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Sample records for monodon baculovirus mbv

  1. Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

    PubMed

    Ramesh Kumar, D; Elumalai, Rajasegaran; Raichur, Ashok M; Sanjuktha, M; Rajan, J J; Alavandi, S V; Vijayan, K K; Poornima, M; Santiago, T C

    2016-07-01

    In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp. PMID:27132538

  2. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

    SciTech Connect

    Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.; Kwang, Jimmy

    2009-09-01

    White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.

  3. Oral Vaccination of Baculovirus-Expressed VP28 Displays Enhanced Protection against White Spot Syndrome Virus in Penaeus monodon

    PubMed Central

    S, Syed Musthaq; Kwang, Jimmy

    2011-01-01

    White Spot Syndrome Virus (WSSV) is an infectious pathogen of shrimp and other crustaceans, and neither effective vaccines nor adequate treatments are currently available. WSSV is an enveloped dsDNA virus, and one of its major envelope proteins, VP28, plays a pivotal role in WSSV infection. In an attempt to develop a vaccine against WSSV, we inserted the VP28 gene into a baculovirus vector tailored to express VP28 on the baculovirus surface under the WSSV ie1 promoter (Bac-VP28). The Bac-VP28 incorporated abundant quantity (65.3 µg/ml) of VP28. Shrimp were treated by oral and immersion vaccination with either Bac-VP28 or wild-type baculovirus (Bac-wt). The treatment was followed by challenge with WSSV after 3 and 15 days. Bac-VP28 vaccinated shrimp showed significantly higher survival rates (oral: 81.7% and 76.7%; immersion: 75% and 68.4%) than Bac-wt or non-treated shrimp (100% mortality). To verify the protective effects of Bac-VP28, we examined in vivo expression of VP28 by immunohistochemistry and quantified the WSSV copy number by qPCR. In addition to that, we quantified the expression levels shrimp genes LGBP and STAT by real-time RT-PCR from the samples obtained from Bac-VP28 vaccinated shrimp at different duration of vaccine regime. Our findings indicate that oral vaccination of shrimp with Bac-VP28 is an attractive preventative measure against WSSV infection that can be used in the field. PMID:22069450

  4. Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test.

    PubMed

    Wangman, Pradit; Longyant, Siwaporn; Chaivisuthangkura, Parin; Sridulyakul, Pattarin; Rukpratanporn, Sombat; Sithigorngul, Paisarn

    2012-08-01

    An immunochromatographic strip test is described for detection of the polyhedrin protein of Penaeus monodon nucleopolyhedrovirus (PemoNPV). The test employs one monoclonal antibody (MAb MBV5) conjugated to colloidal gold to bind to polyhedrin protein and a 1:1:1 mixture of 3 other MAbs (MBV8, 14 and 21) to capture colloidal-gold MAb-protein complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (GAM) antibody is used to capture excess free colloidal-gold conjugated MBV5 to validate test performance. Heating of homogenates of PemoNPV-infected P. monodon postlarvae prepared in PBS for 30min was necessary to maximize T line color intensity, and homogenates of infected postlarvae could still be scored as PemoNPV-positive when diluted 1:64. A strip test result was obtained within 15min of sample application, and although about 200-fold lower than a one-step PCR test for PemoNPV, its detection sensitivity was comparable to a dot blot. Due to its simplicity not reliant on sophisticated equipment or specialized skills, the strip test could be adopted to screen easily for PemoNPV infections at shrimp hatcheries and farms. PMID:22580094

  5. The polyhedra of the occluded baculoviruses of marine decapod crustacea: A unique structure, crystal organization, and proposed model

    PubMed

    Bonami; Aubert; Mari; Poulos; Lightner

    1997-11-01

    The baculoviruses of marine penaeid shrimp, PmSNPV and PvSNPV (MBV type and BP type, respectively), have distinctly different occlusion bodies (OBs) from those of the insect baculoviruses. In contrast to insect baculovirus, the penaeid baculovirus OB is unenveloped and formed by large subunits (SuOBs), as observed by electron microscopy after negative staining. The polyhedrin subunits measure 17 to 23 nm in diameter and appear icosahedral, resembling full and empty viral particles. Although these SuOBs look similar in morphometrics to shrimp parvoviruses, their density, polypeptide composition, and UV spectra are more characteristic of proteins than nucleoproteins. Common to the two shrimp baculovirus OBs that were investigated is the aggregation of three icosahedral SuOBs into a triplet. The observed difference in their crystalline structure is directly related to the way in which triplets attach to each other to form the OB. In the BP-type OB, the triplets form alternating parallel rows in all three dimensions. On the other hand, in the MBV-type OB, four triplets form a hollow sphere which we call a "rosette," the building blocks of the MBV-type OB. We assembled models for the penaeid baculovirus OB as an alternative to those hypothesized for insect baculovirus OBs. Copyright 1997 Academic Press. Copyright 1997 Academic Press PMID:9417978

  6. Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries.

    PubMed

    Joseph, Toms C; James, Roswin; Anbu Rajan, L; Surendran, P K; Lalitha, K V

    2015-09-01

    Viral pathogens appear to exert the most significant constraints on the growth and survival of crustaceans under culture conditions. The prevalence of viral pathogens White Spot Syndrome Virus (WSSV), Hepatopancreatic Parvo Virus (HPV), Monodon Baculo Virus (MBV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Penaeus monodon post-larvae was studied. Samples collected from different hatcheries and also samples submitted by farmers from Kerala were analyzed. Out of 104 samples collected, WSSV was detected in 12.5% of the post-larvae samples. Prevalence of concurrent infections by HPV, MBV and WSSV (either dual or triple infection) was present in 60.6% of the total post-larvae tested. Out of the 51 double positives, 98% showed either HPV or IHHNV infection. HPV or IHHNV was detected in 11 post-larval samples showing triple viral infection. This is the first report of IHHNV from India. Result of this study reveals the lack of efficient screening strategies to eradicate viruses in hatchery reared post-larvae. PMID:26217783

  7. Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries

    PubMed Central

    Joseph, Toms C.; James, Roswin; Anbu Rajan, L.; Surendran, P.K.; Lalitha, K.V.

    2015-01-01

    Viral pathogens appear to exert the most significant constraints on the growth and survival of crustaceans under culture conditions. The prevalence of viral pathogens White Spot Syndrome Virus (WSSV), Hepatopancreatic Parvo Virus (HPV), Monodon Baculo Virus (MBV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Penaeus monodon post-larvae was studied. Samples collected from different hatcheries and also samples submitted by farmers from Kerala were analyzed. Out of 104 samples collected, WSSV was detected in 12.5% of the post-larvae samples. Prevalence of concurrent infections by HPV, MBV and WSSV (either dual or triple infection) was present in 60.6% of the total post-larvae tested. Out of the 51 double positives, 98% showed either HPV or IHHNV infection. HPV or IHHNV was detected in 11 post-larval samples showing triple viral infection. This is the first report of IHHNV from India. Result of this study reveals the lack of efficient screening strategies to eradicate viruses in hatchery reared post-larvae. PMID:26217783

  8. CHARACTERIZATION OF SHRIMP BACULOVIRUS

    EPA Science Inventory

    The research undertaken involved the partial characterization of a baculovirus of the pink shrimp, Penaeus duorarum. The significance of the study is related to the fact that the shrimp baculovirus is morphologically similar to insect vaculoviruses which were considered unique to...

  9. Baculoviruses: Molecular Biology of Mosquito Baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses found in mosquitoes have been assigned to the Nucleopolyhedroviruses and are of growing interest as they may represent a separate branch within the Baculoviridae that existed prior to the split of lepidopteran nucleopolyhedroviruses and granuloviruses. They may also be ancestral to th...

  10. Baculoviruses and nucleosome management

    SciTech Connect

    Volkman, Loy E.

