to bovine viral diarrhoea virus (BVDV is endemic in most cattle-producing countries throughout the world. The key elements of a BVDV control programme are biosecurity, elimination of persistently infected animals and surveillance. Bovine viral diarrhoea (BVD is a notifiable disease in Belgium and an official eradication programme started from January 2015, based on testing ear notches sampled during the official identification and registration of calves at birth. An antigen-capture ELISA test based on the detection of BVDV Erns protein is used. Ear notch sample may also be used to characterize the genotype of the calf when appropriate elution/dilution buffer is added. Both BVDV antigen-ELISA analysis and animal traceability could be performed.With regards to the reference protocol used in the preparation of ear notch samples, alternative procedures were tested in terms of BVDV analytic sensitivity, diagnostic sensitivity and specificity, as well as quality and purity of animal DNA.The Allflex DNA Buffer D showed promising results in BVDV diagnosis and genome analyses, opening new perspectives for the livestock industry by the exploitation of the animal genome. Due to the high number of cattle involved in the Belgian official BVDV eradication programme based on ear notch tags sample, a large database on both BVDV status of newborn calves and cattle genome could be created for subsequent different uses (e.g. traceability, determination of parentage, genetic signatures throughout the genome associated with particular traits evolving through a more integrated animal health.

Innate immune responses of calves during transient infection with a noncytopathic strain of bovine viral diarrhea virus

DEFF Research Database (Denmark)

Muller-Doblies, D.; Arquint, A.; Schaller, P.

2004-01-01

In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared...

Role of bovine herpesvirus type 5 (BoHV-5) in diseases of cattle. Recent findings on BoHV-5 association with genital disease

Science.gov (United States)

Favier, P.A.; Marin, M.S.; Pérez, S.E.

2012-01-01

Bovine herpesvirus type 5 (BoHV-5) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. This virus is a major causative agent of non-suppurative meningoencephalitis in young cattle. It was first isolated in 1962 from a neurological disease outbreak in Australia. BoHV-5 is genetically and antigenically related to bovine herpesvirus type 1 (BoHV-1), a highly prevalent virus responsible for respiratory and genital disease in cattle. Initially, BoHV-5 was considered a subtype of BoHV-1 (BoHV-1.3). However, the exclusive presentation of outbreaks of neurological disease suggested that the virus was a new agent with characteristics of neuropathogenicity. Even though both are neurotropic viruses, only BoHV-5 is capable of replicating extensively in the central nervous system and inducing neurological disease. Occasionally, encephalitis caused by BoHV-1 has been reported. Like other alpha-herpesviruses, BoHV-5 can establish latency in nervous ganglia and, by stress factors or glucocorticoid treatment, latent virus can be reactivated. During episodes of reactivation, the virus is excreted in nasal, ocular and genital secretions and transmitted to other susceptible hosts. Recently, BoHV-5 has been associated with infection of the reproductive tract. The virus has been isolated and the presence of viral DNA has been demonstrated in semen samples from Brazil and Australia and natural transmission of the virus through contaminated semen has also been described. Embryos and oocytes are permissive for BoHV-5 infection and BoHV-5 DNA has been detected in the central nervous system of aborted fetuses. The objective of this review is to compile the limited information on the recent association between BoHV-5 and reproductive disorders in cattle. PMID:26623291

Sensitivity and specificity of the AdenoPlus test for diagnosing adenoviral conjunctivitis.

Science.gov (United States)

Sambursky, Robert; Trattler, William; Tauber, Shachar; Starr, Christopher; Friedberg, Murray; Boland, Thomas; McDonald, Marguerite; DellaVecchia, Michael; Luchs, Jodi

2013-01-01

To compare the clinical sensitivity and specificity of the AdenoPlus test with those of both viral cell culture (CC) with confirmatory immunofluorescence assay (IFA) and polymerase chain reaction (PCR) at detecting the presence of adenovirus in tear fluid. A prospective, sequential, masked, multicenter clinical trial enrolled 128 patients presenting with a clinical diagnosis of acute viral conjunctivitis from a combination of 8 private ophthalmology practices and academic centers. Patients were tested with AdenoPlus, CC-IFA, and PCR to detect the presence of adenovirus. The sensitivity and specificity of AdenoPlus were assessed for identifying cases of adenoviral conjunctivitis. Of the 128 patients enrolled, 36 patients' results were found to be positive by either CC-IFA or PCR and 29 patients' results were found to be positive by both CC-IFA and PCR. When compared only with CC-IFA, AdenoPlus showed a sensitivity of 90% (28/31) and specificity of 96% (93/97). When compared only with PCR, AdenoPlus showed a sensitivity of 85% (29/34) and specificity of 98% (89/91). When compared with both CC-IFA and PCR, AdenoPlus showed a sensitivity of 93% (27/29) and specificity of 98% (88/90). When compared with PCR, CC-IFA showed a sensitivity of 85% (29/34) and specificity of 99% (90/91). AdenoPlus is sensitive and specific at detecting adenoviral conjunctivitis. An accurate and rapid in-office test can prevent the misdiagnosis of adenoviral conjunctivitis that leads to the spread of disease, unnecessary antibiotic use, and increased health care costs. Additionally, AdenoPlus may help a clinician make a more informed treatment decision regarding the use of novel therapeutics. clinicaltrials.gov Identifier: NCT00921895.

Inactivation of infectious bovine rhinotracheitis virus by gamma irradiation

International Nuclear Information System (INIS)

Nonomiya, Takashi; Yamashiro, Tomio; Tsutsumi, Takamasa; Ito, Hitoshi; Ishigaki, Isao.

1990-01-01

Radiation inactivation of Infectious Boivne Rhinotracheitis (IBR) virus was investigated by suspending in a commercial preparation medium (c.p.m.) or IBR antibody free serum and irradiated at room temperature or dry ice frozen condition. Normal pooled serum was also analysed by electrophoresis with cellulose acetate membrane after irradiation at frozen and non-frozen condition. The virus inactivation was determined by MDBK cell line which 50 % tissue culture infectious dose (TCID 50 ) was calculated by Behrens Kaerber method. D 10 value at non-frozen condition in serum was obtained as 1.1-1.2 kGy and that in c.p.m. was 1.3-1.4 kGy. On the other hand, D 10 value was increased to 3.4-3.6 kGy in serum and 3.9 kGy in c.p.m. at frozen condition. On the irradiation effect of bovine serum, four peaks of albumin, α, β and γ-globulin fraction were obtained from non-irradiation and irradiated serum up to 2 kGy at non-frozen condition by electrophoresis. More than 4 kGy irradiation, the peaks of globulin fractions became not clear and at more than 8 kGy, changed to one large peak. On the other hand, these changes of electrophoretic patterns were not observed even at 30 kGy irradiation in frozen condition. From these results, necessary dose was decided as 20-25 kGy at frozen condition for inactivation of IBR virus in serum. (author)

Construction of recombinant DNA clone for bovine viral diarrhea virus

International Nuclear Information System (INIS)

Yeo, S.G.; Cho, H.J.; Masri, S.A.

1992-01-01

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

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Sandra Arenhart

Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Spatial patterns of Bovine Corona Virus and Bovine Respiratory Syncytial Virus in the Swedish beef cattle population

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Björkman Camilla

2010-05-01

Full Text Available Abstract Background Both bovine coronavirus (BCV and bovine respiratory syncytial virus (BRSV infections are currently wide-spread in the Swedish dairy cattle population. Surveys of antibody levels in bulk tank milk have shown very high nationwide prevalences of both BCV and BRSV, with large variations between regions. In the Swedish beef cattle population however, no investigations have yet been performed regarding the prevalence and geographical distribution of BCV and BRSV. A cross-sectional serological survey for BCV and BRSV was carried out in Swedish beef cattle to explore any geographical patterns of these infections. Methods Blood samples were collected from 2,763 animals located in 2,137 herds and analyzed for presence of antibodies to BCV and BRSV. Moran's I was calculated to assess spatial autocorrelation, and identification of geographical cluster was performed using spatial scan statistics. Results Animals detected positive to BCV or BRSV were predominately located in the central-western and some southern parts of Sweden. Moran's I indicated global spatial autocorrelation. BCV and BRSV appeared to be spatially related: two areas in southern Sweden (Skaraborg and Skåne had a significantly higher prevalence of BCV (72.5 and 65.5% respectively; almost the same two areas were identified as being high-prevalence clusters for BRSV (69.2 and 66.8% respectively. An area in south-east Sweden (Kronoberg-Blekinge had lower prevalences for both infections than expected (23.8 and 20.7% for BCV and BRSV respectively. Another area in middle-west Sweden (Värmland-Dalarna had also a lower prevalence for BRSV (7.9%. Areas with beef herd density > 10 per 100 km2 were found to be at significantly higher risk of being part of high-prevalence clusters. Conclusion These results form a basis for further investigations of between-herds dynamics and risk factors for these infections in order to design effective control strategies.

Bovine aortic arch: A novel association with thoracic aortic dilation

International Nuclear Information System (INIS)

Malone, C.D.; Urbania, T.H.; Crook, S.E.S.; Hope, M.D.

2012-01-01

Aim: To investigate whether there is a link between bovine arch and thoracic aortic aneurysm. Materials and methods: Computed tomography (CT) and magnetic resonance imaging (MRI) images of the thorax of 191 patients with dilated thoracic aortas and 391 consecutive, unselected patients as controls were retrospectively reviewed. Bovine arch was considered present if either a shared origin of the left common carotid and innominate arteries or an origin of the left common carotid from the innominate artery was identified. A chi-square test was used to evaluate the significance of differences between subgroups. Results: A trend towards increased prevalence of bovine arch was seen in patients with dilated aortas (26.2%) compared to controls (20.5%, p = 0.12). The association was statistically significant in patients over 70 years old (31.9%, p = 0.019) and when dilation involved the aortic arch (47.6%, p = 0.003). Conclusions: An association between bovine arch and aortic dilation is seen in older patients, and when dilation involves the aortic arch. Bovine arch should be considered a potential risk factor for thoracic aortic aneurysm.

Isolation and identification of a bovine viral diarrhea virus from sika deer in china

OpenAIRE

Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; Zhang, Pengju; Zhang, Lianxue

2011-01-01

Abstract Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical...

Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

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Arainga Mariluz

2012-03-01

Full Text Available Abstract Background Bovine leukemia virus (BLV is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G or reduced (TaxS240P transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting

Using a Herd Profile to Determine Age-Specific Prevalence of Bovine Leukemia Virus in Michigan Dairy Herds

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Ronald J. Erskine

2012-01-01

Full Text Available Enzootic bovine leukosis is a contagious disease of cattle caused by the retrovirus, bovine leukemia virus (BLV and is the most common cause of malignant neoplasm in cattle. In order to facilitate surveillance of this disease in dairy herds, we developed a method to combine ELISA of milk collected during routine production testing with a prescribed sampling of cows that is independent of the proportion of cows within each lactation. In 113 Michigan dairy herds, milk samples from ten cows in each of the 1st, 2nd, 3rd, and ≥4th lactations were analyzed for anti-Bovine Leukemia Virus (BLV antibodies by milk ELISA. For each herd, a BLV herd profile (BHP was calculated as the simple average of the percent of BLV-positive cows within each of the four lactation groups. The mean BHP for all herds was 32.8%, with means of 18.5, 28.8, 39.2, and 44.8% of 1st, 2nd, 3rd, and ≥4th lactation animals infected, respectively. In eight herds, we determined the correlation between the BHP, and true herd prevalence by testing the entire lactating herd (r=0.988,  P<0.0001. The BHP allows discrimination of lactation-specific BLV prevalence within a dairy herd, to help identify risk factors and management plans that may be important in transmission of BLV.

Inactivation of infectious bovine rhinotracheitis virus by gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Nonomiya, Takashi; Yamashiro, Tomio; Tsutsumi, Takamasa (Animal Quarantine Service, Yokohama (Japan)); Ito, Hitoshi; Ishigaki, Isao

1990-10-01

Radiation inactivation of Infectious Boivne Rhinotracheitis (IBR) virus was investigated by suspending in a commercial preparation medium (c.p.m.) or IBR antibody free serum and irradiated at room temperature or dry ice frozen condition. Normal pooled serum was also analysed by electrophoresis with cellulose acetate membrane after irradiation at frozen and non-frozen condition. The virus inactivation was determined by MDBK cell line which 50 % tissue culture infectious dose (TCID{sub 50}) was calculated by Behrens Kaerber method. D{sub 10} value at non-frozen condition in serum was obtained as 1.1-1.2 kGy and that in c.p.m. was 1.3-1.4 kGy. On the other hand, D{sub 10} value was increased to 3.4-3.6 kGy in serum and 3.9 kGy in c.p.m. at frozen condition. On the irradiation effect of bovine serum, four peaks of albumin, {alpha}, {beta} and {gamma}-globulin fraction were obtained from non-irradiation and irradiated serum up to 2 kGy at non-frozen condition by electrophoresis. More than 4 kGy irradiation, the peaks of globulin fractions became not clear and at more than 8 kGy, changed to one large peak. On the other hand, these changes of electrophoretic patterns were not observed even at 30 kGy irradiation in frozen condition. From these results, necessary dose was decided as 20-25 kGy at frozen condition for inactivation of IBR virus in serum. (author).

Evaluation of bovine coronavirus antibody levels, virus shedding, and respiratory disease incidence throughout the beef cattle production cycle

Science.gov (United States)

Objective- Determine how levels of serum antibody to bovine coronavirus (BCV) are related to virus shedding patterns and respiratory disease incidence in beef calves at various production stages. Animals- 890 crossbred beef calves from four separately managed herds at the U.S. Meat Animal Research C...

Ultraviolet-C irradiation for inactivation of viruses in foetal bovine serum.

Science.gov (United States)

Vaidya, Vivek; Dhere, Rajeev; Agnihotri, Snehal; Muley, Ravindra; Patil, Sanjay; Pawar, Amit

2018-07-05

Foetal Bovine Serum (FBS) and porcine trypsin are one of the essential raw materials used in the manufacturing of cell culture based viral vaccines. Being from animal origin, these raw materials can potentially contaminate the final product by known or unknown adventitious agents. The issue is more serious in case of live attenuated viral vaccines, where there is no inactivation step which can take care of such adventitious agents. It is essential to design production processes which can offer maximum viral clearance potential for animal origin products. Ultraviolet-C irradiation is known to inactivate various adventitious viral agents; however there are limited studies on ultraviolet inactivation of viruses in liquid media. We obtained a recently developed UVivatec ultraviolet-C (UV-C) irradiation based viral clearance system for evaluating its efficacy to inactivate selected model viruses. This system has a unique design with spiral path of liquid allowing maximum exposure to UV-C light of a short wavelength of 254 nm. Five live attenuated vaccine viruses and four other model viruses were spiked in tissue culture media and exposed to UV-C irradiation. The pre and post UV-C irradiation samples were analyzed for virus content to find out the extent of inactivation of various viruses. These experiments showed substantial log reduction for the majority of the viruses with few exceptions based on the characteristics of these viruses. Having known the effect of UV irradiation on protein structure, we also evaluated the post irradiation samples of culture media for growth promoting properties using one of the most fastidious human diploid cells (MRC-5). UV-C exposure did not show any notable impact on the nutritional properties of culture media. The use of an UV-C irradiation based system is considered to be promising approach to mitigate the risk of adventitious agents in cell culture media arising through animal derived products. Copyright © 2018 Elsevier Ltd. All

Bovine respiratory syncytial virus (BRSV) pneumonia in beef calf herds despite vaccination

DEFF Research Database (Denmark)

Larsen, Lars Erik; Tegtmeier, C.; Pedersen, E.

2001-01-01

to the outbreak. The clinical signs comprised nasal discharge, pyrexia, cough and increased respiratory rates. A total of 28 calves died in the 2 herds. The laboratory investigations revealed that BRSV was involved and probably initiated both outbreaks. Furthermore, the serological results suggested...... beef herds failed to protect the calves against severe or even fatal BRSV mediated respiratory disease 2 months later.......The present report describes the clinical, pathological, serological and virological findings in calves from 2 larger Danish beef herds experiencing outbreaks of pneumonia. The calves had been vaccinated with an inactivated bovine respiratory syncytial virus (BRSV) vaccine 2 months prior...

A stochastic model for simulation of the economic consequences of bovine virus diarrhoea virus infection in a dairy herd

DEFF Research Database (Denmark)

Sørensen, J.T.; Enevoldsen, Carsten; Houe, H.

1995-01-01

A dynamic, stochastic model simulating the technical and economic consequences of bovine virus diarrhoea virus (BVDV) infections for a dairy cattle herd for use on a personal computer was developed. The production and state changes of the herd were simulated by state changes of the individual cows...... and heifers. All discrete events at the cow level were triggered stochastically. Each cow and heifer was characterized by state variables such as stage of lactation, parity, oestrous status, decision for culling, milk production potential, and immune status for BVDV. The model was controlled by 170 decision...... variables describing biologic and management variables including 21 decision variables describing the effect of BVDV infection on the production of the individual animal. Two markedly different scenarios were simulated to demonstrate the behaviour of the developed model and the potentials of the applied...

Association between the level of antibodies in bulk tank milk and bovine respiratory syncytial virus exposure in the herd.

Science.gov (United States)

Klem, T B; Tollersrud, T; Osterås, O; Stokstad, M

2014-07-12

Antibody levels in bulk tank milk (BTM) against bovine respiratory syncytial virus (BRSV) are used to classify BRSV status of herds. The aim of this study was to investigate how these levels correspond with the time at which the herds were infected. Bulk tank milk, individual milk and serum samples from cows and young stock were investigated using an indirect ELISA. Screenings of BTM from 89 dairy herds during two winter seasons revealed a prevalence of positive herds from 82 per cent to 85 per cent. Eleven herds showed a marked increase in antibody levels between two screenings, indicating new infection. However, two of these herds had been free from BRSV for the last five to seven years. Two newly infected herds were monitored for four years and did not appear to get reinfected. Surprisingly, the BTM antibody levels in these herds remained high throughout the study period, but fluctuated significantly. This shows that the levels of antibodies in BTM can remain high for several years, even in herds where reinfection does not occur. BTM serology is a useful tool in the monitoring of infectious diseases in dairy herds, but has limitations as a diagnostic tool for BRSV infections. British Veterinary Association.

Bovine viral diarrhea virus (BVDV) genetic diversity in Spain: A review

International Nuclear Information System (INIS)

Diéguez, F.J.; Cerviño, M.; Yus, E.

2017-01-01

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus of the family Flaviviridae, causes significant losses in cattle farming worldwide because of reduced milk production, increased mortality of young animals and reproductive, respiratory and intestinal problems. The virus is characterized by an important genetic, and consequently antigenic and pathogenic diversity. Knowing the variability of viral strains present in a population provides valuable information, particularly relevant for control programs development, vaccination recommendations and even identification of likely infection sources. Such information is therefore important at both local and regional levels. This review focuses on the genetic diversity of BVDV isolates infecting cattle in Spain over the last years. According to the published data, the most prevalent BVDV group in Spain was 1b, and to a lesser extent 1d, 1e and 1f. Besides, BVDV-2 has also been found in Spain with several ratified isolates. The studies carried out in Spain also showed increased genetic heterogeneity of BVDV strains, possibly due to a more intensive use of analytical tools available, presenting studies with increasingly greater sample sizes.

Bovine viral diarrhea virus (BVDV) genetic diversity in Spain: A review

Energy Technology Data Exchange (ETDEWEB)

Diéguez, F.J.; Cerviño, M.; Yus, E.

2017-07-01

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus of the family Flaviviridae, causes significant losses in cattle farming worldwide because of reduced milk production, increased mortality of young animals and reproductive, respiratory and intestinal problems. The virus is characterized by an important genetic, and consequently antigenic and pathogenic diversity. Knowing the variability of viral strains present in a population provides valuable information, particularly relevant for control programs development, vaccination recommendations and even identification of likely infection sources. Such information is therefore important at both local and regional levels. This review focuses on the genetic diversity of BVDV isolates infecting cattle in Spain over the last years. According to the published data, the most prevalent BVDV group in Spain was 1b, and to a lesser extent 1d, 1e and 1f. Besides, BVDV-2 has also been found in Spain with several ratified isolates. The studies carried out in Spain also showed increased genetic heterogeneity of BVDV strains, possibly due to a more intensive use of analytical tools available, presenting studies with increasingly greater sample sizes.

Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep

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Burny Arsène

2007-07-01

Full Text Available Abstract Background During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts. Results In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303 replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303 had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset. Conclusion Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression

Clinical report: Detection and management of bovine viral diarrhea virus Type 1b in a large dairy herd

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Case Description: 1,081 newborn calves from a commercial dairy were tested for bovine viral diarrhea virus antigen by pooled RT-PCR as part of a screening program. Ear tissue from twenty six calves initially tested positive and 14 confirmed positive with antigen capture ELISA two weeks later (1.3...

Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV E2 protein.

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Tiansen Li

Full Text Available Bovine viral diarrhea-mucosal disease (BVD-MD is caused by bovine viral diarrhea virus (BVDV, and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3, and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.

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Li, Tiansen; Huang, Meiling; Xiao, Hongran; Zhang, Guoqi; Ding, Jinhua; Wu, Peng; Zhang, Hui; Sheng, Jinliang; Chen, Chuangfu

2017-01-01

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Genetic diversity of bovine viral diarrhoea viruses (BVDV) in Denmark during a 10-year eradication period

DEFF Research Database (Denmark)

Uttenthal, Åse; Stadejek, T.; Nylin, B.

2005-01-01

A 243 base-pair fragment of the 5'- untranslated region (5'-UTR) of bovine viral diarrhoea virus (BVDV) was RT-PCR amplified from tissue samples (after one passage) or from plasma collected from Danish cattle in 1962 (1), 1993 (7), or in 2002-03 (28) when BVD was almost extinct as a result of a 6...... subtype, the samples collected in 2002-2003 belonged to Id (22 samples), 1b (5 samples) and le (I sample) subtypes. In five herds, materials from two animals were obtained for PCR analysis. In four of five herds the sequences of the two viruses were identical, but in one herd the obtained sequences...

Short communication. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV) isolates in Kosovo

OpenAIRE

Izedin Goga; Kristaq Berxholi; Beqe Hulaj; Driton Sylejmani; Boris Yakobson; Yehuda Stram

2014-01-01

Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV) in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of B...

Generation of the Bovine Viral Diarrhea Virus E0 Protein in Transgenic Astragalus and Its Immunogenicity in Sika Deer

OpenAIRE

Gao, Yugang; Zhao, Xueliang; Zang, Pu; Liu, Qun; Wei, Gongqing; Zhang, Lianxue

2014-01-01

The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in tr...

Effects of gastrointestinal parasites on parasite burden, rectal temperature, and antibody titer responses to vaccination and infectious bovine rhinotracheitis virus challenge.

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Schutz, J S; Carroll, J A; Gasbarre, L C; Shelton, T A; Nordstrom, S T; Hutcheson, J P; Van Campen, H; Engle, T E

2012-06-01

Thirty-three colostrum-deprived Holstein bull calves (initial BW of 131 ± 4 kg) were used to determine the effect of timing of anthelmintic administration relative to vaccination on antibody titer response to vaccine component antigens. When calves were at least 3 mo of age, they were sorted randomly into individual pens and assigned to 1 of 3 treatment groups, treatments consisted of 1) dewormed 2 wk before vaccination (DPV), 2) dewormed at the time of vaccination (DV), or 3) control, vaccinated but not dewormed (CONT). All calves were inoculated with infective larvae of brown stomach worms (Ostertagia ostertagi) and intestinal worms (Cooperia spp.) on d 1, 7, 10, 14, and 18 for a total dose of 235,710 infective larvae per calf. Calves (DPV and DV) were dewormed on d 21 or 35 with a 10% fenbendazole suspension at 5 mg/kg of BW. On d 35, all calves were vaccinated with a modified-live virus respiratory vaccine containing IBRV (infectious bovine rhinotracheitis virus), BVDV-1 (bovine viral diarrhea virus genotype 1), BVDV-2 (BVDV genotype 2), PI-3 (parainfluenza-3), and BRSV (bovine respiratory syncytial virus). During the 103-d experiment, weekly fecal egg counts, blood, and rectal temperatures were collected and health status was recorded daily. Blood samples were obtained weekly to determine serum neutralizing (SN) antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 and cytokine levels for IL-4, IL-6, TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-gamma). There was a tendency (P parasite burden and decreased rectal temperature increase after an IBRV challenge. Deworming strategy had no effect on antibody response to vaccination or IBRV challenge.

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

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Maria Schnödt

2016-01-01

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

Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton, via the non-structural protein 3Cpro

DEFF Research Database (Denmark)

Armer, Hannah; Moffat, Katy; Wileman, Thomas

2008-01-01

Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus r....... In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect -tubulin distribution, and the microtubule network remained relatively unaffected....

Reactivity of some mammalian sera with the bovine leukaemia virus env gene polypeptide expressed in Escherichia coli

International Nuclear Information System (INIS)

Slavikova, K.; Zajac, V.

1989-01-01

Sera from bovine leukaemia virus (BLV)-infected cattle and sheep were tested by radioimmunoassay and Western blot for their reactivity with 60,000 protein coded by the env gene of BLV and expressed in Escherichia coli. This protein, antigenically similar to BLV protein, reacted with antibodies against BLV antigens in the sera tested. (author). 3 figs., 1 tab., 13 refs

Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens

International Nuclear Information System (INIS)

Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.

1987-01-01

Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125 I. The 125 I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography

What variables are important in predicting bovine viral diarrhea virus? A random forest approach.

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Machado, Gustavo; Mendoza, Mariana Recamonde; Corbellini, Luis Gustavo

2015-07-24

Bovine viral diarrhea virus (BVDV) causes one of the most economically important diseases in cattle, and the virus is found worldwide. A better understanding of the disease associated factors is a crucial step towards the definition of strategies for control and eradication. In this study we trained a random forest (RF) prediction model and performed variable importance analysis to identify factors associated with BVDV occurrence. In addition, we assessed the influence of features selection on RF performance and evaluated its predictive power relative to other popular classifiers and to logistic regression. We found that RF classification model resulted in an average error rate of 32.03% for the negative class (negative for BVDV) and 36.78% for the positive class (positive for BVDV).The RF model presented area under the ROC curve equal to 0.702. Variable importance analysis revealed that important predictors of BVDV occurrence were: a) who inseminates the animals, b) number of neighboring farms that have cattle and c) rectal palpation performed routinely. Our results suggest that the use of machine learning algorithms, especially RF, is a promising methodology for the analysis of cross-sectional studies, presenting a satisfactory predictive power and the ability to identify predictors that represent potential risk factors for BVDV investigation. We examined classical predictors and found some new and hard to control practices that may lead to the spread of this disease within and among farms, mainly regarding poor or neglected reproduction management, which should be considered for disease control and eradication.

Coinfections of Sudanese dairy cattle with bovine herpes virus 1, bovine viral diarrhea virus, bluetongue virus and bovine herpes virus 4 and their relation to reproductive disorders

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Amira M. Elhassan

2016-12-01

Reults: The meta-analysis of the data indicated high seroprevalence of coinfections with various combinations of these agents; only few animals were singly infected. An infection with BHV-1 was observed to be higher than the prevalence of associations between BHV-1 and the other three viral agents. Prevalence of seropositivities to coinfection with BHV-1/BTV; BHV-1/BVD; BHV-1/BTV/BVD were the highest while seropositivities prevalences that involved BHV-4 were much lower. The highest abortion rates were encountered in coinfections with BHV-1/BVD/BTV (31% and BHV-1/BVD/BTV/BHV-4 (30% while most infertility cases were noticed in coinfection with BHV-1/BVD/BTV (44% and BHV-1/BVD/BTV/BHV-4 (21%, and coinfections with the four viruses were encountered in most of the death after birth cases (25%. Overall mixed infections with BHV-1/BVD/BTV (34% and BHV-1/BVD/BTV/BHV-4 (22.5% were involved in the majority of reproductive problems studied. Conclusion: Mixed infections constitutes the vast majority of cases and are involved in the majority of reproductive disorders investigated. The high prevalence of seropositivity to all of the four viruses should call for an intervention strategy to reduce the impact of these viruses. [J Adv Vet Anim Res 2016; 3(4.000: 332-337

Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model

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Atkinson Mark A

2011-02-01

Full Text Available Abstract Background Alpha-1 antitrypsin (AAT is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA, a mouse model of rheumatoid arthritis (RA. Methods DBA/1 mice were immunized with bovine type II collagen (bCII to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT. Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF, antibodies against both bovine (bCII and mouse collagen II (mCII were tested by ELISA. Results Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially. Conclusion These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

Detection of an untyped strain of bovine respiratory syncytial virus in a dairy herd

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Ingrid Bortolin Affonso

2014-10-01

Full Text Available Bovine respiratory syncytial virus (BRSV causes important lower respiratory tract illness in calves. According to F and G proteins genetic sequences, three BRSV subgroups have been reported and characterized in several countries, showing differences in its distribution. In Brazil, the virus is widely disseminated throughout the herds and the few characterized isolates revealed the solely occurrence of the subgroup B. This study describes the detection and characterization of an untyped BRSV strain from a twenty-days-old calf from a herd without clinical respiratory disease. Nasal swabs were analyzed by RT-nested PCR for the F and G proteins genes. One sample has amplified the F protein gene. Sequencing and subsequent phylogenetic reconstruction were accomplished, revealing that the strain could not be grouped with any other BRSV subgroups reported. This result may suggest that the BRSV is in constantly evolution, even in Brazil, where the vaccination is not a common practice. More detailed studies about BRSV characterization are necessary to know the virus subgroups distribution among the Brazilian herds to recommend appropriated immunoprophylaxis.

New hematological key for bovine leukemia virus-infected Japanese Black cattle.

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Mekata, Hirohisa; Yamamoto, Mari; Kirino, Yumi; Sekiguchi, Satoshi; Konnai, Satoru; Horii, Yoichiro; Norimine, Junzo

2018-02-20

The European Community's (EC) Key, which is also called Bendixen's Key, is a well-established bovine leukemia virus (BLV) diagnostic method that classifies cattle according to the absolute lymphocyte count and age. The EC Key was originally designed for dairy cattle and is not necessarily suitable for Japanese Black (JB) beef cattle. This study revealed the lymphocyte counts in the BLV-free and -infected JB cattle were significantly lower than those in the Holstein cattle. Therefore, applying the EC Key to JB cattle could result in a large number of undetected BLV-infected cattle. Our proposed hematological key, which was designed for JB cattle, improves the detection of BLV-infected cattle by approximately 20%. We believe that this study could help promote BLV control.

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

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

2013-01-01

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

Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

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Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

2016-12-15

We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

International Nuclear Information System (INIS)

Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

2016-01-01

We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

Bovine aortic endothelial cells are susceptible to Hantaan virus infection

International Nuclear Information System (INIS)

Bahr, U.; Muranyi, W.; Mueller, S.; Kehm, R.; Handermann, M.; Darai, G.; Zeier, M.

2004-01-01

Hantavirus serotype Hantaan (HTN) is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS, lethality up to 10%). The natural host of HTN is Apodemus agrarius. Recent studies have shown that domestic animals like cattle are sporadically seropositive for hantaviruses. In the present study, the susceptibility of bovine aortic endothelial cells (BAEC) expressing α V β 3 -integrin to a HTN infection was investigated. Viral nucleocapsid protein and genomic RNA segments were detected in infected BAEC by indirect immunofluorescence assay, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The results of this study strongly support our previous observation on Puumala virus (PUU) that has been propagated efficiently in BAEC. These findings open a new window to contemplate the ecology of hantavirus infection and transmission route from animal to man

Bovine viral diarrhea virus (BVDV genetic diversity in Spain: A review

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Francisco J. Diéguez

2017-07-01

Full Text Available Bovine viral diarrhea virus (BVDV, a member of the genus Pestivirus of the family Flaviviridae, causes significant losses in cattle farming worldwide because of reduced milk production, increased mortality of young animals and reproductive, respiratory and intestinal problems. The virus is characterized by an important genetic, and consequently antigenic and pathogenic diversity. Knowing the variability of viral strains present in a population provides valuable information, particularly relevant for control programs development, vaccination recommendations and even identification of likely infection sources. Such information is therefore important at both local and regional levels. This review focuses on the genetic diversity of BVDV isolates infecting cattle in Spain over the last years. According to the published data, the most prevalent BVDV group in Spain was 1b, and to a lesser extent 1d, 1e and 1f. Besides, BVDV-2 has also been found in Spain with several ratified isolates. The studies carried out in Spain also showed increased genetic heterogeneity of BVDV strains, possibly due to a more intensive use of analytical tools available, presenting studies with increasingly greater sample sizes.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

A Novel Genetic Group of Bovine Hepacivirus in Archival Serum Samples from Brazilian Cattle

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Cláudio W. Canal

2017-01-01

Full Text Available Hepatitis C virus (HCV (genus Hepacivirus; family Flaviviridae is a major human pathogen causing persistent infection and hepatic injury. Recently, emerging HCV-like viruses were described infecting wild animals, such as bats and rodents, and domestic animals, including dogs, horses, and cattle. Using degenerate primers for detecting bovine pestiviruses in a 1996 survey three bovine serum samples showed a low identity with the genus Pestivirus of the Flaviviridae family. A virus could not be isolated in cell culture. The description of bovine hepaciviruses (BovHepV in 2015 allowed us to retrospectively identify the sequences as BovHepV, with a 88.9% nucleotide identity. In a reconstructed phylogenetic tree, the Brazilian BovHepV samples grouped within the bovine HCV-like cluster in a separated terminal node that was more closely related to the putative bovine Hepacivirus common ancestor than to bovine hepaciviruses detected in Europe and Africa.

Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

International Nuclear Information System (INIS)

Fiebach, H.; Uckert, W.; Micheel, B.

1982-01-01

Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle. (author)

Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

Energy Technology Data Exchange (ETDEWEB)

Fiebach, H.; Uckert, W.; Micheel, B. (Akademie der Wissenschaften der DDR, Berlin. Zentralinstitut fuer Krebsforschung)

Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle.

Antigenic variability in bovine viral diarrhea virus (BVDV) isolates from alpaca (Vicugna pacos), llama (Lama glama) and bovines in Chile.

Science.gov (United States)

Aguirre, I M; Quezada, M P; Celedón, M O

2014-01-31

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities where they have been introduced worldwide. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus and mainly to bovine viral diarrhea virus (BVDV). Seventeen Chilean BVDV isolates were analyzed by serum cross neutralization with samples obtained from five llama, six alpacas, three bovines, plus three reference strains belonging to different subgroups and genotypes. The objective was to describe antigenic differences and similarities among them. Antigenic comparison showed significant differences between different subgroups. Consequently, antigenic similarities were observed among isolates belonging to the same subgroup and also between isolates from different animal species belonging the same subgroup. Among the analyzed samples, one pair of 1b subgroup isolates showed significant antigenic differences. On the other hand, one pair of isolates from different subgroups (1b and 1j) shared antigenic similarities indicating antigenic relatedness. This study shows for the first time the presence of antigenic differences within BVDV 1b subgroup and antigenic similarities within 1j subgroup isolates, demonstrating that genetic differences within BVDV subgroups do not necessary corresponds to differences on antigenicity. Copyright © 2013 Elsevier B.V. All rights reserved.