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  11. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    PubMed

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation. PMID:25982944

  12. Baculovirus as a vaccine vector

    PubMed Central

    Lu, Hsin-Yu; Chen, Yi-Hsuan; Liu, Hung-Jen

    2012-01-01

    Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has been utilized as RNA interference mediators, gene delivery vectors, and vaccine vectors for a wide variety of applications. This article focuses on the utilization of baculoviruses as vaccine vectors to prepare antigen or subunit vaccines. PMID:22705893

  13. HSP70 induction during baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are arthropod-specific double-stranded DNA viruses that have been employed as bio-insecticides against crop pests and to produce heterologous proteins in baculovirus expression systems. Although a consensus has emerged on the dominant molecular events driving baculovirus replication i...

  14. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    PubMed

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. PMID:26188128

  15. Book review: Baculovirus Molecular Biology, Second Edition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of cell culture and molecular biology methodologies to the study of baculoviruses has resulted in an explosion of information on this group of insect pathogens. The quantity of the corresponding literature on baculoviruses has reached a level difficult for any one researcher to mast...

  16. Budded baculovirus particle structure revisited.

    PubMed

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2016-02-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs. PMID:26743500

  17. RAPID EFFICIENT PRODUCTION OF BACULOVIRUS EXPRESSION VECTORS

    EPA Science Inventory

    Recombinant baculoviruses have been utilized to produce foreign proteins and have the potential to be safe, efficacious insecticides. solation of recombinant virus is usually by plaque phenotype. ypical recombination rates are less than 1%, thus requiring time consuming inspectio...

  18. RADIOIMMUNOASSAY ANALYSIS OF BACULOVIRUS GRANULINS AND POLYHEDRINS

    EPA Science Inventory

    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera, Trichoplusia ni, and Spodotera frugiperda. Antisera were...

  19. Peptide-mediated interference with baculovirus transduction.

    PubMed

    Mäkelä, Anna R; Närvänen, Ale; Oker-Blom, Christian

    2008-03-20

    Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells. PMID:18294718

  20. [Research Advances in Baculovirus Occlusion-derived Virions].

    PubMed

    Zhu, Shimao; Li, Hui

    2016-01-01

    Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses. PMID:27295890

  1. Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of the 42 baculovirus genomes that have been sequenced to date, some are from closely related viruses that share overall nucleotide sequence identities in excess of 95%. Comparative analysis of genomic sequences from closely related viruses can provide insight into how baculoviruses evolve and adap...

  2. GENETICALLY ENGINEERED BACULOVIRUSES AS AGENTS FOR PEST CONTROL

    EPA Science Inventory

    Baculoviruses constitute one of the largest and most diverse groups of insect pathogenic viruses. Following World War II, research on baculovirus intensified with the realization that these relatively virulent insect-specific viruses were widespread in nature among economically i...

  3. A Circo-Like Virus Isolated from Penaeus monodon Shrimps.

    PubMed

    Pham, Hanh T; Yu, Qian; Boisvert, Maude; Van, Hanh T; Bergoin, Max; Tijssen, Peter

    2014-01-01

    A virus with a circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) genome (PmCV-1) was isolated from Penaeus monodon shrimps in Vietnam. The gene structure of the 1,777-nucleotide (nt) genome was similar to that of circoviruses and cycloviruses, but the nucleic acid and protein sequence identities to these viruses were very low. PMID:24435870

  4. SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp.

    PubMed

    Glenn, K L; Grapes, L; Suwanasopee, T; Harris, D L; Li, Y; Wilson, K; Rothschild, M F

    2005-06-01

    Genetic studies in shrimp have focused on disease, with production traits such as growth left unexamined. Two shrimp species, Litopenaeus vannamei and Penaeus monodon, which represent the majority of US shrimp imports, were selected for single nucleotide polymorphism (SNP) discovery in alpha-amylase (AMY2) and cathepsin-l (CTSL), both candidate genes for growth. In L. vannamei, four SNPs were found in AMY2 and one SNP was found in CTSL. In P. monodon, one SNP was identified in CTSL. The CTSL gene was mapped to linkage group 28 of P. monodon using the female map developed with the Australian P. monodon mapping population. Association analyses for the AMY2 and CTSL genes with body weight (BW) were performed in two L. vannamei populations. While neither gene was found to be significantly associated with BW in these populations, there was a trend in one population towards higher BW for allele G of CTSL SNP C681G. PMID:15932404

  5. Fundamentals of Baculovirus Expression and Applications.

    PubMed

    Kost, Thomas A; Kemp, Christopher W

    2016-01-01

    In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology. PMID:27165326

  6. Chapter 4: Baculoviruses and other occluded insect viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are among the most thoroughly studied insect pathogens. Members of Baculoviridae possess a large, circular double-stranded DNA genome contained within the enveloped nucleoprotein core of a rod-shaped virion. Baculovirus replication is distinguished by the production of two different ...

  7. Hormone activation of baculovirus expressed progesterone receptors.

    PubMed

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  8. A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

    PubMed

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M; Jakubowska, Agata K; Herrero, Salvador

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  9. A novel baculovirus-derived promoter with high activity in the baculovirus expression system

    PubMed Central

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M.; Jakubowska, Agata K.

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  10. [Advances in baculovirus per os infection and per os infectivity factor--A review].

    PubMed

    Li, Hui; Guo, Caiping; Zhu, Shimao

    2015-04-01

    Baculoviruses are a family of arthropod-specific viruses that mainly affect insects of the orders Lepidoptera, Hymenoptera, and Diptera. In nature, baculoviruses establish infection in their hosts orally and a battery of proteins designated as per os infectivity factors play pivotal roles in baculovirus per os infection. This review summarizes the basic characteristics of baculovirus and discusses the main events that baculovirus establishes per os infection, including the evolutionary advantages for baculovirus to initiate infection through the oral route, the binding and fusion of baculovirus virions with insect midgut microvilli and the functional roles of baculovirus per os infectivity factors. These achievements and advances should promise to shed light on the understanding and utilization of baculovirus for bio-control and exogenous gene expression in the future. PMID:26211313

  11. Ancient Coevolution of Baculoviruses and Their Insect Hosts

    PubMed Central

    Herniou, Elisabeth A.; Olszewski, Julie A.; O'Reilly, David R.; Cory, Jenny S.

    2004-01-01

    If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts. PMID:15016845

  12. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  13. Genome Scale Transcriptomics of Baculovirus-Insect Interactions

    PubMed Central

    Nguyen, Quan; Nielsen, Lars K.; Reid, Steven

    2013-01-01

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies. PMID:24226166

  14. Diseases of the eye of farmed shrimp Penaeus monodon.

    PubMed

    Smith, P T

    2000-12-21

    Lesions were found in the eyes of cultured shrimp Penaeus monodon that displayed non-specific signs of disease, including lethargy, dark pigmentation, brown gills, empty midgut, anorexia, white tail muscle, necrosis of uropods and fouled cuticle. Eye lesions were associated with sexual development in moribund shrimp in at least 1 disease event. Suppurative inflammation, granuloma and malacia were observed in histological examination of the eye and the causative agents of lesions appear to be Vibrio spp. and a rod-shaped virus (similar to Lymphoid Organ Virus, Gill-Associated Virus [GAV] and Yellow-Head Virus). Suppurative inflammation was characterised by edema, infiltration of haemocytes and local sites of abscesses. Eyes with granuloma usually appeared white in pond-side examinations, and histology showed that fibrous tissue replaced ommatidia, ganglia and internal structures of the eye. Malacia of the eye was characterised by necrosis of nervous tissue, vacuolation and vascular proliferation in the medulla ganglia. Levels of presumptive Vibrionaceace were high in moribund specimens and Gram-negative rods were observed in some specimens as free particles in the interstitial fluid and haemolymph in the eye. Transmission electron microscopy showed that nerve cells in the fasciculated zone (near the basement membrane) contained cytoplasmic vesicles (1 to 3 microm in diameter) with particles (15 to 26 nm in diameter) and rod-shaped nucleocapsids. The rods were similar to those of GAV and were 130 to 260 nm long, 10 to 16 nm in diameter and had helical symmetry with a screw-like thread (2.4 to 3.5 nm pitch). Also, unidentified enveloped virions, averaging 74 nm in diameter, were observed in cytoplasmic vesicles in the fasciculated zone. In conclusion, it is suggested that bacterial and viral infections of the eye could result in impaired neuroendocrine functions, which may cause a range of clinical signs of disease. PMID:11206731

  15. Baculovirus infection induces heat shock response in vivo and in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are insect pathogens that have been exploited as bio-insecticides for the management of crop pests and for their ability to produce an abundance of heterologous proteins in baculovirus expression systems. Defining the molecular properties of baculovirus strains that broaden host range ...