Seroprevalence of viral and bacterial diseases among the bovines in Himachal Pradesh, India

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Shailja Katoch

2017-12-01

Full Text Available Aim: The study was designed to measure the seroprevalence of viral and bacterial diseases: Infectious bovine rhinotracheitis, bovine viral diarrhea, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, brucellosis, and paratuberculosis among bovine of Himachal Pradesh during the year 2013-2015. Materials and Methods: The serum samples were collected from seven districts of state, namely, Bilaspur, Kangra, Kinnaur, Lahul and Spiti, Mandi, Sirmour, and Solan. The samples were screened using indirect ELISA kits to measure the seroprevalence of viral and bacterial diseases. Results: The overall seroprevalence of infectious bovine rhinotracheitis was 24.24%, bovine viral diarrhea 1.52%, bovine leukemia 9.09%, bovine parainfluenza 57.58%, bovine respiratory syncytial disease 50%, brucellosis 19.69%, and paratuberculosis 9.09% in Himachal Pradesh. The seroprevalence of bovine rhinotracheitis, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, and paratuberculosis in the state varied significantly (p0.01. Multiple seropositivity has been observed in this study. Bovine parainfluenza virus 3 was observed commonly in mixed infection with almost all viruses and bacteria under study. Conclusion: The viral and bacterial diseases are prevalent in the seven districts of Himachal Pradesh investigated in the study. Therefore, appropriate management practices and routine vaccination programs should be adopted to reduce the prevalence of these diseases.

Inactivation of RNA viruses by gamma irradiation

International Nuclear Information System (INIS)

Nonomiya, Takashi; Morimoto, Akinori; Iwatsuki, Kazuo; Tsutsumi, Takamasa; Ito, Hitoshi; Yamashiro, Tomio; Ishigaki, Isao.

1992-01-01

Four kinds of RNA viruses, Bluetongue virus (BT), Bovine Virus Diarrhea-Mucosal Disease virus (BVD·MD), Bovine Respiratory Syncytial virus (RS), Vesicular Stmatitis virus (VS), were subjected to various doses of gamma irradiation to determine the lethal doses. The D 10 values, which are the dose necessary to decimally reduce infectivity, ranged from 1.5 to 3.4 kGy under frozen condition at dry-ice temperature, and they increased to 2.6 to 5.0 kGy under frozen condition at dry-ice temperature. Serum neutralzing antibody titer of Infectious Bovine Rhinotracheitis (IBR) was not adversely changed by the exposure to 36 kGy of gamma-rays under frozen condition. Analysis of electrophoresis patterns of the bovine serum also reveales that the serum proteins were not remarkably affected, even when exposed to 36 kGy of gamma radiation under frozen condition. The results suggested that gamma irradiation under frozen condition is an effective means for inactivating both DNA and RNA viruses without adversely affecting serum proteins and neutralizing antibody titer. (author)

Inactivation of RNA viruses by gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Nonomiya, Takashi; Morimoto, Akinori; Iwatsuki, Kazuo; Tsutsumi, Takamasa (Ministry of Agriculture, Forestry and fisheries, Yokohama, Kanagawa (Japan). Animal Quarantine Service); Ito, Hitoshi; Yamashiro, Tomio; Ishigaki, Isao

1992-09-01

Four kinds of RNA viruses, Bluetongue virus (BT), Bovine Virus Diarrhea-Mucosal Disease virus (BVD[center dot]MD), Bovine Respiratory Syncytial virus (RS), Vesicular Stmatitis virus (VS), were subjected to various doses of gamma irradiation to determine the lethal doses. The D[sub 10] values, which are the dose necessary to decimally reduce infectivity, ranged from 1.5 to 3.4 kGy under frozen condition at dry-ice temperature, and they increased to 2.6 to 5.0 kGy under frozen condition at dry-ice temperature. Serum neutralzing antibody titer of Infectious Bovine Rhinotracheitis (IBR) was not adversely changed by the exposure to 36 kGy of gamma-rays under frozen condition. Analysis of electrophoresis patterns of the bovine serum also reveales that the serum proteins were not remarkably affected, even when exposed to 36 kGy of gamma radiation under frozen condition. The results suggested that gamma irradiation under frozen condition is an effective means for inactivating both DNA and RNA viruses without adversely affecting serum proteins and neutralizing antibody titer. (author).

Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2.

Science.gov (United States)

Walz, Paul H; Riddell, Kay P; Newcomer, Benjamin W; Neill, John D; Falkenberg, Shollie M; Cortese, Victor S; Scruggs, Daniel W; Short, Thomas H

2018-04-23

Bovine viral diarrhea virus (BVDV) is an important viral cause of reproductive disease, immune suppression and clinical disease in cattle. The objective of this study was to compare reproductive protection in cattle against the impacts of bovine viral diarrhea virus (BVDV) provided by three different multivalent vaccines containing inactivated BVDV. BVDV negative beef heifers and cows (n = 122) were randomly assigned to one of four groups. Groups A-C (n = 34/group) received two pre-breeding doses of one of three commercially available multivalent vaccines containing inactivated fractions of BVDV 1 and BVDV 2, and Group D (n = 20) served as negative control and received two doses of saline prior to breeding. Animals were bred, and following pregnancy diagnosis, 110 cattle [Group A (n = 31); Group B (n = 32); Group C (n = 31); Group D (n = 16)] were subjected to a 28-day exposure to cattle persistently infected (PI) with BVDV (1a, 1b and 2a). Of the 110 pregnancies, 6 pregnancies resulted in fetal resorption with no material for testing. From the resultant 104 pregnancies, BVDV transplacental infections were demonstrated in 73 pregnancies. The BVDV fetal infection rate (FI) was calculated at 13/30 (43%) for Group A cows, 27/29 (93%) for Group B cows, 18/30 (60%) for Group C cows, and 15/15 (100%) for Group D cows. Statistical differences were observed between groups with respect to post-vaccination antibody titers, presence and duration of viremia in pregnant cattle, and fetal infection rates in offspring from BVDV-exposed cows. Group A vaccination resulted in significant protection against BVDV infection as compared to all other groups based upon outcome measurements, while Group B vaccination did not differ in protection against BVDV infection from control Group D. Ability of inactivated BVDV vaccines to provide protection against BVDV fetal infection varies significantly among commercially available products; however, in this challenge

Development and application of radioimmunoassay and enzyme immunoassays in microbiological and immunological diagnosis. 3. Comparative studies for the detection of virus antibodies with passive hemagglutination test, radioimmunoassay and enzyme immunoassay, resp

Energy Technology Data Exchange (ETDEWEB)

Lauf, H; Struy, H; Morenz, J [Medizinische Akademie, Magdeburg (German Democratic Republic)

1982-06-01

Radioimmuno- and enzyme immunoassays (solid phase RIA and ELISA) developed by the authors for the determination of antibodies of adeno-2- and parainfluenza-1-viruses are described and the detection sensibility for antibodies is compared with that of the conventional passive hemagglutination test. The sensibility of the radioimmunoassay for the detection of IgG antibodies against adeno-2-viruses is nearly 10 times higher than that of the passive hemagglutination. RIA and ELISA show no essential differences in their detection sensibilities in the detection of IgG antibodies against parainfluenza-1-viruses.

Effects of bovine leukemia virus infection on crossbred and purebred dairy cattle productive performance in Brazil

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Daniela Souza Rajão

2014-02-01

Full Text Available The aim of this study was to evaluate the effects of bovine leukemia virus (BLV infection on productive performance of dairy cattle in Brazil. A total of 158 blood samples from lactating adult cows, purebred Holstein and crossbred Holstein X Zebu, were analyzed by Agar Gel Immunodifusion Test (AGID and leukogram. According to AGID and leukogram results, animals were grouped into three categories: seronegative, seropositive without persistent lymphocytosis, and seropositive with persistent lymphocytosis. Milk production data were compared between groups, according to breed. BLV infected females showed lower milk yield than uninfected ones, both purebred and crossbred ones. There was no difference between milk yield of seropositive cows with or without persistent lymphocytosis. These results indicate an association between BLV infection and reduction of milk production, and this study is the first one to show these effects in crossbred Holstein X Zebu cows.

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.

Synthetic analogues of bovine bactenecin dodecapeptide reduce herpes simplex virus type 2 infectivity in mice

DEFF Research Database (Denmark)

Jenssen, Håvard; Shestakov, Andrey; Hancock, Robert E. W

2013-01-01

We have evaluated the potential of four synthetic peptides (denoted HH-2, 1002, 1006, 1018) with a distant relationship to the host defense peptide bovine bactenecin dodecapeptide for their ability to prevent genital infections with herpes simplex virus type 2 (HSV-2) in mice. All four peptides...... infectious doses of HSV-2. These data show that peptides HH-2 and 1018 have antiviral properties and can be used to prevent genital herpes infection in mice. (C) 2013 Elsevier B.V. All rights reserved....... was introduced in human semen. Two of the peptides proved especially effective in reducing HSV-2 infection also in vivo. When admixed with virus prior to inoculation, both HH-2 and 1018 reduced viral replication and disease development in a genital model of HSV-2 infection in mice, and also when using very high...

Detección del virus de la leucosis bovina en ganado criollo colombiano mediante PCR-anidado Bovine leukemia virus detection in Creole Colombian breeds using nested-PCR

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Darwin Yovanny Hernández-Herrera

2011-12-01

Full Text Available Se evaluó la presencia del virus de la leucosis bovina (VLB en 360 muestras de ADN de ocho razas bovinas criollas: Blanco Orejinegro (BON, Casanareño (CAS, Costeño con Cuernos (CCC, Chino Santandereano (ChS, Caqueteño (CQT, Hartón del Valle (HV, Romosinuano (RS y San Martinero (SM, dos Razas Sintéticas Colombianas: Lucerna (LUC y Velásquez (VEL y dos razas foráneas: Brahmán (B y Holstein (H. Para la detección del pro-virus se amplificó una región del gen env viral, mediante PCR anidada. La presencia del VLB fue mayor en la raza HV seguido por ChS (83.3% y 60% respectivamente, VEL y LUC tuvieron el mismo porcentaje (50%, en CAS, CCC y CQT la presencia del virus fue de 26.7%, 23.3% y 16.7% respectivamente; no se encontró el virus en BON, SM y RS. En las razas foráneas la presencia fue de 83.3% para H y 6.7% para B. Se encontró dependencia altamente significativa entre la presencia del VLB y la raza, el sexo y región de origen de la muestra. El promedio de presencia en las razas criollas fue menor que en las foráneas, menor en los machos que en las hembras y en la región norte que en el suroccidente y el centro del país.Using 360 DNA samples from eight Creole bovine breeds Blanco Orejinegro (BON, Casanareño (CAS, Costeño con Cuernos (CCC, Chino Santandereano (ChS, Caqueteño (CQT, Hartón del Valle (HV, Romosinuano (RS and San Martinero (SM, two synthetic Colombian breeds: Lucerna (LUC and Velásquez (VEL and two introduced breeds Brahmán (B and Holstein (H; the presence of Bovine Leukemia Virus (BLV was evaluated through the amplification of a viral gene region env (provirus detection - nested-PCR. The percentage of presence and independence test were calculated (X². Presence of BLV was higher in HV breed, followed by ChS (83.3% and 60% respectively; VEL and LUC breeds showed the same percentage (50%. In CAS, CCC and CQT the presence of virus was 26.7%, 23.3% y 16.7% respectively. On the other hand, no virus presence was

A RETROSPECTIVE STUDY OF BOVINE ABORTIONS ASSOCIATED WITH BACILLUS-LICHENIFORMIS

DEFF Research Database (Denmark)

Agerholm, J.S.; Krogh, H.V.; Jensen, H.E.

1995-01-01

A retrospective study of bovine abortions associated with Bacillus licheniformis is described. The material consisted of 2445 bovine abortions submitted for diagnostics from 1986 through 1993. Initially, B, licheniformis had been isolated from 81 cases. Sections of these cases were reexamined...... isolations, especially from the placenta, lungs, and abomasal contents, combined with the histological findings points to B, licheniformis abortions as being of haematogenous origin with subsequent transplacental spread to the fetus....

Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of genetic marker alleles associated with a trait indicative of fertility of the bovine subject and/or off-spring

DEFF Research Database (Denmark)

2009-01-01

NOVELTY - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles that are associated with a trait indicative of fertility of the bovine subject and/or off-spring. USE - The methods are useful...... for determining fertility in a bovine subject; and selecting bovine subjects for breeding purposes (all claimed). DETAILED DESCRIPTION - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles...... that are associated with a trait indicative of fertility of the bovine subject and/or off-spring, where the two or more genetic marker alleles are single nucleotide polymorphisms selected from Hapmap60827-rs29019866, ARS-BFGL-NGS-40979, Hapmap47854-BTA-119090, ARS-BFGL-NGS-114679, Hapmap43841-BTA-34601, Hapmap43407...

Short communication. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV isolates in Kosovo

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Izedin Goga

2014-03-01

Full Text Available Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

Science.gov (United States)

Hoferer, Marc; Braun, Anne; Sting, Reinhard

2017-07-01

Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Characterization of thymus-associated lymphoid depletion in bovine calves acutely or persistently infected with bovine viral diarrhea virus 1, bovine viral diarrhea virus 2 or HoBi-like pestivirus.

Science.gov (United States)

Falkenberg, Shollie M; Bauermann, Fernando V; Ridpath, Julia F

2017-11-01

Naïve pregnant cattle exposed to pestiviruses between 40-125 days of gestation can give birth to persistently infected (PI) calves. Clinical presentation and survivability, in PI cattle, is highly variable even with the same pestivirus strain whereas the clinical presentation in acute infections is more uniform with severity of symptoms being primarily a function of virulence of the infecting virus. The aim of this study was to compare thymic depletion, as measured by comparing the area of the thymic cortex to the medulla (corticomedullary ratio), in acute and persistent infections of the same pestivirus isolate. The same general trends were observed with each pestivirus isolate. Thymic depletion was observed in both acutely and persistently infected calves. The average thymic depletion observed in acutely infected calves was greater than that in age matched PI calves. PI calves, regardless of infecting virus, revealed a greater variability in amount of depletion compared to acutely infected calves. A trend was observed between survivability and depletion of the thymus, with PI calves surviving less than 5 weeks having lower corticomedullary ratios and greater depletion. This is the first study to compare PI and acutely infected calves with the same isolates as well as to evaluate PI calves based on survivability. Further, this study identified a quantifiable phenotype associated with potential survivability.

Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains.

Science.gov (United States)

Eberle, Kirsten C; Neill, John D; Venn-Watson, Stephanie K; McGill, Jodi L; Sacco, Randy E

2015-10-01

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1β, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

Viruses and Breast Cancer

Science.gov (United States)

Lawson, James S.; Heng, Benjamin

2010-01-01

Viruses are the accepted cause of many important cancers including cancers of the cervix and anogenital area, the liver, some lymphomas, head and neck cancers and indirectly human immunodeficiency virus associated cancers. For over 50 years, there have been serious attempts to identify viruses which may have a role in breast cancer. Despite these efforts, the establishment of conclusive evidence for such a role has been elusive. However, the development of extremely sophisticated new experimental techniques has allowed the recent development of evidence that human papilloma virus, Epstein-Barr virus, mouse mammary tumor virus and bovine leukemia virus may each have a role in the causation of human breast cancers. This is potentially good news as effective vaccines are already available to prevent infections from carcinogenic strains of human papilloma virus, which causes cancer of the uterine cervix. PMID:24281093

Viruses and Breast Cancer

International Nuclear Information System (INIS)

Lawson, James S.; Heng, Benjamin

2010-01-01

Viruses are the accepted cause of many important cancers including cancers of the cervix and anogenital area, the liver, some lymphomas, head and neck cancers and indirectly human immunodeficiency virus associated cancers. For over 50 years, there have been serious attempts to identify viruses which may have a role in breast cancer. Despite these efforts, the establishment of conclusive evidence for such a role has been elusive. However, the development of extremely sophisticated new experimental techniques has allowed the recent development of evidence that human papilloma virus, Epstein-Barr virus, mouse mammary tumor virus and bovine leukemia virus may each have a role in the causation of human breast cancers. This is potentially good news as effective vaccines are already available to prevent infections from carcinogenic strains of human papilloma virus, which causes cancer of the uterine cervix

Viruses and Breast Cancer

Energy Technology Data Exchange (ETDEWEB)

Lawson, James S., E-mail: james.lawson@unsw.edu.au; Heng, Benjamin [School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney (Australia)

2010-04-30

Viruses are the accepted cause of many important cancers including cancers of the cervix and anogenital area, the liver, some lymphomas, head and neck cancers and indirectly human immunodeficiency virus associated cancers. For over 50 years, there have been serious attempts to identify viruses which may have a role in breast cancer. Despite these efforts, the establishment of conclusive evidence for such a role has been elusive. However, the development of extremely sophisticated new experimental techniques has allowed the recent development of evidence that human papilloma virus, Epstein-Barr virus, mouse mammary tumor virus and bovine leukemia virus may each have a role in the causation of human breast cancers. This is potentially good news as effective vaccines are already available to prevent infections from carcinogenic strains of human papilloma virus, which causes cancer of the uterine cervix.

Effects of guidelines on adeno-tonsillar surgery on the clinical behaviour of otorhinolaryngologists in Italy

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Motta Giovanni

2013-01-01

Full Text Available Abstract Background Several guidelines on adeno-tonsillar disease have been proposed in recent years and some discrepancies in relation both to clinical manifestations and indications for surgical treatment have emerged. The aim of the study was to verify what influence (adeno-tonsillectomy guidelines have had on the clinical behaviour of ENT specialists in Italy. Our study is a retrospective and multi-centre case series with chart review. Methods The survey involved 14,770 children, aged between the ages of 2 and 11, who had undergone adeno-tonsillar surgery between 2002 and 2008 in fourteen Italian tertiary and secondary referral centres. Anova test was used for the statistical analysis, assuming p Results The frequency of adeno-tonsillar surgeries did not change significantly (p>0.05 during the study period and following the Italian policy document publication. Overall, adeno-tonsillectomy was the most frequent intervention (64.1%, followed by adenoidectomy (31.1% and tonsillectomy (4.8%. The indications for surgery did not change significantly for each of the operations (p>0.05, with the exception of adeno-tonsillectomy in case of feverish episodes due to acute recurrent tonsillitis ≥ 5 without nasal obstruction (decreased p= 0.010 , even when the feverish episodes due to acute recurrent tonsillitis were Conclusions The recommendations first developed in Italy in a 2003 policy document and then resumed in guidelines in 2008, were not implemented by ENT units involved in the survey. The study highlights the fact that the indications for adeno-tonsillar operations are based on the overall clinical presentation (comorbidity rather than on a single symptom. Guidelines are necessary to give coherent recommendations based on both the findings obtained through randomized controlled trials and the data collected from observational studies.

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

Science.gov (United States)

Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2015-04-01

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of border disease virus (BDV) on serological surveillance within the bovine virus diarrhea (BVD) eradication program in Switzerland.

Science.gov (United States)

Kaiser, V; Nebel, L; Schüpbach-Regula, G; Zanoni, R G; Schweizer, M

2017-01-13

In 2008, a program to eradicate bovine virus diarrhea (BVD) in cattle in Switzerland was initiated. After targeted elimination of persistently infected animals that represent the main virus reservoir, the absence of BVD is surveilled serologically since 2012. In view of steadily decreasing pestivirus seroprevalence in the cattle population, the susceptibility for (re-) infection by border disease (BD) virus mainly from small ruminants increases. Due to serological cross-reactivity of pestiviruses, serological surveillance of BVD by ELISA does not distinguish between BVD and BD virus as source of infection. In this work the cross-serum neutralisation test (SNT) procedure was adapted to the epidemiological situation in Switzerland by the use of three pestiviruses, i.e., strains representing the subgenotype BVDV-1a, BVDV-1h and BDSwiss-a, for adequate differentiation between BVDV and BDV. Thereby the BDV-seroprevalence in seropositive cattle in Switzerland was determined for the first time. Out of 1,555 seropositive blood samples taken from cattle in the frame of the surveillance program, a total of 104 samples (6.7%) reacted with significantly higher titers against BDV than BVDV. These samples originated from 65 farms and encompassed 15 different cantons with the highest BDV-seroprevalence found in Central Switzerland. On the base of epidemiological information collected by questionnaire in case- and control farms, common housing of cattle and sheep was identified as the most significant risk factor for BDV infection in cattle by logistic regression. This indicates that pestiviruses from sheep should be considered as a source of infection of domestic cattle and might well impede serological BVD surveillance.

Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

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Bedeković Tomislav

2011-12-01

Full Text Available Abstract Background Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described. Findings Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells. Conclusions Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.

Severity of Bovine Tuberculosis Is Associated with Co-Infection with Common Pathogens in Wild Boar

Science.gov (United States)

Risco, David; Serrano, Emmanuel; Fernández-Llario, Pedro; Cuesta, Jesús M.; Gonçalves, Pilar; García-Jiménez, Waldo L.; Martínez, Remigio; Cerrato, Rosario; Velarde, Roser; Gómez, Luis; Segalés, Joaquím; Hermoso de Mendoza, Javier

2014-01-01

Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszky's disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under

Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar.

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David Risco

Full Text Available Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa, a recognized reservoir of bovine tuberculosis (bTB in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes, or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs, was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2, Aujeszky's disease virus (ADV, swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures

Comparison of the virucidal efficiency of peracetic acid, potassium monopersulfate and sodium hypochlorite on hepatitis A and enteric cytopathogenic bovine orphan virus.

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Martin, H; Soumet, C; Fresnel, R; Morin, T; Lamaudière, S; Le Sauvage, A L; Deleurme, K; Maris, P

2013-10-01

The virucidal activity of peroxy-products was evaluated and compared with sodium hypochlorite using the EN 14675 European suspension test and a surface test developed in our laboratory. The classical approach on infectivity of viruses was complemented with a prospective approach on virus genomes. Both infectivity tests were adapted and/or developed to determine the activity of disinfectants against reference bovine enterovirus type 1 [enteric cytopathogenic bovine orphan virus (ECBO)] and resistant hepatitis A virus (HAV) in conditions simulating practical use. Similar concentrations of active chlorine were virucidal against both viruses, either at 0·062% using the suspension test or at 0·50-1% using the surface test. However, for potassium monopersulfate and peracetic acid products, concentrations of approximately three times (3%) to 72 times (9%) higher were necessary against HAV than ECBO when determined with the suspension test. With the surface test, 4-8% peroxy-products were virucidal against HAV, either 16 times more peroxy-products concentrations than against ECBO. No significant impact on the targeted area of the viral genome measured by real-time RT-PCRs was obtained for ECBO and HAV suspensions treated with disinfectants, even with doses higher than the minimal virucidal concentrations. Sodium hypochlorite, but not peroxy-products, had similar activity against ECBO and HAV. No relation could be established between infectivity tests and genome destruction. This is the first comparative study that investigates with novel suspension and surface tests the reduction of infectivity and genome destruction of two resistant viruses by peroxy-compounds. The results and conclusions collected with European standards are discussed. © 2013 The Society for Applied Microbiology.

Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches.

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Hongyan Xia

Full Text Available The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.

Radiographic and radionuclide lung perfusion imaging in healthy calves and calves naturally infected with bovine respiratory syncytial virus

International Nuclear Information System (INIS)

Verhoeff, J.; Brom, W.E. van den; Ingh, T.S.G.A.M. van den

1992-01-01

Nine calves between three and 18 weeks old with serologically confirmed natural bovine respiratory syncytial virus infection were examined clinically, radiographically and by radionuclide lung perfusion imaging. The results were compared with those from seven healthy calves. The diseased calves were euthanased and examined pathologically, virologically and bacteriologically. The clinical signs indicated that the disease was in an acute stage. Radiography of the diseased animals revealed cysts, corresponding morphologically with bullous emphysema, and infiltrations roughly corresponding in distribution with atelectatic and, or, pneumonic areas. Radionuclide lung perfusion imaging revealed no perfusion shifts between the left and right lungs and a normal perfusion pattern in five of the nine diseased calves. The abnormalities in the perfusion patterns of three calves were probably caused by anatomical disorders such as cysts and pleural adhesions, but no cause of the abnormality could be found in one calf. These findings suggest that in calves infected with bovine respiratory syncytial virus, the normal perfusion pattern is maintained until anatomical disorders occur. The pathological examination and radiography revealed that the cranioventral lung fields were particularly poorly ventilated. This finding and the normal perfusion pattern indicate that these parts of the lungs are probably the sites where shuntings and perfusion-ventilation mismatchings occur

Use of DNA from milk tank for diagnosis and typing of bovine leukaemia virus

International Nuclear Information System (INIS)

Felmer, R.; Zuniga, J.; Recabal, M.; Floody, H.

2005-01-01

With the aim of achieving a better understanding of the epidemiology of Bovine leukaemia virus (BLV) infection, we investigated the suitability of milk tank samples for effecting molecular epidemiology studies of BLV in a southern area of Chile. As part of a serological survey for BLV antibodies carried out in 280 herds, we selected 33 strong positive samples, from which DNA was isolated to perform a BLV-specific nested PCR. Using RFLP analysis, all 33 PCR products could be assigned to the known Australian or the Belgium subgroups. A phylogenetic tree resulting from the comparison of these sequences demonstrates the relations and differences among and within the subgroups. (author)

Bovine viral diarrhea virus type 2 impairs macrophage responsiveness to toll-like receptor ligation with the exception of toll-like receptor 7

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Bovine viral diarrhea virus (BVDV) is a member of the Flaviviradae family. BVDV isolates are classified into two biotypes based on the development of cytopathic (cp) or non-cytopathic (ncp) effects in epithelial cell culture. In addition, BVDV isolates are further separated into species, BVDV1 and 2...

Morphology and Molecular Composition of Purified Bovine Viral Diarrhea Virus Envelope.

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Nathalie Callens

2016-03-01

Full Text Available The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV, a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

Science.gov (United States)

Nekouei, Omid; Durocher, Jean; Keefe, Greg

2016-01-01

This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

OpenAIRE

Su, Feifei; Myers, Valerie D.; Knezevic, Tijana; Wang, JuFang; Gao, Erhe; Madesh, Muniswamy; Tahrir, Farzaneh G.; Gupta, Manish K.; Gordon, Jennifer; Rabinowitz, Joseph; Ramsey, Frederick V.; Tilley, Douglas G.; Khalili, Kamel; Cheung, Joseph Y.; Feldman, Arthur M.

2016-01-01

Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expre...

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

DEFF Research Database (Denmark)

Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

2015-01-01

Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

Persistent Bovine Viral Diarrhea Virus infection in domestic and wild small ruminants and camelids including the mountain goat (Oreamnos americanus

Directory of Open Access Journals (Sweden)

Danielle Darracq Nelson

2016-01-01

Full Text Available Bovine viral diarrhea virus (BVDV is a Pestivirus best known for causing a variety of disease syndromes in cattle, including gastrointestinal disease, reproductive insufficiency, immunosuppression, mucosal disease, and hemorrhagic syndrome. The virus can be spread by transiently infected individuals and by persistently infected animals that may be asymptomatic while shedding large amounts of virus throughout their lifetime. BVDV has been reported in over 40 domestic and free-ranging species, and persistent infection has been described in eight of those species: white-tailed deer, mule deer, eland, mousedeer, mountain goats, alpacas, sheep, and domestic swine. This paper reviews the various aspects of BVDV transmission, disease syndromes, diagnosis, control, and prevention, as well as examines BVDV infection in domestic and wild small ruminants and camelids including mountain goats (Oreamnos americanus.

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

Directory of Open Access Journals (Sweden)

Loy John Dustin

2013-01-01

Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

A new analgesia regimen after (adeno) tonsillectomy in children: a pilot study.

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Syed, M I; Magos, T A; Singh, J; Montague, M L

2016-12-01

The objective was to ascertain the efficacy of a new analgesic regimen introduced in children undergoing (adeno)tonsillectomy in view of the ban on codeine use in children codeine, albeit one should bear in mind that parental concerns and adverse effects of the drug were seen in a minority of patients (n = 11) and anaesthetists were reluctant to prescribe the drug in cases of severe OSA or associated central apnoeas (n = 7). © 2015 John Wiley & Sons Ltd.

Cryo-gamma radiation inactivation of bovine herpesvirus type-1

Science.gov (United States)

Degiorgi, C. Fernández; Smolko, E. E.; Lombardo, J. H.

1999-07-01

The radioresistance of bovine herpesvirus-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), suspended in free serum Glasgow-MEM medium and frozen at -78°C was studied. The number of surviving virus at a given dose of gamma-radiation was determined by a plaque assay system. D 10 values were calculated before and after removal of cell debris. The D 10 values obtained were 4.72 kGy and 7.31 kGy before and after removal of cell debris, respectively. Our results indicate that the inactivated viral particles could be used for vaccine preparation or diagnostic reagents.

Retention and topology of the bovine viral diarrhea virus glycoprotein E2.

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Radtke, Christina; Tews, Birke Andrea

2017-10-01

Pestiviruses are enveloped viruses that bud intracellularly. They have three envelope glycoproteins, E rns , E1, and E2. E2 is the receptor binding protein and the main target for neutralizing antibodies. Both E rns and E2 are retained intracellularly. Here, E2 of the bovine viral diarrhea virus (BVDV) strain CP7 was used to study the membrane topology and intracellular localization of the protein. E2 is localized in the ER and there was no difference between E2 expressed alone or in the context of the viral polyprotein. The mature E2 protein was found to possess a single span transmembrane anchor. For the mapping of a retention signal CD72-E2 fusion proteins, as well as E2 alone were analysed. This confirmed the importance of the transmembrane domain and arginine 355 for intracellular retention, but also revealed a modulating effect on retention through the cytoplasmic tail of the E2 protein, especially through glutamine 370. Mutants with a strong impact on retention were tested in the viral context and we were able to rescue BVDV with certain mutations that in E2 alone impaired intracellular retention and lead to export of E2 to the cells surface.

Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

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Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

2014-06-01

Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular assays for targeting human and bovine enteric viruses in coastal waters and their application for library-independent source tracking

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Fong, T.-T.; Griffin, Dale W.; Lipp, E.K.

2005-01-01

Rapid population growth and urban development along waterways and coastal areas have led to decreasing water quality. To examine the effects of upstream anthropogenic activities on microbiological water quality, methods for source-specific testing are required. In this study, molecular assays targeting human enteroviruses (HEV), bovine enteroviruses (BEV), and human adenoviruses (HAdV) were developed and used to identify major sources of fecal contamination in the lower Altamaha River, Georgia. Two-liter grab samples were collected monthly from five tidally influenced stations between July and December 2002. Samples were analyzed by reverse transcription- and nested-PCR. PCR results were confirmed by dot blot hybridization. Eleven and 17 of the 30 surface water samples tested positive for HAdV and HEV, respectively. Two-thirds of the samples tested positive for either HEV or HAdV, and the viruses occurred simultaneously in 26% of samples. BEV were detected in 11 of 30 surface water samples. Binary logistic regression analysis showed that the presence of both human and bovine enteric viruses was not significantly related to either fecal coliform or total coliform levels. The presence of these viruses was directly related to dissolved oxygen and streamflow but inversely related to water temperature, rainfall in the 30 days preceding sampling, and chlorophyll-?? concentrations. The stringent host specificity of enteric viruses makes them good library-independent indicators for identification of water pollution sources. Viral pathogen detection by PCR is a highly sensitive and easy-to-use tool for rapid assessment of water quality and fecal contamination when public health risk characterization is not necessary. Copyright ?? 2005, American Society for Microbiology. All Rights Reserved.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

International Nuclear Information System (INIS)

Iourin, Oleg; Harlos, Karl; El Omari, Kamel; Lu, Weixian; Kadlec, Jan; Iqbal, Munir; Meier, Christoph; Palmer, Andrew; Jones, Ian; Thomas, Carole; Brownlie, Joe; Grimes, Jonathan M.; Stuart, David I.

2012-01-01

The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described. Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress

Genome analysis of an atypical bovine pestivirus from fetal bovine serum.

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Gao, Shandian; Du, Junzheng; Tian, Zhancheng; Xing, Shanshan; Chang, Huiyun; Liu, Guangyuan; Luo, Jianxun; Yin, Hong

2016-08-01

We report the complete genome sequence of a bovine pestivirus LVRI/cont-1 originated from a commercial batch of fetal bovine serum. Its complete genome consists of 12,282 nucleotides (nt), which contain an open reading frame (ORF) of 11,700 bp flanked by 5' and 3' untranslated regions (383 and 199 bp). The size of the 5'UTR and the individual protein coding region of LVRI/cont-1 are identical to those of the reference virus Th/04_KhonKaen, but it has a deletion of the first 56 nt in the 3'UTR. Alignment of the complete nucleotide sequence and phylogenetic analysis indicate that this viral isolate belongs to atypical pestiviruses.

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

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Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

2016-01-15

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

International Nuclear Information System (INIS)

Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O.; Tumpey, Terrence M.; Pushko, Peter

2016-01-01

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

Science.gov (United States)

Passos-Castilho, Ana Maria; Marchand, Claude; Archambault, Denis

2018-02-01

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Pathological studies on bovine viral diarrhea

International Nuclear Information System (INIS)

Elkady, A.A.M.A.

2002-01-01

Bovine viral diarrhea virus (BVDV) is classified as an RNA virus in the family flavin viride and is a member of the genus pest virus (Collet et al 1989). BVDV has a worldwide distribution and infections in cattle populations (Kahrs et al 1970). It was recognized since 50 years ago, the initial description of an acute enteric disease of cattle in North America, which was characterized by outbreaks of diarrhea and erosive of digestive tract (Olafsonp et al 1946). The disease and causative agent were named bovine viral diarrhea (B V D ) and (B V DV), respectively. This virus was subsequently associated with a sporadically occurring and highly fatal enteric disease that was termed mucosal disease (M D), (Ramsey and Chivers 1953). The initial isolate of BVDV did not produce cytopathic effect in cell culture, whereas an isolate from MD did produce cytopathic effects (Lee et al 1957). In vitro characteristic of non cytopathic or sytopathic effects of BVDV is referred to as the biotype of the virus. It has now been established that MD occurs only when xattle that are born immuno tolerant to and persistently infected with a noncyropathic BVDV become super infected with a cytopathic BVDV. The knowledge of the molecular biology. Pathogenesis and epidemiology of BVDV has greatly evolved in the past 10-15 years and has provided a better understanding of this complex infectious agent. Infection with BVDV can result in a wide spectrum of diseases ranging from subclinical infection s to a highly fatal from known as mucosal disease (ND). The clinical response to infection depends on multiple interactive factors. Host factors that influence the clinical outcome of BVDV infection include whether the host is immunocompetent or immuno tolerant to BVDV, pregnancy status, gestational age of the fetus, immune status (passively derived or actively derived from previous infection or vaccination) and concurrent level of environmental stress

A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle

NARCIS (Netherlands)

Schrijver, R.S.; Langedijk, J.P.M.; Middel, W.G.J.; Kramps, J.A.; Rijsewijk, F.A.M.; Oirschot, van J.T.

1998-01-01

Bovine respiratory syncytial virus (BRSV) strains are tentatively divided in subgroups A, AB and B, based on antigenic differences of the G protein. A Dutch BRSV strain (Waiboerhoeve: WBH), could not be assigned to one of the subgroups, because the strain did not react with any monoclonal antibody

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

Science.gov (United States)

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

2017-01-01

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

Feed intake and weight changes in Bos indicus-Bos taurus crossbred steers following Bovine Viral Diarrhea Virus Type 1b challenge under production conditions

Science.gov (United States)

Bovine viral diarrhea virus (BVDV) has major impacts on beef cattle production worldwide, but the understanding of host animal genetic influence on illness is limited. This study evaluated rectal temperature, weight change and feed intake in Bos indicus crossbred steers (n = 366) that were challenge...

Surface properties, more than size, limiting convective distribution of virus-sized particles and viruses in the central nervous system.