  16. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    SciTech Connect

    Chen, Hong-Zhang; Wu, Carol P.; Chao, Yu-Chan; Liu, Catherine Yen-Yen

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  17. Establishment of the BacMam system using silkworm baculovirus.

    PubMed

    Imai, Atsutoshi; Tadokoro, Takashi; Kita, Shunsuke; Horiuchi, Masataka; Fukuhara, Hideo; Maenaka, Katsumi

    2016-09-16

    The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. PMID:27480929

  18. Barriers to success: How baculoviruses establish efficient systemic infections

    PubMed Central

    Passarelli, A. Lorena

    2011-01-01

    The mechanisms used by baculoviruses to exit the midgut and cause systemic infection of their insect hosts have been debated for decades. After being ingested, baculoviruses reach the midgut, where several host barriers need to be overcome in order to establish successful infection. One of these barriers is the basal lamina, a presumably virus-impermeable extracellular layer secreted by the epithelial cells lining the midgut and trachea. This review discusses new evidence that demonstrates how these viruses breach the basal lamina and establish efficient systemic infections. The biochemical mechanisms involved in dismantling basal lamina during baculovirus infection may also provide new insights into the process of basal lamina remodeling in invertebrate and vertebrate animals. PMID:21300392

  19. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  20. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  1. [Expression and characterization of rabies virus nucleoprotein in baculovirus].

    PubMed

    Cao, Lei; Tang, Qing; Tao, Xiao-yan; Huang, Ying; Du, Jia-liang; Zhang, Shou-feng; Hu, Rong-liang

    2010-09-01

    To construct a recombinant expression plasmid Bacmid-N containing the N gene of Rabies Virus, the N gene of RV CVS-11 strain was cloned into the baculovirus shuttle vector (Bacmid). Recombinant Baculovirus AcMNPV-N (P1 Viral stock) was obtained by transfecting the Bacmid-N into the insect cell line of Sf9. The expressed nucleoprotein was identified and analysised by ELISA, FA, SDS-PAGE and Western blot assays. The results showed that the NP protein was expressed intracellularly and had a good antigenicity, which would be potentially used for further study on the diagnostic reagent of rabies virus detection. PMID:21043133

  2. Bioinformatics analysis of codon usage patterns and influencing factors in Penaeus monodon nudivirus.

    PubMed

    Tyagi, Anuj; Singh, Niraj K; Gurtler, Volker; Karunasagar, Indrani

    2016-02-01

    Penaeus monodon nudivirus (PmNV) is one of the most important and most commonly reported shrimp viruses. In the present study, codon usage of PmNV was studied in detail. Based on effective number of codons (ENC) values, strong to low codon usage bias was observed in PmNV genes. Nucleotide composition-ENC correlation analysis and the GC3 versus ENC relationship indicated that compositional constraint has a major effect on codon usage of PmNV. At the whole-genome level, relative synonymous codon usage (RSCU) analysis showed almost complete antagonism between the codon usage pattern of PmNV and its host P. monodon. However, codon adaptive index (CAI) values indicated that forces of selective/translational constraints have been able to overcome this antagonism in some genes. PMID:26586333

  3. Origin of Ecdysosteroid UDP-glycosyltransferases of Baculoviruses through Horizontal Gene Transfer from Lepidoptera

    PubMed Central

    Hughes, Austin L.

    2014-01-01

    Baculoviruses infecting Lepidoptera (butterflies and moths) encodes an enzyme known as ecdysosteroid UDP-glycosyltransferase (EGT), which inactivates insect host ecdysosteroid hormones, thereby preventing molt and pupation and permitting a build-up of the viral population within the host. Baculovirus EGT shows evidence of homology to insect UDP-glycosyltransferases, and a phylogenetic analysis supported the closest relative of baculovirus EGT are the UGT33 and UGT34 families of lepidopteran UDP-glycosyltransferases. The phylogenetic analysis thus supported that baculovirus EGT arose by horizontal gene transfer of a UDP-glycosyltransferase from a lepidopteran host, an event that occurred 70 million years ago at the earliest but possibly much more recently. Three amino acid replacements unique to baculovirus EGTs and conserved in all available baculovirus sequences were identified in the N-terminal region of the molecule. Because of their conservation, these amino acids are candidates for playing an important functional role in baculovirus EGT function. PMID:24834437

  4. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  5. Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Wang, Y; Fu, M J; Zhao, C; Bao, W Y; Zhou, F L; Yang, Q B; Jiang, S G; Qiu, L H

    2016-01-01

    MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation. PMID:26909956

  6. Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon.

    PubMed

    Babu, Divya T; Antony, Swapna P; Joseph, Simi P; Bright, Ann Rose; Philip, Rosamma

    2013-03-01

    A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed. PMID:23262396

  7. Hybrid of baculovirus and galactosylated PEI for efficient gene carrier.

    PubMed

    Kim, You-Kyoung; Choi, Jae Young; Jiang, Hu-Lin; Arote, Rohidas; Jere, Dhananjay; Cho, Myung-Haing; Je, Yeon Ho; Cho, Chong-Su

    2009-04-25

    Baculovirus, containing an appropriate eukaryotic promoter, is considered an attractive approach for an efficient and safe gene delivery vehicle. However, the drawbacks of baculovirus, such as the lack of specificity and the inactivation of baculovirus by the complement system in human serum, negatively affect efficient gene delivery. Therefore, a hybrid system utilizing the positive aspects of both viral and non-viral vector systems would be useful to overcome the obstacles of either system alone. In this study, we constructed a hybrid system composed of baculovirus (B) and galactosylated polyethylenimine (GP)/DNA complexes through electrostatic interaction. The resulting GP/B hybrid had suitable physicochemical properties and low cytotoxicity for use in gene therapy. Furthermore, the GP/B significantly enhanced transduction efficiency and showed good cell-specificity compared to either viral or non-viral vector systems. These results suggest that the GP/B hybrid system can be used in gene therapy to enhance transduction efficiency and hepatocyte specificity. PMID:19272627

  8. Maximizing in vivo production of Agrotis ipsilon (Hufnagel) baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The black cutworm, Agrotis ipsilon (Hufnagel), is a pest causing damage to a variety plants of from turf to row crops. A recently discovered baculovirus has the potential to be developed as a biological pesticide to provide targeted control of this insect pest. Initial field trials in turf grass and...

  9. BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

    EPA Science Inventory

    The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

  10. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  11. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    PubMed

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  12. Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei.

    PubMed

    Tuyen, N X; Verreth, J; Vlak, J M; de Jong, M C M

    2014-11-01

    White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R0 for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R0 between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon. PMID:25189688

  13. Baculovirus expressed virus-like particles of Pea eation mosaic virus vary in size and encapsidate baculovirus mRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica m...