Science.gov (United States)

Chen, Michael Y; Hoffer, Alan; Morrison, Paul F; Hamilton, John F; Hughes, Jeffrey; Schlageter, Kurt S; Lee, Jeongwu; Kelly, Brandon R; Oldfield, Edward H

2005-08-01

Achieving distribution of gene-carrying vectors is a major barrier to the clinical application of gene therapy. Because of the blood-brain barrier, the distribution of genetic vectors to the central nervous system (CNS) is even more challenging than delivery to other tissues. Direct intraparenchymal microinfusion, a minimally invasive technique, uses bulk flow (convection) to distribute suspensions of macromolecules widely through the extracellular space (convection-enhanced delivery [CED]). Although acute injection into solid tissue is often used for delivery of oligonucleotides, viruses, and liposomes, and there is preliminary evidence that certain of these large particles can spread through the interstitial space of the brain by the use of convection, the use of CED for distribution of viruses in the brain has not been systematically examined. That is the goal of this study. Investigators used a rodent model to examine the influence of size, osmolarity of buffering solutions, and surface coating on the volumetric distribution of virus-sized nanoparticles and viruses (adeno-associated viruses and adenoviruses) in the gray matter of the brain. The results demonstrate that channels in the extracellular space of gray matter in the brain are large enough to accommodate virus-sized particles and that the surface characteristics are critical determinants for distribution of viruses in the brain by convection. These results indicate that convective distribution can be used to distribute therapeutic viral vectors in the CNS.

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

International Nuclear Information System (INIS)

Qualley, Dominic F.; Sokolove, Victoria L.; Ross, James L.

2015-01-01

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

Energy Technology Data Exchange (ETDEWEB)

Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

2015-03-13

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.

Determining resistance to mastitis in a bovine subject involves detecting presence or absence of genetic marker associated with trait indicative of mastitis resistance of the bovine subject and/or off-spring from it

DEFF Research Database (Denmark)

2010-01-01

NOVELTY - Determining (m1) resistance to mastitis in a bovine subject, involves detecting in a sample from the bovine subject the presence or absence of at least one genetic marker that is associated with at least one trait indicative of mastitis resistance of the bovine subject and/or off......-spring from it, where the genetic marker is located on the bovine chromosome BTA11 in the region flanked by and including the zeta-chain associated protein 70kD (ZAP70) and CD8B genes, where the presence or absence of the genetic marker is indicative of mastitis resistance. USE - For determining resistance...... to mastitis in a bovine subject for determining mastitis resistance in a bovine subject; for detecting the presence or absence in a bovine subject of at least one genetic marker associated with resistance to mastitis; and for estimating breeding value in respect of susceptibility to mastitis in a bovine...

A quantitative risk-analysis for introduction of Bovine Viral Diarrhoea Virus in the Netherlands through cattle imports.

Science.gov (United States)

Santman-Berends, I M G A; Mars, M H; Van Duijn, L; Van den Broek, K W H; Van Schaik, G

2017-10-01

Many countries have implemented control programmes aiming to eradicate Bovine Viral Diarrhoea Virus (BVDV). After obtaining the free status, a risk of re-introduction of the virus through import may remain. Therefore the risk of introduction of BVDV through cattle imports in the Netherlands was quantified and the effectiveness of subsequent intervention measures was assessed. Data, literature and expert opinion were used to estimate values for input parameters to feed a stochastic simulation model. The probability that BVDV was imported was differentiated into persistently infected (PI) cattle, trojan cows that transmitted the virus vertically resulting in a PI foetus (TR) and transient infected cattle (TI). The import risk was stratified to beef, dairy, small scale, suckler, trade, veal and young stock herds. The intervention scenarios that were evaluated consisted of virus testing, a combination of virus testing and antibody testing in pregnant cows, abolishment of imports from high risk countries (i.e. countries with a BVDV prevalence >15%) and a combination of import restrictions and testing prior to import. Each year, 334 (5th and 95th percentile: 65-902) Dutch cattle herds were estimated to be infected with BVDV through import. Veal herds account for most infections associated with import (87%), whereas in the other herd types, only 9 beef, 6 dairy, 2 small scale, 16 suckler, 10 trade and 2 young stock herds are infected through imports per year. Import of PI cattle is the most important risk for introduction in veal herds, while import of TR cows is the main source of BVDV introduction in dairy, small scale and suckler herds. With the intervention scenarios, the number of BVDV infected herds in the Netherlands could be reduced to 81 and 58 herds per year when respectively virus testing or a combination of virus and antibody testing was applied or to 108 herds when import from high risk countries was abolished. With the scenario in which both import from high

Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads.

Science.gov (United States)

Hayashi, Takumi; Mekata, Hirohisa; Sekiguchi, Satoshi; Kirino, Yumi; Mitoma, Shuya; Honkawa, Kazuyuki; Horii, Yoichiro; Norimine, Junzo

2017-09-12

The bovine MHC (BoLA) class II DRB3 alleles are associated with polyclonal expansion of lymphocytes caused by bovine leukemia virus (BLV) infection in cattle. To examine whether the DRB3*0902 allele, one of the resistance-associated alleles, is associated with the proviral load, we measured BLV proviral load of BLV-infected cattle and clarified their DRB3 alleles. Fifty-seven animals with DRB3*0902 were identified out of 835 BLV-infected cattle and had significantly lower proviral load (Pclass II DRA/DRB3*0902 molecule plays an important immunological role in suppressing viral replication, resulting in resistance to the disease progression.

Effects of dietary source and intake of energy on immune competence and the response to an infectious bovine rhinotracheitis virus (IBRV) challenge in cattle

Science.gov (United States)

Objectives were to evaluate how dietary energy intake and source affect immune competence and response to an infectious bovine rhinotracheitis virus (IBRV) challenge in cattle. Forty-eight crossbred beef steers were stratified by body weight within 2 periods and randomized to 1 of 3 dietary treatmen...

Rhabdomyolysis Associated with Parainfluenza Virus

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Miltiadis Douvoyiannis

2013-01-01

Full Text Available Influenza virus is the most frequently reported viral cause of rhabdomyolysis. A 7-year-old child is presented with rhabdomyolysis associated with parainfluenza type 2 virus. Nine cases of rhabdomyolysis associated with parainfluenza virus have been reported. Complications may include electrolyte disturbances, acute renal failure, and compartment syndrome.

In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1 rat-bovine chimeric antibody against bovine leukemia virus infection.

Directory of Open Access Journals (Sweden)

Asami Nishimori

Full Text Available Programmed death-1 (PD-1, an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1 in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12. Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL stage. Cultivation of peripheral blood mononuclear cells (PBMCs isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

Bovine herpes virus-1 (BoHV-1 detection in dairy cattle with reproductive problems in Sudan

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Amira Mohamed Elhassan

2015-06-01

Full Text Available The present work aimed to observe the infection pattern of Bovine herpes virus-1 (BoHV-1 in dairy cattle with reproductive problems in Sudan. A total of 140 samples comprising of vaginal swab (n=97, placenta (n=15, whole blood (n=19, uterine fluid (n=1, and serum (n=8 were collected from 16 dairy herds showing particularly high rate of abortion and infertility in Khartoum State. The samples were used for virus isolation, and were tested by Enzyme-Linked Immunosorbent Assay (ELISA and polymerase chain reaction (PCR. No virus could be isolated from the samples inoculated for isolation in cell culture. Out of 80 specimens tested by ELISA, 7 (8.75% were found to be positive, and one sample was doubtful. Using PCR, 11 (10.7% out of 103 samples were found to be positive. When comparing between two methods for DNA extraction, the DNA extracted by commercial kit was found to be better in quality as compared to the DNA extracted using phenol/chloroform/isoamyl-alcohol method. The study confirmed the presence of BoHV-1 in cattle farms with reproductive problems in Sudan.

The vaccines for Bovine Herpesvirus Type 1: A review | Zhao ...

African Journals Online (AJOL)

Bovine herpesvirus type 1 (BoHV-1) is the pathogen of Infectious Bovine Rhinothracheitis (IBR) disease, causing great economic losses in the livestock industry. Vaccine is a powerful means to control the virus. Here, the review described the currently available knowledge regarding to the advance in the field of BoHV-1 ...

Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries

DEFF Research Database (Denmark)

Vintov, J.; Aarestrup, Frank Møller; Zinn, C. E.

2003-01-01

This study was conducted to investigate the diversity of phage types and associations between penicillin resistance and phage types among 815 Staphylococcus aureus isolates from bovine mastitis in nine European countries and USA. All isolates were examined for susceptibility to antimicrobial agents...... associated with penicillin resistance in contrast to phage group I (P = 0.0023) and phage complex-80 (P = 0.0066). This study confirms that a large number of phage types of S. aureus cause bovine mastitis, but that some types predominate. In addition, these findings could indicate that the use of penicillin...... in the bovine environment has selected for specific types of S. aureus in countries with a high frequency of resistance....

Bovine lymphocytic leukemia: studies of etiology, pathogenesis and mode of transmission. Progress report No. 18, June 1975--June 1978

Energy Technology Data Exchange (ETDEWEB)

Sorensen, D.K.

1978-07-01

The primary objective of this research is to elucidate the cause(s) and early pathogenesis of the adult form of lymphosarcoma in cattle. Consequently, a major portion of our research is centered around experimental transmission of this disease. Bovine leukemia is believed to be caused by an oncogenic RNA virus designated bovine leukemia virus (BLV). We have consistently demonstrated the presence of BLV particles in leukemic cattle and cattle with a persistent lymphocytosis, but never in normal cattle. These BLV particles have been partially purified and highly concentrated to provide a potent inoculum used to inoculate 12 late stage bovine fetuses (in utero) and two newborn calves. Our current study involves extensive monitoring of these inoculated animals to detect early precancerous changes and obtain a detailed description of the events occurring early in the pathogenesis of bovine lymphosarcoma. From our ongoing monitoring study we will be able to detail when, in what sequence, and to what extent each parameter changes in the course of lymphosarcoma development. In addition to our transmission and monitoring studies we are examining various lymphocyte subpopulations in an attempt to determine which cell type is responsible for BLV production and to determine if this same cell type carries the nuclear pocket abnormality associated with the adult form of bovine lymphosarcoma. (ERB)

Vaccinia virus as a subhelper for AAV replication and packaging

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Andrea R Moore

Full Text Available Adeno-associated virus (AAV has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

Bovine lymphocytic leukemia: studies of etiology, pathogenesis, and mode of transmission. Progress report No. 19, June 1978-June 1979

Energy Technology Data Exchange (ETDEWEB)

Sorensen, D.K.

1979-07-01

Bovine leukemia is believed to be caused by an oncogenic RNA virus designated bovine leukemia virus (BLV). The presence of BLV particles in lymphocyte cultures from leukemic cattle and cattle with a persistent lymphocytosis has been consistentily demonstrated. Concentrated, cell free, BLV preparations were used to inoculate 12 late stage bovine fetuses (in utero) and two newborn calves. Current studies involve extensive monitoring of these inoculated animals to detect precancerous changes and obtain a detailed description of the events preceding the development of lymphosarcoma. Ongoing monitoring studies will provide a complete record of all changes in the various leukemia associated parameters. We will then be able to detail when, in what sequence, and to what extent each parameter changes in the course of lymphosarcoma development. Fourteen animals were successfully inoculated during the study. Eleven remain alive, and comprise the current monitoring program. All eleven of these animals are definitely infected with BLV, and in nine the infection has substantially progressed with respect to the parameters being monitored. In addition to transmission and monitoring studies, various lymphocyte subpopulations were examined to determine which cell type(s) are involved in the pathogenesis of bovine lymphosarcoma. These studies have conclusively established that B-lymphocytes are the target cells for BLV infection and that they carry the morphologic nuclear abnormality associated with this disease.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

Science.gov (United States)

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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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.

Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

Science.gov (United States)

Hoover, Spencer; Striker, Rob

2008-04-01

The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

Isolation and identification of a bovine viral diarrhea virus from sika deer in china.

Science.gov (United States)

Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; Zhang, Pengju; Zhang, Lianxue

2011-02-25

Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China) using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE), indirect immunoperoxidase test (IPX) and electron microscopy(EM). The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV) and 3 strains of border disease virus(BDV) in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

Isolation and identification of a bovine viral diarrhea virus from sika deer in china

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Wang Nan

2011-02-01

Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE, indirect immunoperoxidase test (IPX and electron microscopy(EM. The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV and 3 strains of border disease virus(BDV in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

Histopathological and molecular study of Neospora caninum infection in bovine aborted fetuses

Institute of Scientific and Technical Information of China (English)

Amir Kamali; HesamAdin Seifi; Ahmad Reza Movassaghi; Gholam Reza Razmi; Zahra Naseri

2014-01-01

To estimate the extent to which abortion in dairy cows was associated with of Neospora caninum (N. caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran. Methods: Polymerase chain reaction (PCR) test was used to detect Neospora infection in the brain of 395 bovine aborted fetuses from 9 provinces of Iran. In addition, the brains of aborted fetuses were taken for histopathological examination. To identify the risk factors associated with neosporosis, data analysis was performed by SAS. Results: N. caninum was detected in 179 (45%) out of 395 fetal brain samples of bovine aborted fetuses using PCR. Among the PCR-positive brain samples, only 56 samples were suited for histopathological examination. The characteristic lesions of Neospora infection including non-suppurative encephalitis were found in 16 (28%) of PCR-positive samples. The risk factors including season, parity of dam, history of bovine virus diarrhea and infectious bovine rhinotracheitis infection in herd, cow’s milk production, herd size and fetal appearance did not show association with the infection. This study showed that Neospora caused abortion was significantly more in the second trimester of pregnancy than other periods. In addition, a significant association was observed between Neospora infection and stillbirth. Conclusions: The results showed N. caninum infection was detected in high percentage of aborted fetuses. In addition, at least one fourth of abortions caused by Neospora infection. These results indicate increasing number of abortions associated with the protozoa more than reported before in Iran.

Histopathological and molecular study of Neospora caninum infection in bovine aborted fetuses

Institute of Scientific and Technical Information of China (English)

Amir; Kamali; Hesam; Adin; Seifi; Ahmad; Reza; Movassaghi; Gholam; Reza; Razmi; Zahra; Naseri

2014-01-01

Objective:To estimate the extent to which abortion in dairy cows was associated with of Neospom caninum(N.caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran.Methods:Polymerase chain reaction(PCR) test was used to detect Neospora infection in the brain of 395 bovine aborted fetuses from 9 provinces of Iran.In addition,the brains of aborted fetuses were taken for histopathological examination.To identify the risk factors associated with neosporosis,data analysis was performed by SAS.Results:N.caninum was detected in 179(45%) out of 395 fetal brain samples of bovine aborted fetuses using PCR.Among the PCR-positive brain samples,only 56 samples were suited for histopathological examination.The characteristic lesions of Neospora infection including non-suppurative encephalitis were found in 16(28%) of PCR-positive samples.The risk factors including season,parity of dam,history of bovine virus diarrhea and infectious bovine rhinotracheitis infection in herd,cow’s milk production,herd size and fetal appearance did not show association with the infection.This study showed that Neospora caused abortion was significantly more in the second trimester of pregnancy than other periods.In addition,a significant association was observed between Neospora infection and stillbirth.Conclusions:The results showed N.caninum infection was detected in high percentage of aborted fetuses.In addition,at least one fourth of abortions caused by Neospora infection.These results indicate increasing number of abortions associated with the protozoa more than reported before in Iran.

Bovine leukemia virus infection in cattle of China: Association with reduced milk production and increased somatic cell score.

Science.gov (United States)

Yang, Y; Fan, W; Mao, Y; Yang, Z; Lu, G; Zhang, R; Zhang, H; Szeto, C; Wang, C

2016-05-01

The main objective of this study was to investigate the individual cow effect of bovine leukemia virus (BLV) infection on milk production and somatic cell score (SCS). The fluorescence resonance energy transfer (FRET) quantitative PCR established in this study and a commercial ELISA kit revealed that 49.1% of dairy cattle (964/1,963) from 6 provinces of China and 1.6% of beef cattle (22/1,390) from 15 provinces were BLV positive. In a detailed study of 105 cows, BLV was found most commonly in buffy coat samples that also had highest copy numbers (10(4.75±1.56) per mL); all cows negative for BLV in buffy coat samples were also negative in vaginal swab, milk, and fecal samples. Copy numbers of BLV were 10(2.90±0.42)/gram of feces, 10(0.83±0.62)/mL of milk, and 10(2.18±0.81) per vaginal swab. The BLV-positive cows had significantly lower milk production in the early (26.8 vs. 30.9kg) and middle stages of lactation (22.2 vs. 26.1kg) in animals with ≥4 parities than the BLV-negative cows; they also had significantly higher SCS in early and middle lactation stages (early=5.2 vs. 4.3; middle=4.9 vs. 3.9) in animals with ≥4 parities. Milk production and SCS did not significantly differ between the BLV-infected and -uninfected cows when they were in the late lactation stage or in animals with ≤3 parities. Taken together, our results indicate that BLV infections are widespread in the dairy farms of China. Vaginal secretions and feces may be involved in BLV transmission. A BLV infection may result in reduced milk yield and increased SCS in a parity and lactation stage-restricted manner. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

International Nuclear Information System (INIS)

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu; Kohara, Junko; Hirata, Toh-ichi; Okada, Kosuke; Ohashi, Kazuhiko; Onuma, Misao

2005-01-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-α and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-α-induced responses, in this study we examined the TNF-α-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-α (rTNF-α) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5 + or sIgM + cells and these cells showed resistance to TNF-α-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-α-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection

Bovine aortic arch and idiopathic pulmonary artery aneurysm associated with bronchial compression

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Süleyman Sezai Yıldız

2015-09-01

Full Text Available The left common carotid artery originating from the brachiocephalic trunk is termed the bovine aortic arch. Although it is the third most-common normal variant found in 9% humans, the origin of this term remains unclear. Until now, It has not been reported in the literature bovine aortic arch togetherness with pulmonary aneurysm and bronchial compression. Herein, we present a case with bovine aorta arch and pulmonary artery aneurysm associated with bronchial compression, which is incidentally detected by X-ray film. A 56-year-old Caucasian female admitted to the cardiology clinic with complaint of chest pain. Physical examination was unremarkable. Blood biochemistry values and cardiac markers were in normal range. Chest radiography revealed a widened mediastinum and prominent pulmonary conus with no active pulmonary disease. A subsequent transthoracic echocardiography revealed left ventricular hypertrophy, left atrial enlargement (diameter: 41 mm, mild mitral and tricuspid valve insufficiency, dilatation of main pulmonary artery (parasternal short-axis view diameter: 33 mm, normal pulmonary artery pressure and normal left ventricular systolic function. Computed tomography revealed bovine aortic arch associated with pulmonary artery aneurysm (diameter: 53 mm. And left main bronch of trachea was critically squeezed by aortic arch. Aortic and pulmonary vascular anomalies should be considered in patients with chest pain. And, identification with imaging modalities is important for prevention of chronic and irreversible complications.

BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status

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Jimba Mayuko

2012-09-01

Full Text Available Abstract Background Bovine leukemia virus (BLV is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR method using Coordination of Common Motifs (CoCoMo primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests. Results BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lew et al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA-DRB3 genotypes. Conclusions Our results suggest that the quantitative measurement of proviral load by BLV

Determination of the minimal fusion peptide of bovine leukemia virus gp30

International Nuclear Information System (INIS)

Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert; Charloteaux, Benoit

2007-01-01

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide

An experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus (BRSV) based on one combined exposure of calves

DEFF Research Database (Denmark)

Tjørnehøj, Kirsten; Uttenthal, Åse; Viuff, B.

2003-01-01

Bovine respiratory syncytial virus (BRSV) has been recognised as an important pathogen in calf pneumonia for 30 years, but surprisingly few effective infection models for studies of the immune response and the pathogenesis in the natural host have been established. We present a reproducible...... disease. This model is a valuable tool for the study of the pathogenesis of BRSV and for vaccine efficacy studies....

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

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Hongmei Wang

Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

Genetic diversity of Brazilian bovine pestiviruses detected between 1995 and 2014

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Pestivirus infections in ruminants result in significant economic losses worldwide. The etiological agents are three species from the genus Pestivirus, family Flaviviridae, including Bovine Viral Diarrhea Virus type 1 (BVDV-1), BVDV-2, Border Disease Virus (BDV), and an atypical pestivirus named HoB...

Methicillin resistant staphylococci associated with bovine mastitis and their zoonotic importance

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S. Vishnupriya

2014-06-01

Full Text Available Aim: The present study was conducted to determine the zoonotic importance of methicillin resistant staphylococci associated with bovine mastitis and their potential role in transmission to animal handlers. Materials and Methods: A total of 158 milk samples from bovine mastitis cases and 126 nasal swabs from the animal handlers were sampled in and around Pondicherry (Southern India. The Presence of Staphylococcal organism was confirmed by PCR amplification using the genus specific primers and among the isolated Staphylococci; methicillin resistance was identified by genetic amplification of mec A methicillin resistant gene. Then the amplified gene from the bacteria expressing the mecA gene (PBP2a (~2kb fragment was further sequenced using four sets of primer pairs and aligned for determining their genetic relatedness between the sequences. Both phenotypic and genotypic analysis was carried out for the six MRS isolates (three bovine and three human in this study. Results: Out of 158 mastitis milk samples; 96 and 19 bovine isolates were found to be positive for Staphylococcal genus specific PCR and methicillin resistant (mecA gene PCR, respectively. Similarly, Out of 126 human nasal swabs, 64 and 13 human isolates were found to be positive for Staphylococcal genus specific PCR and mec A gene PCR, respectively. Among the 160 staphylococcal isolates (Bovine and Human origin; 51 were identified as coagulase-positive staphylococci (CPS and remaining as coagulase-negative staphylococci (CONS. The results obtained in this study revealed the presence of many species of Staphylococci but the predominant species were Staphylococcus aureus and S. epidermidis. The Sequence analysis of the mec A gene of human isolates obtained in this study had a maximum identity (99% -100% with the bovine isolates. Conclusion: The phenotypic and genotypic analysis carried out for the six MRS (Methicillin Resistant Staphylococci isolates in this study were indistinguishable

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4 Strains Isolated from In Vitro Bovine Embryos production in Argentina.

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Erika González Altamiranda

Full Text Available Bovine herpesvirus 4 (BoHV-4 is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these

Genomic regions associated with bovine milk fatty acids in both summer and winter milk samples

NARCIS (Netherlands)

Bouwman, A.C.; Visker, M.H.P.W.; Arendonk, van J.A.M.; Bovenhuis, H.

2012-01-01

Background - In this study we perform a genome-wide association study (GWAS) for bovine milk fatty acids from summer milk samples. This study replicates a previous study where we performed a GWAS for bovine milk fatty acids based on winter milk samples from the same population. Fatty acids from

Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle.

Science.gov (United States)

Crawshaw, Timothy R; Brown, Ian H; Essen, Steve C; Young, Stuart C L

2008-10-01

Sporadic cases of an acute fall in milk production, "milk drop", were investigated in a Holstein Friesian dairy herd in Devon. The investigation was a case control study with two controls per case. Paired blood samples demonstrated that rising antibody titres to human influenza A/England/333/80 (H1N1) and human influenza A/Eng/427/88 (H3N2) were associated with an acute fall in milk production. Rising titres to bovine respiratory syncytial virus (BRSV), bovine virus diarrhoea virus (BVD), infectious bovine rhinotracheitis (IBR) and parainfluenza virus 3 (PI3) were not associated with an acute fall in milk production. Cases with rises in antibody to influenza A had significantly higher respiratory scores and rectal temperatures than their controls. The mean loss of milk production for the cases with rises in antibody to influenza A compared to their controls was 159.9L. This study provides further evidence that influenza A persists in cattle and causes clinical disease.

Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

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Twizere, J C; Kerkhofs, P; Burny, A; Portetelle, D; Kettmann, R; Willems, L

2000-11-01

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

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Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

2018-04-11

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Epstein-Barr virus-associated lymphomas.

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

2017-10-19

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

Real-Time Detection of a Virus Using Detection Dogs.

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Angle, T Craig; Passler, Thomas; Waggoner, Paul L; Fischer, Terrence D; Rogers, Bart; Galik, Patricia K; Maxwell, Herris S

2015-01-01

Viral infections are ubiquitous in humans, animals, and plants. Real-time methods to identify viral infections are limited and do not exist for use in harsh or resource-constrained environments. Previous research identified that tissues produce unique volatile organic compounds (VOC) and demonstrated that VOC concentrations change during pathologic states, including infection, neoplasia, or metabolic disease. Patterns of VOC expression may be pathogen specific and may be associated with an odor that could be used for disease detection. We investigated the ability of two trained dogs to detect cell cultures infected with bovine viral diarrhea virus (BVDV) and to discriminate BVDV-infected cell cultures from uninfected cell cultures and from cell cultures infected with bovine herpes virus 1 (BHV 1) and bovine parainfluenza virus 3 (BPIV 3). Dogs were trained to recognize cell cultures infected with two different biotypes of BVDV propagated in Madin-Darby bovine kidney cells using one of three culture media. For detection trials, one target and seven distractors were presented on a scent wheel by a dog handler unaware of the location of targets and distractors. Detection of BVDV-infected cell cultures by Dog 1 had a diagnostic sensitivity of 0.850 (95% CI: 0.701-0.942), which was lower than Dog 2 (0.967, 95% CI: 0.837-0.994). Both dogs exhibited very high diagnostic specificity (0.981, 95% CI: 0.960-0.993) and (0.993, 95% CI: 0.975-0.999), respectively. These findings demonstrate that trained dogs can differentiate between cultured cells infected with BVDV, BHV1, and BPIV3 and are a realistic real-time mobile pathogen sensing technology for viral pathogens. The ability to discriminate between target and distractor samples plausibly results from expression of unique VOC patterns in virus-infected and -uninfected cells.

Bioreactor production of recombinant herpes simplex virus vectors.

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Knop, David R; Harrell, Heather

2007-01-01

Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

DEFF Research Database (Denmark)

Pandya, Mital; Rasmussen, Michael; Hansen, Andreas

2015-01-01

Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation......, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular...... pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network...

The role of viral population diversity in adaptation of bovine coronavirus to new host environments.

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Monica K Borucki

Full Text Available The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing and 38,000×(Illumina. The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

Bovine Herpes Virus 1 (BHV-1) and Herpes Simplex Virus Type 1 (HSV-1) Promote Survival of Latently Infected Sensory Neurons, in Part by Inhibiting Apoptosis

Science.gov (United States)

Jones, Clinton

2013-01-01

α-Herpesvirinae subfamily members, including herpes simplex virus type 1 (HSV-1) and bovine herpes virus 1 (BHV-1), initiate infection in mucosal surfaces. BHV-1 and HSV-1 enter sensory neurons by cell-cell spread where a burst of viral gene expression occurs. When compared to non-neuronal cells, viral gene expression is quickly extinguished in sensory neurons resulting in neuronal survival and latency. The HSV-1 latency associated transcript (LAT), which is abundantly expressed in latently infected neurons, inhibits apoptosis, viral transcription, and productive infection, and directly or indirectly enhances reactivation from latency in small animal models. Three anti-apoptosis genes can be substituted for LAT, which will restore wild type levels of reactivation from latency to a LAT null mutant virus. Two small non-coding RNAs encoded by LAT possess anti-apoptosis functions in transfected cells. The BHV-1 latency related RNA (LR-RNA), like LAT, is abundantly expressed during latency. The LR-RNA encodes a protein (ORF2) and two microRNAs that are expressed in certain latently infected neurons. Wild-type expression of LR gene products is required for stress-induced reactivation from latency in cattle. ORF2 has anti-apoptosis functions and interacts with certain cellular transcription factors that stimulate viral transcription and productive infection. ORF2 is predicted to promote survival of infected neurons by inhibiting apoptosis and sequestering cellular transcription factors which stimulate productive infection. In addition, the LR encoded microRNAs inhibit viral transcription and apoptosis. In summary, the ability of BHV-1 and HSV-1 to interfere with apoptosis and productive infection in sensory neurons is crucial for the life-long latency-reactivation cycle in their respective hosts. PMID:25278776

Serological evidence of Hobi-like virus circulation in Argentinean water buffalo

Science.gov (United States)

Hobi-like pestiviruses (also known as bovine viral diarrhea virus 3) have been sporadically reported from naturally infected cattle in Brazil, Asia and Europe. Although Hobi-like viruses seem to be endemic in Brazilian bovines and buffalo, they have not been studied in the other countries of South A...

Genetic association of marbling score with intragenic nucleotide variants at selection signals of the bovine genome.

Science.gov (United States)

Ryu, J; Lee, C

2016-04-01

Selection signals of Korean cattle might be attributed largely to artificial selection for meat quality. Rapidly increased intragenic markers of newly annotated genes in the bovine genome would help overcome limited findings of genetic markers associated with meat quality at the selection signals in a previous study. The present study examined genetic associations of marbling score (MS) with intragenic nucleotide variants at selection signals of Korean cattle. A total of 39 092 nucleotide variants of 407 Korean cattle were utilized in the association analysis. A total of 129 variants were selected within newly annotated genes in the bovine genome. Their genetic associations were analyzed using the mixed model with random polygenic effects based on identical-by-state genetic relationships among animals in order to control for spurious associations produced by population structure. Genetic associations of MS were found (Pdirectional selection for greater MS and remain selection signals in the bovine genome. Further studies of fine mapping would be useful to incorporate favorable alleles in marker-assisted selection for MS of Korean cattle.

Bovine leukaemia virus genotypes 5 and 6 are circulating in cattle from the state of São Paulo, Brazil.

Science.gov (United States)

Gregory, Lilian; Carrillo Gaeta, Natália; Araújo, Jansen; Matsumiya Thomazelli, Luciano; Harakawa, Ricardo; Ikuno, Alice A; Hiromi Okuda, Liria; de Stefano, Eliana; Pituco, Edviges Maristela

2017-12-01

Enzootic bovine leucosis (EBL) is a silent disease caused by a retrovirus [bovine leukaemia virus (BLV)]. BLV is classified into almost 10 genotypes that are distributed in several countries. The present research aimed to describe two BLV gp51 env sequences of strains detected in the state of São Paulo, Brazil and perform a phylogenetic analysis to compare them to other BLV gp51 env sequences of strains around the world. Two bovines from different herds were admitted to the Bovine and Small Ruminant Hospital, School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil. In both, lymphosarcoma was detected and the presence of BLV was confirmed by nested PCR. The neighbour-joining algorithm distance method was used to genotype the BLV sequences by phylogenetic reconstruction, and the maximum likelihood method was used for the phylogenetic reconstruction. The phylogeny estimates were calculated by performing 1000 bootstrap replicates. Analysis of the partial envelope glycoprotein (env) gene sequences from two isolates (25 and 31) revealed two different genotypes of BLV. Isolate 25 clustered with ten genotype 6 isolates from Brazil, Argentina, Thailand and Paraguay. On the other hand, isolate 31 clustered with two genotype 5 isolates (one was also from São Paulo and one was from Costa Rica). The detected genotypes corroborate the results of previous studies conducted in the state of São Paulo, Brazil. The prediction of amino acids showed substitutions, particularly between positions 136 and 150 in 11 out of 13 sequences analysed, including sequences from GenBank. BLV is still important in Brazil and this research should be continued.

Vaccination of cattle against bovine viral diarrhoea

NARCIS (Netherlands)

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

1999-01-01

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

Evaluating enzootic bovine leukemia virus infection by means of molecular probe compared with the results of serological tests

International Nuclear Information System (INIS)

Reichert, M.; Grundbock, J.; Rulka, J.; Kozaczynska, B.; Stec, J.

1994-01-01

The present studies were aimed at determining the relation between the finding obtained by means of serological tests and the specific molecular probe. Serological tests were performed according to the methods recommended by the Polish Ministry of Agriculture; ELISA was run with ''Bioveta'' and ''Rhone Merieux'' kits and the AGID test was performed with EBL antigen made in our laboratory. The molecular probe was prepared from the previously cloned provirus DNA of EBL virus. The EBL provirus was detected in 28 samples taken from 44 randomly selected cows in three herds on which a leukemia eradication programme was in process. Three sera out of 28 positive reacting animals were negative in AGID test and only one serum in ELISA. The results indicate that the use of a specific molecular probe has some advantages in the diagnosis of latent virus infections. Besides, it can be applied in the studies on the pathogenesis of enzootic bovine leukemia. (author). 13 refs, 1 fig., 1 tab

Emerging animal viruses: real threats or simple bystanders?

Directory of Open Access Journals (Sweden)

Eduardo Furtado Flores

2013-10-01

Full Text Available The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV, is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae, tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV, was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The

Dimerization of BTas is required for the transactivational activity of bovine foamy virus

International Nuclear Information System (INIS)

Tan Juan; Qiao Wentao; Xu Fengwen; Han Hongqi; Chen Qimin; Geng Yunqi

2008-01-01

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activity on the LTR and IP. Furthermore, BTas (Δ46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV

Engineered Viruses as Genome Editing Devices

Science.gov (United States)

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-01-01

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

Acute and latent infection by bovine herpesvirus type 2 in a guinea pig model.

Science.gov (United States)

Torres, Fabrício Dias; Cargnelutti, Juliana Felipetto; Masuda, Eduardo Kenji; Weiblen, Rudi; Flores, Eduardo Furtado

2010-02-01

Bovine herpetic mammillits is a self-limiting cutaneous disease of the udder and teats of cows associated with bovine herpesvirus 2 (BoHV-2) whose pathogenesis is poorly understood. This article describes the use of guinea pigs (Cavia porcellus) to study the pathogenesis of BoHV-2 infection. Twelve weanling female guinea pigs inoculated subcutaneously with BoHV-2 in the genitalia and teats developed local hyperemia, edema, vesicles, ulcers and scabs. Infectious virus was recovered between days 3 and 7 post-infection (pi) from the genital area (9/12) and teats (1/12); and all inoculated animals seroconverted (virus-neutralizing titers of 16-128). Histological examination of lesions revealed lymphoplasmacytic perivascular infiltrates and intranuclear inclusion bodies in keratinocytes. PCR examination of tissues collected at day 35 pi detected latent viral DNA predominantly in lumbosacral spinal segments. In another experiment, eight females inoculated with BoHV-2 in the genitalia and treated with dexamethasone (Dx) at day 35 pi developed mild to moderate local signs, yet no virus could be recovered from lesions. PCR examination of spinal segments from these animals confirmed the presence of latent viral DNA. These results demonstrate that guinea pigs are susceptible to BoHV-2 infection and therefore may be used to study selected aspects of BoHV-2 biology.

Frequency of antibodies against bovine herpesvirus type 1 (BoHV-1 in beef cattle not vaccinated

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Ermilton Junio Pereira de Freitas

2014-06-01

Full Text Available Bovine herpesvirus type 1 (BoHV-1, is responsible for clinical manifestations such as infectious bovine rhinotracheitis, abortion, conjunctivitis, infectious pustular vulvovaginitis and balanoposthitis. This virus has been responsible for major losses in different productive and reproductive herds in the country. Thus, the objective of this study was to estimate the frequency of antibodies against BoHV-1 in beef heifers not vaccinated in Microregion of Imperatriz, Maranhao, and identify the age group most affected by the virus, as well as a study of factors associated with virus infection and to evaluate the indirect ELISA using the serum neutralization (SN as a reference standard. The study was conducted in 48 herds, cutting, distributed in 12 counties of Microregion of Imperatriz. The samples were collected from female cattle stratified into three age groups, ? 12 months, between 12 and 36 months and ? 36 months of age. The samples were subjected to two serological tests, ELISA and SN. In each herd, an epidemiological questionnaire was applied in order to obtain information on management and reproductive sanitary, for the study of risk factors. The frequency of antibodies against BoHV-1 in Microregion of Imperatriz was 63.23%, and the municipalities of Açailândia Buritirana showed the highest frequencies, both with 80.44%, the most affected age group, the Microregion, was animals aged ? 36 months (69.65%. Based on the results we can conclude that the frequency of antibodies against BoHV-1 is high, between the age groups most affected were the animals aged ? 36 months were considered risk factors for virus transmission, return to estrus (OR=1.874, recovery of animals from other states / region (OR=1.365 and the creation of goat / sheep associated with bovine (OR=1.348, the indirect ELISA technique showed moderate concordance when compared to SN technique, which is the gold standard technique for diagnosis of BoHV-1.