  14. Invasion of Asian tiger shrimp, Penaeus monodon Fabricius, 1798, in the western north Atlantic and Gulf of Mexico

    USGS Publications Warehouse

    Fuller, Pam L.; Knott, David M.; Kingsley-Smith, Peter R.; Morris, James A.; Buckel, Christine A.; Hunter, Margaret E.; Hartman, Leslie D.

    2014-01-01

    After going unreported in the northwestern Atlantic Ocean for 18 years (1988 to 2006), the Asian tiger shrimp, Penaeus monodon, has recently reappeared in the South Atlantic Bight and, for the first time ever, in the Gulf of Mexico. Potential vectors and sources of this recent invader include: 1) discharged ballast water from its native range in Asia or other areas where it has become established; 2) transport of larvae from established non-native populations in the Caribbean or South America via ocean currents; or 3) escape and subsequent migration from active aquaculture facilities in the western Atlantic. This paper documents recent collections of P. monodon from the South Atlantic Bight and the Gulf of Mexico, reporting demographic and preliminary phylogenetic information for specimens collected between North Carolina and Texas from 2006 through 2012. The increased number of reports in 2011 and 2012, ranging from 102 mm to 298 mm total length, indicates that an adult population is present in densities sufficient for breeding, which is indicative of incipient establishment. Based on these reports of P. monodon, its successful invasion elsewhere, and its life history, we believe that this species will become common in the South Atlantic Bight and Gulf of Mexico in less than 10 years. Penaeus monodon is an aggressive predator in its native range and, if established, may prey on native shrimps, crabs, and bivalves. The impacts of an established P. monodon population are potentially widespread (e.g., alterations in local commercial fisheries, direct and indirect pressures on native shrimp, crab and bivalve populations, and subsequent impacts on the populations of other predators of those organisms) and should be considered by resource managers. The impacts of P. monodon on native fauna and the source(s) or vector(s) of the invasion, however, remain unknown at this time.

  15. The Baculovirus Uses a Captured Host Phosphatase to Induce Enhanced Locomotory Activity in Host Caterpillars

    PubMed Central

    Katsuma, Susumu; Koyano, Yasue; Kang, WonKyung; Kokusho, Ryuhei; Kamita, Shizuo George; Shimada, Toru

    2012-01-01

    The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme. PMID:22496662

  16. [Estimate the safety of baculovirus insecticides: the history and the status Quo].

    PubMed

    Xiao, H Z; Qi, Y P; Yang, F H

    2001-05-01

    This article reviews the evaluation of security of baculovirus used as a pesticide. Since 1970s, the scholars have done a lot of experiments with kinds of baculovirus to test the security of a large number kinds of living things even our human beings. Almost all experiments proved that baculovirus is secure, but some experiments came to different conclusions. These gave rise to great debate twice when they were published, but these conclusions have been proved to be wrong with later test by other scientists or the author himself. Since 90s baculovirus have been used a great deal as the vector to express the foreign gene. Some of them reported the expression in mammalian cells, which brought the suspicious of the baculovirus safety. This article made an analysis and a conclusion about them. Also, this article laid emphasis on the security of recombination baculovirus with the results of the security experiment of self-made recombination baculovirus pesticide. In the last analysis, this article draws a conclusion that the baculovirus and recombinant baculovirus insecticides are secure. PMID:11517591

  17. Characterization, expression and silencing by RNAi of p53 from Penaeus monodon.

    PubMed

    Dai, Wenting; Qiu, Lihua; Zhao, Chao; Fu, Mingjun; Ma, Zhenhua; Zhou, Falin; Yang, Qibin

    2016-06-01

    The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon. PMID:27112755

  18. Characterization of Intestinal Bacteria in Wild and Domesticated Adult Black Tiger Shrimp (Penaeus monodon)

    PubMed Central

    Rungrassamee, Wanilada; Klanchui, Amornpan; Maibunkaew, Sawarot; Chaiyapechara, Sage; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

    2014-01-01

    The black tiger shrimp (Penaeus monodon) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated P. monodon broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely i) Proteobacteria (Vibrio, Photobacterium, Novosphingobium, Pseudomonas, Sphingomonas and Undibacterium), ii) Firmicutes (Fusibacter), and iii) Bacteroidetes (Cloacibacterium). The shared bacterial members in P. monodon from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of Vibrio and Photobacterium in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp. PMID:24618668

  19. Use of Whole Genome Sequence Data To Infer Baculovirus Phylogeny

    PubMed Central

    Herniou, Elisabeth A.; Luque, Teresa; Chen, Xinwen; Vlak, Just M.; Winstanley, Doreen; Cory, Jennifer S.; O'Reilly, David R.

    2001-01-01

    Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses. PMID:11483757

  20. Visualization of Intracellular Pathways of Engineered Baculovirus in Mammalian Cells

    PubMed Central

    Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

    2014-01-01

    Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy. PMID:24457070

  1. [Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector].

    PubMed

    Chen, Qi; Zhang, Shou-Feng; Liu, Ye; Fu, Yun-Hong; Sun, Cheng-Long; Yang, Yang; Gong, Ting; Song, Fei-Fei; Hu, Rong-Liang

    2012-09-01

    To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine. PMID:23233923

  2. Antibiotics in South Indian coastal sea and farmed prawns (Penaeus monodon).

    PubMed

    Palaniyappan, Venkatesh; Nagalingam, Arun Kumar; Ranganathan, Hari Prasad; Kandhikuppam, Krishnamoorthy Bharathi; Kothandam, Hari Prasath; Vasu, Soumya

    2013-01-01

    Sulphonamides and chloramphenicol antibiotics were analysed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in sea and farmed prawn (Penaeus monodon) samples obtained from the coastal region of southern India during 2011-2012. Average recoveries were 77-99% and precision was between 1% and 8%. The results revealed that in sea prawn samples neither of the two antibiotics was detected, but in farmed samples from coastal Andhra Pradesh some sulphonamides were detected in a concentration range greater than the maximum residual limit as set by Council Directive 2377/90 EC. PMID:24779904

  3. Melanosis in Penaeus monodon: Involvement of the Laccase-like Activity of Hemocyanin.

    PubMed

    Bris, Cédric Le; Cudennec, Benoit; Dhulster, Pascal; Drider, Djamel; Duflos, Guillaume; Grard, Thierry

    2016-01-27

    In shrimp, the development of postmortem melanosis resulting from phenoloxidase activities leads to important economic losses. Phenoloxidase enzymes include catechol oxidases, laccases, and tyrosinases, but hemocyanin is also capable of phenoloxidase activities. These activities have been explored in Penaeus monodon, using different substrates. Results highlighted that tyrosinase-specific substrates were little oxidized, whereas hydroquinone (laccase-specific substrate) was more highly oxidized than l-DOPA (nonspecific substrate) in the pereopods and pleopods. Global phenoloxidase activity, assayed with l-DOPA, did not appear thermally stable over time and probably resulted from phenoloxidase enzymes. Conversely, the laccase-like activity assayed with hydroquinone was thermally stable over time, reflecting the thermal stability of hemocyanin. Independently of the anatomical compartment, the temperature, or the substrate, the highest activities were assayed in the cuticular compartments. This study demonstrates the complexity of phenoloxidase activities in P. monodon, and the importance of considering all the activities, including laccase-like activities such as that of hemocyanin. PMID:26671070