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

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

2017-07-18

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

Molecular cloning, expression and characterization of bovine UQCC and its association with body measurement traits

DEFF Research Database (Denmark)

Liu, Yongfeng; Zan, Linsen; Zhao, Shuanping

2010-01-01

effects on the BL (p = 0.0047) and CD (p = 0.0454. Regarding association analysis of combination of the two SNPs, there are significant effects on the BL (p = 0.0215), CD (p = 0.0282) and PBW (p = 0.0329) in the total population. The results suggest that the UQCC gene is a candidate gene of body...... measurement traits in bovine reproduction and breeding, and provide data for establishing of an animal model using cattle to study big animal body type....... models of height as well as other stature indexes. We have cloned the cDNA sequence coding UQCC gene in bovine. Genomic structural analysis indicated that bovine UQCC shares a high similarity with human UQCC. Furthermore, Real-Time PCR analysis show that the expression of bovine UQCC is remarkably...

Implementation of polymerase chain reaction (PCR and Real-Time PCR in quick identification of bovine herpesvirus 1

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Milić Nenad

2010-01-01

Full Text Available Examinations were performed on 65 samples of nasal smeas taken from calves and young cows with clinical symptoms of respiratory infection to determine the presence of the bovine herpes virus 1 using parallel implementation of molecular and standard methods of virological diagnostics. The appearance of a cytopathogenic effect (CPE was not established in inoculated cell lines 24h, 48h and 72h following inoculation, or after two successive passages of the examined material sample through these cell lines. The application of polymerize chain reaction (PCR using a primer for glucoprotein B and thymidine - kinasis, established the presence of bovine herpes virus 1 nucleic acid in one sample of a bovine nasal smear, while the presence of this virus was established in three samples in an examination of the nasal smear samples using the Real-Time PCR method.

Association between phage types and antimicrobial resistance among bovine isolates of Staphylococcus aureus in 10 countries

DEFF Research Database (Denmark)

Vintov, J.; Aarestrup, Frank Møller; Zinn, C. E.

2003-01-01

This study was conducted to investigate the diversity of phage types and associations between penicillin resistance and phage types among 815 Staphylococcus aureus isolates from bovine mastitis in nine European countries and USA. All isolates were examined for susceptibility to antimicrobial agents...... associated with penicillin resistance in contrast to phage group I (P = 0.0023) and phage complex-80 (P = 0.0066). This study confirms that a large number of phage types of S. aureus cause bovine mastitis, but that some types predominate. In addition, these findings could indicate that the use of penicillin...... in the bovine environment has selected for specific types of S. aureus in countries with a high frequency of resistance. (C) 2003 Elsevier B.V. All rights reserved....

Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus

DEFF Research Database (Denmark)

Larska, Magdalena; Polak, Mirosław P.; Liu, Lihong

2013-01-01

Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental...... infection of calves with atypical pestivirus, BVDV-1 and a combination of both viruses have been examined by routine and new diagnostic tests to validate their robustness and sensitivity. As expected, virus neutralization tests using homologous virus were able to differentiate the two groups infected...... by BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect...

Bovine respiratory syncytial virus : immunopathology and vaccine evaluation

NARCIS (Netherlands)

Antonis, A.F.G.

2010-01-01

Human and bovine RSVs cause severe disease in humans and in cattle respectively. They have been recognised as important respiratory pathogens in the last five decades, and this has resulted in significant research activities on the pathogenesis and intervention strategies around the world.

77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

Science.gov (United States)

2012-05-21

... Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY: Animal and Plant Health... derived from bovines with regard to bovine spongiform encephalopathy. This action will allow interested... importation of live bovines and products derived from bovines with regard to bovine spongiform encephalopathy...

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

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Rahpaya, Sayed Samim; Tsuchiaka, Shinobu; Kishimoto, Mai; Oba, Mami; Katayama, Yukie; Nunomura, Yuka; Kokawa, Saki; Kimura, Takashi; Kobayashi, Atsushi; Kirino, Yumi; Okabayashi, Tamaki; Nonaka, Nariaki; Mekata, Hirohisa; Aoki, Hiroshi; Shiokawa, Mai; Umetsu, Moeko; Morita, Tatsushi; Hasebe, Ayako; Otsu, Keiko; Asai, Tetsuo; Yamaguchi, Tomohiro; Makino, Shinji; Murata, Yoshiteru; Abi, Ahmad Jan; Omatsu, Tsutomu; Mizutani, Tetsuya

2018-05-31

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum ; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes.

Science.gov (United States)

Kubiça, Thaís F; Alves, Sydney H; Weiblen, Rudi; Lovato, Luciane T

2014-01-01

The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 μg mL(-1)) and 1,8-cineole (CC50 = 2996.10 μg mL(-1)) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains

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Dario Amilcar Malacari

2018-04-01

Full Text Available Non-cytopathic (ncp type 2 bovine viral diarrhea virus (BVDV-2 is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124 compared to a high-virulence reference strain (NY-93, using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

Generation of the bovine viral diarrhea virus e0 protein in transgenic astragalus and its immunogenicity in sika deer.

Science.gov (United States)

Gao, Yugang; Zhao, Xueliang; Zang, Pu; Liu, Qun; Wei, Gongqing; Zhang, Lianxue

2014-01-01

The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

Generation of the Bovine Viral Diarrhea Virus E0 Protein in Transgenic Astragalus and Its Immunogenicity in Sika Deer

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Yugang Gao

2014-01-01

Full Text Available The bovine viral diarrhea virus (BVDV, a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR, transcription was verified by reverse transcription- (RT- PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

Clinical infection control in gene therapy : A multidisciplinary conference

NARCIS (Netherlands)

Evans, ME; Jordan, CT; Chang, SMW; Conrad, C; Gerberding, JL; Kaufman, HL; Mayhall, CG; Nolta, JA; Pilaro, AM; Sullivan, S; Weber, DJ; Wivel, NA

2000-01-01

Gene therapy is being studied for the treatment of a variety of acquired and inherited disorders. Retroviruses, adenoviruses, poxviruses, adeno-associated viruses, herpesviruses, and others are being engineered to transfer genes into humans. Treatment protocols using recombinant viruses are being

Bovine neonatal pancytopenia--comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK).

Science.gov (United States)

Euler, Kerstin N; Hauck, Stefanie M; Ueffing, Marius; Deeg, Cornelia A

2013-01-23

Bovine neonatal pancytopenia (BNP) is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV) was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney) cells, the cell line used for production of the associated vaccine. By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK

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Euler Kerstin N

2013-01-01

Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney cells, the cell line used for production of the associated vaccine. Results By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. Conclusions The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

Bovine mastitis may be associated with the deprivation of gut Lactobacillus.

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Ma, C; Zhao, J; Xi, X; Ding, J; Wang, H; Zhang, H; Kwok, L Y

2016-02-01

Bovine mastitis is an economical important microbial disease in dairy industry. Some recent human clinical trials have shown that oral probiotics supplementation could effectively control clinical mastitis, suggesting that the mechanism of mastitis protection might be achieved via the host gut microbiota. We aimed to test our hypothesis that bovine mastitis was related to changes in both the mammary and gut microbial profiles. By quantitative PCR, the milk and faecal microbial profiles of cows with low (1×10 6 cells/ml) somatic cell count (SCC) were compared. Firstly, we observed drastic differences in both the milk and faecal microbial compositions at genus and Lactobacillus-species levels between the two groups. Secondly, the pattern of faecal microbial community changes of mastitis cows was similar to that of the milk, characterised by a general increase in the mastitis pathogens (Enterococcus, Streptococcus and Staphylococcus) and deprivation of Lactobacillus and its members (L. salivarius, L. sakei, L. ruminis, L. delbrueckii, L. buchneri, and L. acidophilus). Thirdly, only the faecal lactobacilli, but not bifidobacteria correlated with the milk microbial communities and SCC. Our data together hint to a close association between bovine mastitis, the host gut and milk microbiota.

Human papilloma virus vaccine associated uveitis.

Science.gov (United States)

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

2014-03-01

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

Immortalized sheep microglial cells are permissive to a diverse range of ruminant viruses.

Science.gov (United States)

Stanton, James B; Swanson, Beryl; Orozco, Edith; Muñoz-Gutiérrez, Juan F; Evermann, James F; Ridpath, Julia F

2017-12-01

Ruminants, including sheep and goats (small ruminants), are key agricultural animals in many parts of the world. Infectious diseases, including many viral diseases, are significant problems to efficient production of ruminants. Unfortunately, reagents tailored to viruses of ruminants, and especially small ruminants, are lacking compared to other animals more typically used for biomedical research. The purpose of this study was to determine the permissibility of a stably immortalized, sheep microglial cell line to viruses that are reported to infect ruminants: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), small ruminant lentiviruses (SRLV), and bovine respiratory syncytial virus (BRSV). Sublines A and H of previously isolated, immortalized, and characterized (CD14-positive) ovine microglial cells were used. Bovine turbinate cells and goat synovial membrane cells were used for comparison. Cytopathic changes were used to confirm infection of individual wells, which were then counted and used to calculate the 50% tissue culture infectious dose. Uninoculated cells served as negative controls and confirmed that the cells were not previously infected with these viruses using polymerase chain reaction (PCR). Inoculation of the two microglial cell sublines with laboratory and field isolates of BVDV, BoHV-1, and BRSV resulted in viral infection in a manner similar to bovine turbinate cells. Immortalized microglia cells are also permissive to SRLV, similar to goat synovial membrane cells. These immortalized sheep microglial cells provide a new tool for the study of ruminant viruses in ruminant microglial cell line.

Primary surveys on molecular epidemiology of bovine viral diarrhea virus 1 infecting goats in Jiangsu province, China.

Science.gov (United States)

Mao, Li; Li, Wenliang; Yang, Leilei; Wang, Jianhui; Cheng, Suping; Wei, Yong; Wang, Qiusheng; Zhang, Wenwen; Hao, Fei; Ding, Yonglong; Sun, Yinhua; Jiang, Jieyuan

2016-09-05

Bovine viral diarrhea virus (BVDV) is a pathogen of domestic and wildlife animals worldwide and is associated with several diseases. In China, there are many reports about genotyping of BVDV strains originated from cattle and pigs, and some of them focused on the geographical distributions of BVDV. Currently, the goat industry in Jiangsu province of China is under going a rapid expansion. Most of these goat farms are backyard enterprises and in close proximity to pig and cattle farms. However, there was very limited information about BVDV infections in goats. The objective of this study was to assess the frequency of BVDV infections of goats, the relationship of these infections to clinical signs and determine what BVDV genotypes are circulating in Jiangsu province. From 236 goat sera collected from six regions in Jiangsu province between 2011 and 2013, BVDV-1 was identified in 29 samples from the five regions by RT-PCR. The BVDV-1 infections occurred with/without clinical signs. Eight different BVDV-1 strains were identified from these positive samples based on the 5'-untranslated region (5'-UTR) sequences, and further clustered into four BVDV-1 subtypes on the phylogenetic analysis. Three were BVDV-1b, two BVDV-1m, two BVDV-1o, and one BVDV-1p, respectively. To our knowledge, this is the first report to investigate the occurrence of BVDV and the genotypes of BVDV infecting goats in China. The results indicated that BVDV-1 infections were indeed present and the viruses were with genetic variations in Chinese goat herds. The information would be very useful for prevention and control of BVDV-1 infections in China.

Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle.

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Natalie Dow

Full Text Available Bovine viral diarrhea virus (BVDV, a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

Evaluation of commercial ELISA kits for detection of antibodies against bovine atypical pestivirus

DEFF Research Database (Denmark)

Larska, Magdalena; Polak, Mirosław P.; Uttenthal, Åse

A group of emerging bovine pestiviruses becomes a possible threat to Bovine Viral diarrhea virus (BVDV) control and eradication programs in the countries of their origin and in the new continents due to the lack of validated detection methods. The use of ELISA kits may be acheaper, time saving...... and less laborious option allowing screening for antibodies in large populations. Since test specific for emerging and new BVDV strains are still under preparation, the purpose of this work was to evaluate available BVDV antibody ELISA assays for their ability to detect antibodies against Hobi-like viruses....... Analysis of a panel of sera obtained from calves experimentally inoculated with Hobi-like virus (isolated from a calf from Thailand) and BVDV type 1 strain using five different ELISA kits in comparison to neutralization test was performed. The specificity and sensitivity of the tests depended greatly...

Bovine lymphocytic leukemia: studies of etiology, pathogenesis and mode of transmission. Progress report No. 17, July 1976--October 1977

Energy Technology Data Exchange (ETDEWEB)

Sorensen, D.K.

1977-07-22

The primary objective of the proposed research will be elucidation of the etiology and pathogenesis of bovine leukemia. We have consistently demonstrated C-type particles in mitogen stimulated lymphocyte cultures from leukemic cows and cows with a persistent lymphocytosis. These particles have been concentrated and partially purified by continuous flow, density gradient, ultracentrifugation. Newborn calves and late stage bovine fetuses have been inoculated with these concentrated cell free preparations. Our current study involves extensive monitoring of these inoculated animals to detect early pre-cancerous changes. The following parameters are being measured: the serological titer against a bovine leukemia associated antigen; the percentage of lymphocytes showing nuclear pockets; the percentage of mitogen stimulated lymphocytes with C-type particles adherent to their surface; the percentage of B-lymphocytes in the peripheral circulation; the complete blood count; and the quantity of bovine leukemia virus (BLV) production as determined by the syncytia induction assay. Additional proposals include: using the monitoring parameters to study animals with the juvenile and thymic forms of leukemia; the examination of adult lymphosarcoma cases to determine which tissues harbor BLV; and lymphocyte subpopulation work to further define which cell types are associated with BLV production and tumor formation.

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

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

2007-09-01

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

Association between Genes BoLA-DRB3.2*8 and BoLA-DRB3.2*12 with Resistance and BoLA-DRB3.2*16 with Susceptibility to Infection by Bovine Leukemia Virus

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C Úsuga-Monroy*, JJ Echeverri Zuluaga and A López-Herrera

2016-11-01

Full Text Available The Bovine Leukemia Virus (BLV is a retrovirus that affects the immune system of cattle as their target cells are B lymphocytes. Some polymorphisms at the BoLA-DRB 3.2 gene have been associated with resistance/susceptibility to diseases. The objective of this research was to determine the polymorphisms at the BoLA-DRB 3.2 gene and associate them with resistance (R, neutrality (N or susceptibility (S to BLV in a Holstein cow population.500 blood samples were taken. Nested PCR was performed for detecting BLV virus and PCR-RFLP was performed to identify alleles of gene BoLA-DRB 3.2. Susceptibility was determined using odds ratio (OR and P value. According to their genotype, cows were classified in homozygous (R/R, N/N, or S/S and heterozygous (R/N, R/S, N/S. BLV molecular prevalence was 44%. The most frequent allele was BoLA-DRB3.2*22 (16.8%, alleles associated with resistance to BLV were BoLA-DRB3.2*8 (OR=1.489; P<0.10 and BoLA-DRB3.2*12 (OR=3.897; P<0.10 and allele BoLA-DRB3.2*16 (OR=0.710; P<0.10 was associated with susceptibility. Allele BoLA-DRB3.2*8 had the highest allelic frequency for negative cows (0.19. 63.7% of cows with genotype RN and 70% of cows with genotype RR were resistant to infection by BLV. Alleles R and S have a dominant effect on allele N (P<0.05. The use of reliable diagnostic techniques in conjunction with identification of resistant or susceptible animals can monitor the progress of the disease in dairy herds. Alleles BoLA-DRB3.2*8 and *12 were positively related to the disease and therefore cows have low risk of infection, unlike allele BoLA-DRB3.2*16 which was negatively related and animals have high risk for the disease.

Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior.

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Souza, F N; Piepers, S; Della Libera, A M M P; Heinemann, M B; Cerqueira, M M O P; De Vliegher, S

2016-04-01

Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifer's teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

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Van den Broeke, A; Cleuter, Y; Beskorwayne, T; Kerkhofs, P; Szynal, M; Bagnis, C; Burny, A; Griebel, P

2001-02-01

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

Biochemical map of polypeptides specified by foot-and-mouth disease virus.

OpenAIRE

Grubman, M J; Robertson, B H; Morgan, D O; Moore, D M; Dowbenko, D

1984-01-01

Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical pol...

Prevalence and associated risk factors for bovine tick infestation in two districts of lower Punjab, Pakistan.

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Sajid, Muhammad Sohail; Iqbal, Zafar; Khan, Muhammad Nisar; Muhammad, Ghulam; Khan, Muhammad Kasib

2009-12-01

Bovine tick infestation is still a serious nuisance to livestock and the dairy industry of Pakistan. The current paper reports the prevalence and associated risk factors for bovine tick infestation in the districts Layyah and Muzaffargarh of lower Punjab, Pakistan. A questionnaire-based survey was conducted to identify and to quantify variation in the prevalence of bovine tick infestation with respect to host (age, species, sex, and breed) and environmental (geographical area and climate) determinants. Multiple stage cluster random sampling was used and 3500 cattle and buffaloes from the two districts were selected. Prevalence of bovine tick infestation was significantly higher (OR=1.95; p2025; 47.3%). Hyalomma anatolicum anatolicum was the major tick species (33.5%; 1173/3500), followed by Rhipicephalus sanguineus (13%; 456/3500). The highest monthly prevalence in both the districts was found in July. Ticks were not found in Layyah from November to March and in Muzaffargarh from December to March. The average number of ticks was proportional to the prevalence of infestation. Also, tick infestation in a 7cmx7cm dewlap of the animal was proportional to that of the rest of body. Prevalence of tick infestation was associated (p<0.05) with district, host species and breed. In cattle, prevalence of tick infestation was associated (p<0.05) with age and sex of host. The results of this study provide better understanding of disease epidemiology in the study districts, which will help for planning of control strategies.

Polyomavirus specific cellular immunity: from BK-virus-specific cellular immunity to BK-virus-associated nephropathy ?

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manon edekeyser

2015-06-01

Full Text Available In renal transplantation, BK-virus-associated nephropathy has emerged as a major complication, with a prevalence of 5–10% and graft loss in >50% of cases. BK-virus is a member of the Polyomavirus family and rarely induces apparent clinical disease in the general population. However, replication of polyomaviruses, associated with significant organ disease, is observed in patients with acquired immunosuppression, which suggests a critical role for virus-specific cellular immunity to control virus replication and prevent chronic disease. Monitoring of specific immunity combined with viral load could be used to individually assess the risk of viral reactivation and virus control. We review the current knowledge on BK-virus specific cellular immunity and, more specifically, in immunocompromised patients. In the future, immune-based therapies could allow us to treat and prevent BK-virus-associated nephropathy.

[Bovine herpesvirus 4 (BoHV-4): general aspects of the biology and status in Argentina].

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Morán, Pedro E; Pérez, Sandra E; Odeón, Anselmo C; Verna, Andrea E

2015-01-01

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle with respiratory infections, vulvovaginitis, mastitis, abortions, endometritis and from apparently healthy animals throughout the world. Although it has not yet been established as causal agent of a specific disease entity, it is primarily associated with reproductive disorders of cattle. This virus can infect a wide range of species, either in vivo or in vitro. Two groups of prototype strains were originated from the first isolates: the DN599-type strains (American group) and the Movar-type strains (European group). In Argentina, BoHV-4 was isolated and characterized in 2007 from vaginal discharge samples taken from cows that had aborted. So far, more than 40 isolates, mainly associated with aborting bovine females have been registered in our country. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

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Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2015-04-19

Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

Not all cows are epidemiologically equal: quantifying the risks of bovine viral diarrhoea virus (BVDV) transmission through cattle movements.

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Gates, M Carolyn; Humphry, Roger W; Gunn, George J; Woolhouse, Mark E J

2014-10-17

Many economically important cattle diseases spread between herds through livestock movements. Traditionally, most transmission models have assumed that all purchased cattle carry the same risk of generating outbreaks in the destination herd. Using data on bovine viral diarrhoea virus (BVDV) in Scotland as a case example, this study provides empirical and theoretical evidence that the risk of disease transmission varies substantially based on the animal and herd demographic characteristics at the time of purchase. Multivariable logistic regression analysis revealed that purchasing pregnant heifers and open cows sold with a calf at foot were associated with an increased risk of beef herds being seropositive for BVDV. Based on the results from a dynamic within-herd simulation model, these findings may be partly explained by the age-related probability of animals being persistently infected with BVDV as well as the herd demographic structure at the time of animal introductions. There was also evidence that an epidemiologically important network statistic, "betweenness centrality" (a measure frequently associated with the potential for herds to acquire and transmit disease), was significantly higher for herds that supplied these particular types of replacement beef cattle. The trends for dairy herds were not as clear, although there was some evidence that open heifers and open lactating cows were associated with an increased risk of BVDV. Overall, these findings have important implications for developing simulation models that more accurately reflect the industry-level transmission dynamics of infectious cattle diseases.

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

Science.gov (United States)

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

1999-01-01

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

Real-Time Detection of a Virus Using Detection Dogs

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Craig eAngle

2016-01-01

Full Text Available Viral infections are ubiquitous in humans, animals, and plants. Real-time methods to identify viral infections are limited and do not exist for use in harsh or resource-constrained environments. Previous research identified that tissues produce unique volatile organic compounds (VOC and demonstrated that VOC concentrations change during pathologic states including infection, neoplasia, or metabolic disease. Patterns of VOC expression may be pathogen-specific and may be associated with an odor that could be used for disease detection.We investigated the ability of two trained dogs to detect cell cultures infected with bovine viral diarrhea virus (BVDV and to discriminate BVDV-infected cell cultures from uninfected cell cultures and from cell cultures infected with bovine herpes virus 1 (BHV 1 and bovine parainfluenza virus 3 (BPIV 3. Dogs were trained to recognize cell cultures infected with two different biotypes of BVDV propagated in MDBK cells using one of three culture media. For detection trials, one target and seven distractors were presented on a scent wheel by a dog handler unaware of the location of targets and distractors.Detection of BVDV- infected cell cultures by Dog 1 had a diagnostic sensitivity of 0.850 (95% CI: 0.701 - 0.942, which was lower than Dog 2 (0.967, 95% CI: 0.837 - 0.994. Both dogs exhibited very high diagnostic specificity (0.981, 95% CI: 0.960 - 0.993 and (0.993, 95% CI: 0.975 - 0.999, respectively.These findings demonstrate that trained dogs can differentiate between cultured cells infected with BVDV, BHV1, and BPIV3 and are a realistic real-time mobile pathogen sensing technology for viral pathogens. The ability to discriminate between target and distractor samples plausibly results from expression of unique VOC patterns virus-infected and uninfected cells.

Infection of endothelial cells by common human viruses.

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Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Prevalence of bovine leukemia virus (BLV infection in the northeast of Iran

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Shalaleh Mousavi

2014-06-01

Full Text Available The purpose of this study was to determine the prevalence of bovine leukemia virus (BLV in Khorasan Razavi and Khorasan Shomali provinces which are the main provinces located in the northeast of Iran. Total number of 429 blood samples were collected from industrial dairy herds. The samples were categorized based on province, age (2-3, 4-6, and 7-10 years old, calving (≤ 2, 3-5, and > 5 and herd size (≤ 100, 101-250, and > 250 and examined by indirect ELISA. The results of this study showed that 109 (25.4% out of 429 serum samples were BLV seropositive. The BLV prevalence among cattle of dairy herds of Khorasan Razavi and Khorasan Shomali provinces were 29.8% and 1.5%, respectively. The results showed that the number of seropositive animals was increased significantly with the age (p 5 was 15.5%, 33.0% and 42.9%, respectively, with a significant difference between calving ≤ 2 and > 5 (p 250 was 19.7%, 14.3% and 42.1%, respectively, which was significantly higher in herds with more than 250 cattle (p < 0.05. This study revealed that BLV infection in dairy herds of northeast of Iran was influenced by geographical location (province, age, calving and herd size.

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

International Nuclear Information System (INIS)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-01-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [ 3 H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating

HoBi-like viruses: an emerging group of pestiviruses

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The genus Pestivirus is composed by four important pathogens of livestock: bovine viral diarrhea virus types 1 and 2 (BVDV-1 and BVDV-2), classical swine fever virus (CSFV) and border disease virus of sheep (BDV). BVDV are major pathogens of cattle and infection results in significant economic losse...

Evaluation of experimental vaccines for bovine viral diarrhea in bovines, ovines and guinea pigs Evaluación de vacunas experimentales para la diarrea viral bovina en bovinos, ovinos y cobayos

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F. Fernández

2009-06-01

Full Text Available The bovine viral diarrhea virus (BVDV infection control should be based on elimination of persistently infected animals and on immunization through vaccination, to prevent fetal infection. However, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. This study evaluated the humoral immune response against homologous and heterologous strains of 7 inactivated BVDV vaccines, in bovines and two experimental models (ovine and guinea pig which might be used to test candidate vaccines. Vaccines formulated with BVDV Singer, Oregon, NADL, and multivalent, induced seroconversion in the three animal models studied, reaching antibody titres higher than 2. Vaccine containing 125C -genotype 2- only induced a low antibody response in ovine, while VS-115 NCP vaccine was not immunogenic. Furthermore, bovine sera at 60 dpv presented homologous as well as heterologous antibody response, indicating a high degree of cross-reactivity among the strains studied. However, when bovine sera were tested against the Argentine field strain 00-693, they showed the lowest levels of cross-reactivity, suggesting the need of continued surveillance to identify and characterize emerging field BVDV strains. Finally, optimal correlations between bovine-ovine and bovine-guinea pig models were observed, indicating that two alternative species could replace bovines when testing the immunogenicity of BVDV candidate vaccines.El control del virus de la diarrea viral bovina (VDVB se basa en la eliminación de animales persistentemente infectados, y la inmunización de hembras para prevenir infecciones fetales. La eficiencia de estas vacunas es variable por su baja inmunogenicidad. Se evaluó la respuesta inmune humoral contra virus homólogos y heterólogos de 7 vacunas experimentales inactivadas del VDVB en bovinos y en dos modelos experimentales (ovinos y cobayos. Las vacunas conteniendo VDVB Singer, Oregon, NADL y polivalentes indujeron seroconversión en

Clinical presentation resembling mucosal disease associated with 'HoBi'-like pestivirus in a field outbreak

Science.gov (United States)

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV) And Border disease virus (BDV). Recently, atypical pestiviruses (‘HoBi’-like pestiviruses) were iden...

Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

Science.gov (United States)

Castro, Eliana F; Fabian, Lucas E; Caputto, María E; Gagey, Dolores; Finkielsztein, Liliana M; Moltrasio, Graciela Y; Moglioni, Albertina G; Campos, Rodolfo H; Cavallaro, Lucía V

2011-06-01

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

Science.gov (United States)

Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

2012-01-01

Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

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

Science.gov (United States)

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

2018-05-01

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

Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV in four bovine species in China.

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Mingliang Deng

Full Text Available To determine the nationwide status of persistent BVDV infection in different bovine species in China and compare different test methods, a total of 1379 serum samples from clinical healthy dairy cattle, beef cattle, yaks (Bos grunniens, and water buffalo (Bubalus bubalis were collected in eight provinces of China from 2010 to 2013. The samples were analyzed using commercial antibody (Ab and antigen (Ag detection kits, and RT-PCR based on the 5'-UTR and Npro gene sequencing. Results showed that the overall positive rates for BVDV Ab, Ag and RT-PCR detection were 58.09% (801/1379, 1.39% (14/1010, and 22.64% (146/645, respectively, while the individual positive rates varied among regions, species, and farms. The average Ab-positive rates for dairy cattle, beef cattle, yaks, and water buffalo were 89.49% (298/333, 63.27% (248/392, 45.38% (236/520, and 14.18% (19/134, respectively, while the Ag-positive rates were 0.00% (0/116, 0.77% (3/392, 0.82% (3/368, and 5.97% (8/134, respectively, and the nucleic acid-positive rates detected by RT-PCR were 32.06% (42/131, 13.00% (26/200, 28.89% (52/180, and 19.40% (26/134, respectively. In addition, the RT-PCR products were sequenced and 124 5'-UTR sequences were obtained. Phylogenetic analysis of the 5'-UTR sequences indicated that all of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-1b (33.06%, BVDV-1m (49.19%, or a new cluster, designated as BVDV-1u (17.74%. Phylogenetic analysis based on Npro sequences confirmed this novel subtype. In conclusion, this study revealed the prevalence of BVDV-1 in bovine species in China and the dominant subtypes. The high proportion of bovines with detectable viral nucleic acids in the sera, even in the presence of high Ab levels, revealed a serious threat to bovine health.

Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China.

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Deng, Mingliang; Ji, Sukun; Fei, Wentao; Raza, Sohail; He, Chenfei; Chen, Yingyu; Chen, Huanchun; Guo, Aizhen

2015-01-01

To determine the nationwide status of persistent BVDV infection in different bovine species in China and compare different test methods, a total of 1379 serum samples from clinical healthy dairy cattle, beef cattle, yaks (Bos grunniens), and water buffalo (Bubalus bubalis) were collected in eight provinces of China from 2010 to 2013. The samples were analyzed using commercial antibody (Ab) and antigen (Ag) detection kits, and RT-PCR based on the 5'-UTR and Npro gene sequencing. Results showed that the overall positive rates for BVDV Ab, Ag and RT-PCR detection were 58.09% (801/1379), 1.39% (14/1010), and 22.64% (146/645), respectively, while the individual positive rates varied among regions, species, and farms. The average Ab-positive rates for dairy cattle, beef cattle, yaks, and water buffalo were 89.49% (298/333), 63.27% (248/392), 45.38% (236/520), and 14.18% (19/134), respectively, while the Ag-positive rates were 0.00% (0/116), 0.77% (3/392), 0.82% (3/368), and 5.97% (8/134), respectively, and the nucleic acid-positive rates detected by RT-PCR were 32.06% (42/131), 13.00% (26/200), 28.89% (52/180), and 19.40% (26/134), respectively. In addition, the RT-PCR products were sequenced and 124 5'-UTR sequences were obtained. Phylogenetic analysis of the 5'-UTR sequences indicated that all of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-1b (33.06%), BVDV-1m (49.19%), or a new cluster, designated as BVDV-1u (17.74%). Phylogenetic analysis based on Npro sequences confirmed this novel subtype. In conclusion, this study revealed the prevalence of BVDV-1 in bovine species in China and the dominant subtypes. The high proportion of bovines with detectable viral nucleic acids in the sera, even in the presence of high Ab levels, revealed a serious threat to bovine health.

Epizootic of ovine congenital malformations associated with Schmallenberg virus infection.

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van den Brom, R; Luttikholt, S J M; Lievaart-Peterson, K; Peperkamp, N H M T; Mars, M H; van der Poel, W H M; Vellema, P

2012-02-01

Epizootic outbreaks of congenital malformations in sheep are rare and have, to the best of our knowledge, never been reported before in Europe. This paper describes relevant preliminary findings from the first epizootic outbreak of ovine congenital malformations in the Netherlands. Between 25 November and 20 December 2011, congenital malformations in newborn lambs on sheep farms throughout the country were reported to the Animal Health Service in Deventer. Subsequently, small ruminant veterinary specialists visited these farms and collected relevant information from farmers by means of questionnaires. The deformities varied from mild to severe, and ewes were reported to have given birth to both normal and deformed lambs; both male and female lambs were affected. Most of the affected lambs were delivered at term. Besides malformed and normal lambs, dummy lambs, unable to suckle, were born also on these farms. None of the ewes had shown clinical signs during gestation or at parturition. Dystocia was common, because of the lambs' deformities. Lambs were submitted for post-mortem examination, and samples of brain tissue were collected for virus detection. The main macroscopic findings included arthrogryposis, torticollis, scoliosis and kyphosis, brachygnathia inferior, and mild-to-marked hypoplasia of the cerebrum, cerebellum and spinal cord. Preliminary data from the first ten affected farms suggest that nutritional deficiencies, intoxication, and genetic factors are not likely to have caused the malformations. Preliminary diagnostic analyses of precolostral serum samples excluded border disease virus, bovine viral diarrhoea virus, and bluetongue virus. In December 2011, samples of brain tissue from 54 lambs were sent to the Central Veterinary Institute of Wageningen University Research, Lelystad. Real-time PCR detected the presence of a virus, provisionally named the Schmallenberg virus, in brain tissue from 22 of the 54 lambs, which originated from seven of eight

Clinical and virological characteristics of calves experimentally infected with a Brazilian isolate of bovine viral diarrhea virus type 1a

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Luana Marchi Quadros

Full Text Available ABSTRACT: To study the pathogenicity of the Brazilian bovine viral diarrhea virus (BVDV type 1a 241.10 isolate, four calves were intranasally inoculated with a viral suspension containing 107.2 TCID50 mL-1. One calf was left uninoculated and kept in contact with the other calves to investigate viral transmissibility. After inoculation, the animals were monitored daily for clinical signs of infection. The presence of the virus in the blood and nasal secretions was confirmed by virus isolation in cell culture. White blood cells were quantified prior to and every 3 days after infection, and the presence of antibodies was checked every 7 days, starting at day 0 until day 42 post-inoculation (pi. After infection, nasal and ocular serous secretions were observed between days 1 and 5 pi, along with a mild cough from days 2 to 4 pi; however, no severe clinical signs were present. Body temperature was slightly elevated between days 4 and 6 pi. The control calf did not develop any of the signs observed in the infected animals. Cell culture-mediated virus isolation confirmed viremia between days 4 and 8 pi and the presence of the virus in the nasal secretions between days 1 and 10 pi. All infected animals showed a decrease in white blood cell count. Antibodies could be detected from day 14 pi, and these levels remained high until day 35 pi. The control calf had no viremia, viral presence in nasal secretions, or positive serology, indicating the absence of viral transmission. Thus, isolate BVDV 1a 241.10 has low pathogenicity and transmissibility but retains immunosuppressive capacity.

Survivability of porcine epidemic diarrhea virus (PEDV) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions.

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Pujols, Joan; Segalés, Joaquim

2014-12-05

Bovine plasma was inoculated with porcine epidemic diarrhea virus (PEDV) at an average final titer of 4.2 log10 TCID50/mL to determine the effect of spray drying on viral inactivation. Using a laboratory scale drier, inoculated plasma was spray dried at 200 °C inlet temperature and either 70 or 80 °C throughout substance. Both liquid and dried samples were subjected to three passages on VERO cell monolayers to determine PEDV infectivity. Results indicated liquid samples contained infective virus, but none of the spray dried samples were infectious. Also, survivability of PEDV inoculated on spray dried bovine plasma (SDBP) and stored at 4, 12 or 22 °C was determined for 7, 14 and 21 days. Commercial SDBP powder was inoculated with PEDV to an average final titer of 2.8 log10 TCID50/g. Five samples per time and temperature conditions were subjected to three passages on VERO cell monolayers to determine PEDV infectivity. The virus was non-infectious for all samples stored at 22 °C at 7, 14 and 21 days. PEDV was infective in 1 out of 5 samples stored at 12 °C at 7 days, but none of the samples stored for 14 and 21 days were infectious in cell culture. For samples stored at 4 °C, 4 out of 5 samples were infectious at 7 days, 1 out of 5 samples were infectious at 14 days, but none were infectious at 21 days. In summary, PEDV was not infectious on cell culture within 7 days when stored at room temperature and within 21 days when stored at refrigerated temperature. Copyright © 2014 Elsevier B.V. All rights reserved.

Circulation of multiple subtypes of bovine viral diarrhoea virus type 1 with no evidence for HoBi-like pestivirus in cattle herds of southern Italy.

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Lanave, G; Decaro, N; Lucente, M S; Guercio, A; Cavaliere, N; Purpari, G; Padalino, I; Larocca, V; Antoci, F; Marino, P A; Buonavoglia, C; Elia, G