  4. Impaired telomerase activity hinders proliferation and in vitro transformation of Penaeus monodon lymphoid cells.

    PubMed

    Jayesh, P; Vrinda, S; Priyaja, P; Philip, Rosamma; Singh, I S Bright

    2016-08-01

    Retaining terminal transferase activity of telomerase, the ribonucleoprotein enzyme which add telomeric repeats on chromosome end is thought to be required to prevent cellular ageing. Additionally, telomerase considered as a marker for cell proliferation and immortalization in eukaryotes. We examined telomerase activity in tissues and lymphoid cell culture of Penaeus monodon. Along with telomerase activity, telomere repeats and an attempt on identification of telomerase reverse transcriptase (PmTERT) were made. Telomeric repeat amplification protocol revealed that telomerase-dependent telomeric lengthening has been taking place in P. monodon and the adult tissues were retaining this capacity throughout their lifespan with the highest activity in ovary, testis and lymphoid organ. However, telomerase activity could not be detected in lymphoid cells in culture. The canonical telomeric repeats added by telomerase of lymphoid tissue extract were identified as TTAGG, but pentameric repeats GGTTA and AGGTT were also added by the telomerase. PmTERT protein sequence (partial) shared 100 % identity with the TERT sequence of Daphnia pulex, 27 % sequence identity with Purple sea urchin and 24-25 % with Zebra fish. Undetectable telomerase activity in lymphoid cell culture supports the hypothesis that the inadequate telomerase activity or gene expression may be a reason that prevents neoplastic transformation and spontaneous immortalization of the cells in vitro. Thus, it is envisaged that telomerase activation in lymphoid cells may surmount cellular ageing for in vitro transformation and cell line establishment. PMID:26084784

  5. Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infec...

  6. Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression.

    PubMed

    Chen, Chiu-Ling; Luo, Wen-Yi; Lo, Wen-Hsin; Lin, Kun-Ju; Sung, Li-Yu; Shih, Yung-Shen; Chang, Yu-Han; Hu, Yu-Chen

    2011-12-01

    MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis. PMID:21732325

  7. Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

  8. Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials

    PubMed Central

    Kwang, Timothy Weixin; Zeng, Xinhui; Wang, Shu

    2016-01-01

    Over the past two decades, baculoviruses have become workhorse research tools for transient transgene expression. Although they have not yet been used directly as a gene therapy vector in the clinical setting, numerous preclinical studies have suggested the highly promising potential of baculovirus as a delivery vector for a variety of therapeutic applications including vaccination, tissue engineering, and cancer treatment. As such, there is growing interest in using baculoviruses as human gene therapy vectors, which has led to advances in baculovirus bioprocessing methods. This review provides an overview of the current approaches for scaled-up amplification, concentration, purification, and formulation of AcMNPV baculoviruses, and highlights the key regulatory requirements that must be met before gene therapy clinical trials can be initiated. PMID:26858963

  9. Baculovirus vectors for antiangiogenesis-based cancer gene therapy.

    PubMed

    Luo, W-Y; Shih, Y-S; Lo, W-H; Chen, H-R; Wang, S-C; Wang, C-H; Chien, C-H; Chiang, C-S; Chuang, Y-J; Hu, Y-C

    2011-09-01

    Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors. PMID:21701531

  10. Modularity and evolutionary constraints in a baculovirus gene regulatory network

    PubMed Central

    2013-01-01

    Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates

  11. Overexpression of PNGase at from baculovirus-infected insect cells.

    PubMed

    Ftouhi Paquin, N; Tarentino, A L; Plummer, T H

    1998-11-01

    Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme. PMID:9790895

  12. Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected Penaeus monodon

    PubMed Central

    Jeena, K.; Prasad, K. Pani; Pathan, Mujahid Khan; Babu, P. Gireesh

    2012-01-01

    White spot syndrome virus (WSSV) is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF) 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc) was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i) was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry. PMID:25049679

  13. Interaction of Vibrio spp. with the Inner Surface of the Digestive Tract of Penaeus monodon

    PubMed Central

    Soonthornchai, Wipasiri; Chaiyapechara, Sage; Jarayabhand, Padermsak; Söderhäll, Kenneth; Jiravanichpaisal, Pikul

    2015-01-01

    Several species of Vibrio are the causative agent of gastroenteritis in humans. In aquaculture, Vibrio harveyi (Vh) and V. parahaemolyticus (Vp) have long been considered as shrimp pathogens in freshwater, brackish and marine environments. Here we show by using scanning electron microscopy (SEM) that Penaeus monodon orally inoculated with each of these two pathogens via an Artemia diet had numerous bacteria attached randomly across the stomach surface, in single and in large biofilm-like clusters 6 h post-infection. A subsequent marked proliferation in the number of V. harveyi within the biofilm-like formations resulted in the development of infections in the stomach, the upper and middle midgut, but neither in the posterior midgut nor the hindgut. SEM also revealed the induced production of peritrichous pili-like structures by the Vp attaching to the stomach lining, whilst only a single polar fibre was seen forming an apparent physical bridge between Vh and the host’s epithelium. In contrast to these observations, no such adherences or linkages were seen when trials were conducted with non-pathogenic Vibrio spp. or with Micrococcus luteus, with no obvious resultant changes to the host’s gut surface. In naive shrimp, the hindgut was found to be a favorable site for bacteria notably curved, short-rod shaped bacteria which probably belong to Vibrio spp. Data from the current study suggests that pathogens of P. monodon must be able to colonize the digestive tract, particularly the stomach, where chitin is present, and then they use an array of virulent factors and enzymes to infect their host resulting in disease. Oral infection is a better way of mimicking natural routes of infection; investigating the host-bacteria interactions occurring in the digestive tract may lead to new strategies for the prevention or control of bacterial infections in penaeids. PMID:26285030

  14. Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)

    PubMed Central

    2013-01-01

    Background Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. Results A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. Conclusion The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp. PMID:23414329

  15. Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

    PubMed

    Chaudhari, Aparna; Gireesh-Babu, P; Tripathi, Gayatri; Sabnis, Supriya; Dhamotharan, K; Vardarajan, Remya; Kumari, Kavita; Dasgupta, Subrata; Rajendran, K V

    2015-05-01

    The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment. PMID:26040024

  16. Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus monodon.

    PubMed

    Vrinda, S; Jasmin, C; Sivakumar, K C; Jose, Blessy; Philip, Rosamma; Bright Singh, I S

    2016-07-01

    Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed. PMID:27179884

  17. Effect of the anti-lipopolysaccharide factor isoform 3 (ALFPm3) from Penaeus monodon on Vibrio harveyi cells.

    PubMed

    Jaree, Phattarunda; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

    2012-12-01

    The anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) has previously been shown to have very active in vitro antimicrobial activity against a broad range of Gram-positive and Gram-negative bacteria, certain fungi and viruses, including known pathogens of P. monodon shrimp. With respect to the strong bactericidal effect on Gram-negative and Gram-positive bacteria, the ALFPm3 binds to their principal cell wall components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), with a high affinity. The aim of this study was, therefore, to reveal the effects of treating ALFPm3 on membrane of Vibrio harveyi, a P. monodon pathogenic Gram-negative bacterium. The recombinant (r)ALFPm3 protein was found to localize on the V. harveyi cells in vivo, followed by inducing membrane permeabilization and leakage of cytoplasmic components. Moreover, the effect of rALFPm3 treatment on the bacterial cell morphology was confirmed by scanning and transmission electron microscopy. Membrane disruption and damage, bleb and pore formation, and the leakage of cytoplasmic contents were all clearly observed. Taken together, these results suggested that ALFPm3 effectively kills bacteria through bacterial membrane permeabilization. PMID:23000267

  18. CO-OCCLUSION AND PERSISTENCE OF A BACULOVIRUS MUTANT LACKING THE POLYHEDRIN GENE

    EPA Science Inventory

    A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. oinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Aucographa californica nuclear polyhedrosis...

  19. Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

    2015-01-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  20. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  1. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  2. Reaching the melting point: Degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination.

    PubMed

    Ishimwe, Egide; Hodgson, Jeffrey J; Clem, Rollie J; Passarelli, A Lorena

    2015-05-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in "melting" or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  3. Baculovirus: Molecular Insights on Their Diversity and Conservation

    PubMed Central

    Miele, Solange Ana Belen; Garavaglia, Matías Javier; Belaich, Mariano Nicolás; Ghiringhelli, Pablo Daniel

    2011-01-01

    The Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp. In this study, genome sequences from 57 baculoviruses were analyzed to reevaluate the number and identity of core genes and to understand the distribution of the remaining coding sequences. Thirty one core genes with orthologs in all genomes were identified along with other 895 genes differing in their degrees of representation among reported genomes. Many of these latter genes are common to well-defined lineages, whereas others are unique to one or a few of the viruses. Phylogenetic analyses based on core gene sequences and the gene composition of the genomes supported the current division of the Baculoviridae into 4 genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus. PMID:21716740

  4. Baculovirus expression system and method for high throughput expression of genetic material

    DOEpatents

    Clark, Robin; Davies, Anthony

    2001-01-01

    The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

  5. A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon.

    PubMed

    Cimino, Elisabeth J; Owens, Leigh; Bromage, Erin; Anderson, Trevor A

    2002-07-01

    The induction of hsps by stress in Cherax quadricarinatus and Penaeus monodon was investigated using SDS-PAGE, Western blotting and ELISA techniques. Western blotting showed the presence of an immuno-reactive protein to mouse alpha-human hsp70 IgG1 monoclonal antibody at a mass of 86 kDa (hsp86) in pleopod samples but was not sensitive enough to detect differences in response to stress. An enzyme-linked immunosorbent assay (ELISA) was developed using this antibody for the detection of hsp86 in the pleopods of C. quadricarinatus and P. monodon. Using this assay, significantly higher levels of hsp86 were detected in hyperthermally stressed C. quadricarinatus (21 to 70 g) and P. monodon (14 to 32 g) and hypoosmotically stressed P. monodon (14 to 32 g). Male C. quadricarinatus and P. monodon were thermally stressed with an increase in temperature from 24 to 33 degrees C for a period of 2 h then a recovery period of 6 h. SDS-PAGE gels of thermally stressed C. quadricarinatus and P. monodon samples revealed an increase in protein band intensity at 97 kDa (C. quadricarinatus) and 43 and 35 kDa in P. monodon. A 25 kDa mass protein was induced in C. quadricarinatus when thermally stressed. P. monodon were osmotically stressed with a decrease from 31 to 15 ppt for 2 h with a recovery of 6 h. SDS-PAGE gels revealed increased intensity of bands at 35 and 43 kDa and a 100 kDa band was induced demonstrating a wide range response of protein profile to stress in these species. SDS-PAGE gels of both species investigated also revealed an apparent reduction in band intensity of the haemocyanin subunits in stressed samples. The ELISA described here constitutes the first quantitative assay for the detection of a hsp in crustaceans and the following investigations are believed to be the first to describe the response of hsps to stress in C. quadricarinatus and P. monodon. In doing so, they provide a sound basis for future studies of the role of hsps in physiological functions in

  6. The Host Specificities of Baculovirus per os Infectivity Factors

    PubMed Central

    Song, Jingjiao; Wang, Xi; Hou, Dianhai; Huang, Huachao; Liu, Xijia; Deng, Fei; Wang, Hualin; Arif, Basil M.; Hu, Zhihong; Wang, Manli

    2016-01-01

    Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host’s midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses. PMID:27454435

  7. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

    PubMed Central

    Luckow, V A; Lee, S C; Barry, G F; Olins, P O

    1993-01-01

    The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells. Images PMID:8392598

  8. Iflavirus increases its infectivity and physical stability in association with baculovirus.

    PubMed

    Jakubowska, Agata K; Murillo, Rosa; Carballo, Arkaitz; Williams, Trevor; van Lent, Jan W M; Caballero, Primitivo; Herrero, Salvador

    2016-01-01

    Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus-virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides. PMID:26966651

  9. Iflavirus increases its infectivity and physical stability in association with baculovirus

    PubMed Central

    Jakubowska, Agata K.; Murillo, Rosa; Carballo, Arkaitz; Williams, Trevor; van Lent, Jan W.M.; Caballero, Primitivo

    2016-01-01

    Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides. PMID:26966651

  10. Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity

    PubMed Central

    Ge, Jingping; An, Qi; Gao, Dongni; Liu, Ying; Ping, Wenxiang

    2016-01-01

    Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses. PMID:27063566

  11. Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity.

    PubMed

    Ge, Jingping; An, Qi; Gao, Dongni; Liu, Ying; Ping, Wenxiang

    2016-01-01

    Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses. PMID:27063566

  12. Two plasmolipins from the black tiger shrimp, Penaeus monodon and their response to virus pathogens.

    PubMed

    Vatanavicharn, Tipachai; Pongsomboon, Siriporn; Tassanakajon, Anchalee

    2012-10-01

    Two isoforms of plasmolipin were initially identified from the black tiger shrimp (Penaeus monodon) EST database and completed using 50 RACE to reveal complete cDNAs of 558 bp (PmPLP1) and 537 bp(PmPLP2) with 87% nucleotide sequence identity. The deduced amino acid sequences contained four-transmembrane domains and showed the highest amino acid identity (49% and 51%, respectively) to the honey bee (Apis mellifera) chemokine-like factor (CKLF), with a very similar hydrophobic pattern to other plasmolipins. Transcripts of PmPLP1 and PmPLP2 were observed in all tested shrimp tissues with the highest expression levels in the gill and epipodite for PmPLP1 and in the hemocytes and antennal gland for PmPLP2. PmPLP1 transcript levels were significantly upregulated in hemocytes at 24 and 72 h post infection (hpi) with yellow head virus (YHV) (7.4- and 14.7- fold, respectively), but only after 72 hpi by white spot syndrome virus (WSSV). In contrast, PmPLP2 was only slightly (but statistically significant)up-regulated with YHV and WSSV. Thus, PmPLPs have the potential to be a part of viral infection mechanisms or defense response. This is the first characterization of a plasmolipin gene in crustaceans. PMID:22766100

  13. Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation

    PubMed Central

    Brady, Philip; Elizur, Abigail; Williams, Richard; Cummins, Scott F.; Knibb, Wayne

    2012-01-01

    In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-β-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction. PMID:22355268

  14. Cellular responses of the tiger shrimp Penaeus monodon haemocytes after lipopolysaccharide injection.

    PubMed

    Xian, Jian-An; Zhang, Xiu-Xia; Guo, Hui; Wang, Dong-Mei; Wang, An-Li

    2016-07-01

    This study was aimed at investigating the in vivo effects of lipopolysaccharide (LPS) injection on Penaeus monodon haemocytes at a cellular level. Cellular responses of LPS-injected shrimp were analysed using flow cytometry. Results showed that LPS injection caused total haemocyte count (THC) and count of large cells (semigranular and granular cells) decline. In LPS-injected shrimp, percentage of large cells decreased at the initial stage, and returned to the original level later. After LPS infection, non-specific esterase activity, reactive oxygen species (ROS) production and nitric oxide (NO) production in haemocytes were significantly induced, while apoptotic cell ratio of haemocytes increased. PO activity in plasma increased in shrimp received LPS at 2 μg g(-1) after 3-12 h and at 8 μg g(-1) after 3-6 h, and then returned to the initial levels. These results demonstrated that LPS induced immune responses on haemocytes, including production of ROS and NO, and release of esterase and PO. On the other hand, THC reduction might be due to the ROS/NO-induced apoptosis. Haemocyte apoptosis which would eliminate damaged or weak cells and contribute to haemocyte renewal, may be a defending strategie against pathogens. PMID:27134076

  15. Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

    PubMed

    Sarmiento, Katreena P; Panes, Vivian A; Santos, Mudjekeewis D

    2016-08-01

    Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp. PMID:27335260

  16. Synthesis of silver nanoparticles by coastal plant Prosopis chilensis (L.) and their efficacy in controlling vibriosis in shrimp Penaeus monodon

    NASA Astrophysics Data System (ADS)

    Kandasamy, Kathiresan; Alikunhi, Nabeel M.; Manickaswami, Gayathridevi; Nabikhan, Asmathunisha; Ayyavu, Gopalakrishnan

    2013-02-01

    The present work investigated the effect of leaf extract from coastal plant Prosopis chilensis on synthesis of silver nanoparticles using AgNO3 as a substrate and to find their antibacterial potential on pathogenic Vibrio species in the shrimp, Penaeus monodon. The leaf extract could be able to produce silver nanoparticles, as evident by gradual change in colour of the reaction mixture consisted of the extract and 1 mM AgNO3 to dark brown. The silver nanoparticles exhibited 2 θ values corresponding to the presence of silver nanocrystal, as evident by X-ray diffraction spectrum. The peaks corresponding to flavanones and terpenoids were found to be stabilizing agents of the nanoparticles, as revealed by Fourier transform infrared spectroscopy. The size of silver nanoparticles ranged from 5 to 25 nm with an average of 11.3 ± 2.1 nm and was mostly of spherical in shape, as confirmed by transmission electron microscopy. The silver nanoparticles were found to inhibit Vibrio pathogens viz., Vibrio cholerae, V. harveyi, and V. parahaemolyticus and this antibacterial effect was better than that of leaf extract, as proved by disc diffusion assay. The nanoparticles were then tested in the shrimp Penaeus monodon challenged with the four species of Vibrio pathogens for 30 days. The shrimps fed with silver nanoparticles exhibited higher survival, associated with immunomodulation in terms of higher haemocyte counts, phenoloxidase and antibacterial activities of haemolymph of P. monodon which is on par with that of control. Thus, the present study proved the possibility of using silver nanoparticles produced by coastal Prosopis chilensis as antibacterial agent in controlling vibriosis.

  17. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  18. Evolutionary analysis of the ubiquitin gene of baculovirus and insect hosts.

    PubMed

    Ma, S S; Zhang, Z; Xia, H C; Chen, L; Yang, Y H; Yao, Q; Chen, K P

    2015-01-01

    Baculovirus is the only virus that has been found to encode the ubiquitin protein. In this study, ubiquitin sequences from 16 insects and 49 viruses were collected and compared. The resulting sequences were aligned with virus genomes. Then MAGE 5.0, k-estimated software, as well as other software programs were used for systemic evolutionary, selection pressure, and evolutionary distance analysis. The results of the pairwise ratio of non-synonymous to synonymous substitution values and evolutionary distances showed that ubiquitin from baculovirus and insect hosts have been under purifying selection during evolution and are thus evolutionarily conserved. Moreover, genes from insect hosts were more conserved than those in baculovirus. Analysis of the non-synonymous to synonymous substitution rates at each site and entropy calculations revealed the evolutionary status of every site in the ubiquitin genes of baculovirus and their hosts. Genome locations and phylogenetic trees indicated that granuloviruses and non-photosynthetic vegetation evolved, and granulovirus evolution was more similar to that of insect hosts. Our results suggest that the ubiquitin gene in baculovirus may have been acquired through horizontal transfer from the host. PMID:26345932

  19. Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method

    PubMed Central

    Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

    2015-01-01

    Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions. PMID:26490731

  20. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.

    PubMed

    van Oers, Monique M; Pijlman, Gorben P; Vlak, Just M

    2015-01-01

    In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth 'anniversary' of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements. PMID:25246703

  1. Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

    SciTech Connect

    Changela, Anita; Martin, Alexandra; Shuman, Stewart; Mondragon, Alfonso

    2010-03-05

    Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 {angstrom} resolution. BVP adopts the characteristic cysteine-phosphatase {alpha}/{beta} fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif {sup 118}HCTHGXNRT{sup 126} in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

  2. Highly efficient baculovirus-mediated multigene delivery in primary cells

    PubMed Central

    Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S.; Xie, Ye; Boyce, Frederick M.; Davis, M. Wayne; Neuhauss, Stephan C. F.; Taylor, Verdon; Ballmer-Hofer, Kurt; Berger, Imre; Berger, Philipp

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells. PMID:27143231

  3. Highly efficient baculovirus-mediated multigene delivery in primary cells.

    PubMed

    Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S; Xie, Ye; Boyce, Frederick M; Davis, M Wayne; Neuhauss, Stephan C F; Taylor, Verdon; Ballmer-Hofer, Kurt; Berger, Imre; Berger, Philipp

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells. PMID:27143231

  4. Quality control and analytical methods for baculovirus-based products.

    PubMed

    Roldão, António; Vicente, Tiago; Peixoto, Cristina; Carrondo, Manuel J T; Alves, Paula M

    2011-07-01

    Recombinant baculoviruses (rBac) are used for many different applications, ranging from bio-insecticides to the production of heterologous proteins, high-throughput screening of gene functions, drug delivery, in vitro assembly studies, design of antiviral drugs, bio-weapons, building blocks for electronics, biosensors and chemistry, and recently as a delivery system in gene therapy. Independent of the application, the quality, quantity and purity of rBac-based products are pre-requisites demanded by regulatory authorities for product licensing. To guarantee maximization utility, it is necessary to delineate optimized production schemes either using trial-and-error experimental setups ("brute force" approach) or rational design of experiments by aid of in silico mathematical models (Systems Biology approach). For that, one must define all of the main steps in the overall process, identify the main bioengineering issues affecting each individual step and implement, if required, accurate analytical methods for product characterization. In this review, current challenges for quality control (QC) technologies for up- and down-stream processing of rBac-based products are addressed. In addition, a collection of QC methods for monitoring/control of the production of rBac derived products are presented as well as innovative technologies for faster process optimization and more detailed product characterization. PMID:21784235

  5. A formulated double-stranded RNA diet for reducing Penaeus monodon densovirus infection in black tiger shrimp.

    PubMed

    Chimwai, Chaweewan; Tongboonsong, Punnee; Namramoon, Orathai; Panyim, Sakol; Attasart, Pongsopee

    2016-02-01

    Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in the aquaculture industry in Thailand. Significant reductions in levels of PmDNV as assessed by PCR analysis of shrimp hepatopancreas were seen in both prophylactic and curative experiments after feeding shrimp with a formulated diet containing mixed inactivated bacteria harboring dsRNAs corresponding to the PmDNV ns1 and vp genes. Significant reductions of approximately 88% (prophylactic) and 64% (curative) of PmDNV were observed, suggesting that this diet has a high potential for application in commercial aquaculture for reducing PmDNV associated stunted growth of shrimp. PMID:26767321

  6. Baculovirus cyclobutane pyrimidine dimer photolyases show a close relationship with lepidopteran host homologues.

    PubMed

    Biernat, M A; Ros, V I D; Vlak, J M; van Oers, M M

    2011-08-01

    Cyclobutane pyrimidine dimer (CPD) photolyases repair ultraviolet (UV)-induced DNA damage using blue light. To get insight in the origin of baculovirus CPD photolyase (phr) genes, homologues in the lepidopteran insects Chrysodeixis chalcites, Spodoptera exigua and Trichoplusia ni were identified and characterized. Lepidopteran and baculovirus phr genes each form a monophyletic group, and together form a well-supported clade within the insect photolyases. This suggests that baculoviruses obtained their phr genes from an ancestral lepidopteran insect host. A likely evolutionary scenario is that a granulovirus, Spodoptera litura GV or a direct ancestor, obtained a phr gene. Subsequently, it was horizontally transferred from this granulovirus to several group II nucleopolyhedroviruses (NPVs), including those that infect noctuids of the Plusiinae subfamily. PMID:21477200

  7. A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency

    PubMed Central

    Toth, Ann M.; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L.

    2014-01-01

    Insect cells are often glycoengineered using DNA constructs encoding foreign glyocoenzymes under the transcriptional control of the baculovirus immediate early promoter, ie1. However, we recently found that the delayed early baculovirus promoter, 39K, provides inducible and higher levels of transgene expression than ie1 after baculovirus infection (Lin and Jarvis, 2013). Thus, the purpose of this study was to assess the utility of the 39K promoter for insect cell glycoengineering. We produced two polyclonal transgenic insect cell populations in parallel using DNA constructs encoding foreign glycoenzymes under either ie1 (Sfie1SWT) or 39K (Sf39KSWT) promoter control. The surface of Sfie1SWT cells was constitutively sialylated, whereas the Sf39KSWT cell surface was only strongly sialylated after baculovirus infection, indicating Sf39KSWT cells were inducibly-glycoengineered. All nine glycogene-related transcript levels were induced by baculovirus infection of Sf39KSWT cells and most reached higher levels in Sf39KSWT than in Sfie1SWT cells at early times after infection. Similarly, galactosyltransferase activity, sialyltransferase activity, and sialic acid levels were induced and reached higher levels in baculovirus-infected Sf39KSWT cells. Finally, two different recombinant glycoproteins produced by baculovirus-infected Sf39KSWT cells had lower proportions of paucimannose-type and higher proportions of sialylated, complex-type N-glycans than those produced by baculovirus-infected Sfie1SWT cells. Thus, the 39K promoter provides baculovirus-inducible expression of foreign glycogenes, higher glycoenzyme activity levels, and higher human-type N-glycan processing efficiencies than the ie1 promoter, indicating that this delayed early baculovirus promoter has great utility for insect cell glycoengineering. PMID:24768688

  8. Effect of guava leaves on growth and the non-specific immune response of Penaeus monodon.

    PubMed

    Yin, Xiao-Li; Li, Zhuo-Jia; Yang, Keng; Lin, Hei-Zhao; Guo, Zhi-Xun

    2014-09-01

    Guava (Psidium guajava L.) leaf extracts have antiviral and antibacterial activity against shrimp pathogens such as yellow-head virus (YHV), white spot syndrome virus (WSSV), and Vibrio harveyi, which make it a potential water disinfectant for use in shrimp culture. In this study, the safety of guava leaf supplementation in shrimp was evaluated by studying its influence on growth and the non-specific immune response of Penaeus monodon. Six diets containing different levels of guava leaves (0% [basal diet], 0.025% [G1], 0.05% [G2], 0.1% [G3], 0.2% [G4], and 0.4% [G5]) were fed to groups of shrimp (1.576 ± 0.011 g body weight) in triplicate for 56 days. Growth performance (final body weight, WG, PWG, SGR) of shrimp fed guava leaf diets was significantly higher (P < 0.05) than that of shrimp fed on the basal diet. The G1 diet resulted in the highest body weight gain (308.44%), followed by the G2 (295.45%), G3 (283.05%), G5 (281.29%), G4 (276.11%), and finally the basal diet (214.58%). Survival of shrimp in the G1 diet group was higher than that of shrimp in the control and the other experimental groups; however, no statistical differences (P > 0.05) were found. Dietary supplementation with guava leaf improved the activities of prophenoloxidase (PO) and nitric oxide synthase (NOS) in serum, and of superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LSZ) both in serum and hepatopancreas of shrimp. In the experimental groups, the activities of these enzymes followed a similar pattern of change; they increased initially at low levels of dietary supplementation and then decreased with increasing concentrations of dietary guava leaf. Serum PO and SOD activities in shrimp fed the G1 diet reached 7.50 U ml(-1) and 178.33 U ml(-1), respectively, with PO activity being significantly higher than in controls. In shrimp fed the G1 diet, SOD, ACP, and AKP activities in hepatopancreas were significantly higher than in the controls, reaching

  9. In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

    PubMed

    Wang, Meng; Zheng, Zhenhua; Meng, Jin; Wang, Han; He, Man; Zhang, Fuxian; Liu, Yan; Hu, Bin; He, Zike; Hu, Qinxue; Wang, Hanzhong

    2015-09-01

    Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 μg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios. PMID:26117660

  10. Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

    PubMed Central

    2010-01-01

    Background Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. Results The two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR. Conclusions This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14

  11. Use of Glacial Fronts by Narwhals (Monodon monoceros) in West Greenland

    NASA Astrophysics Data System (ADS)

    Laidre, K. L.

    2015-12-01

    Glacial fronts in Greenland are known to be important summer habitat for narwhals (Monodon monoceros), as freshwater runoff and sediment discharge may aggregate prey at the terminus. We investigated the importance of glacial habitat characteristics in determining narwhal visitation. Narwhals (n=18) were instrumented with satellite transmitters in September 1993-1994 and 2006-2007 in Melville Bay, West Greenland. Daily narwhal locations were interpolated using a correlated random walk based on observed filtered locations and associated positional error. We also compiled a database on physical features of 41 glaciers along the northwest Greenland coast. This covered the entire coastal region with narwhal activity. Parameters included glacier ice velocity (km/yr) from radar satellite data, glacier front advance and retreat, and glacier width (km) at the ice-ocean interface derived using front position data digitized from 20-100m resolution radar image mosaics and Landsat imagery. We also quantified relative volumes and extent of glacial ice discharge, thickness of the glacial ice at the terminus (m), and water depth at the terminus (m) from gravity and airborne radar data, sediment flux from satellite-based analysis, and freshwater runoff from a regional atmospheric climate model (RACMO2.3). We quantified whale visits to glaciers at three distances (5, 7, and 10 km) and conducted proximity analyses on annual and monthly time steps. We estimated 1) narwhal presence or absence, 2) the number of 24 h periods spent at glaciers, and 3) the fraction of study animals that visited each glacier. The use of glacial habitat by narwhals expanded to the north and south between the 1990s (n=9 unique glaciers visited) and the 2000s (n=30 visited), likely due to loss of summer fast ice and later fall freeze-up trends (3.5 weeks later since 1979). We used a generalized linear mixed effects framework to quantify the glacier and fjord habitat characteristics preferred by narwhals.

  12. High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India.

    PubMed

    Biju, Narayanan; Sathiyaraj, Ganesan; Raj, Mithun; Shanmugam, Venu; Baskaran, Babu; Govindan, Umamaheswari; Kumaresan, Gayathri; Kasthuriraju, Karthick Kannan; Chellamma, Thampi Sam Raj Yohannan

    2016-08-01

    Hepatopancreatic microsporidiosis in cultivated Litopenaeus vannamei and Penaeus monodon is caused by the newly emerged pathogen Enterocytozoon hepatopenaei (EHP). It has been detected in shrimp cultured in China, Vietnam and Thailand and is suspected to have occurred in Malaysia and Indonesia and to be associated with severely retarded growth. Due to retarded shrimp growth being reported at farms in the major grow-out states of Tamilnadu, Andhra Pradesh and Odisha in India, shrimp were sampled from a total of 235 affected ponds between March 2014 and April 2015 to identify the presence of EHP. PCR and histology detected a high prevalence of EHP in both P. monodon and L. vannamei, and infection was confirmed by in situ hybridization using an EHP-specific DNA probe. Histology revealed basophilic inclusions in hepatopancreas tubule epithelial cells in which EHP was observed at various developmental stages ranging from plasmodia to mature spores. The sequence of a region of the small subunit rDNA gene amplified by PCR was found to be identical to EHP sequences deposited in GenBank. Bioassays confirmed that EHP infection could be transmitted orally to healthy shrimp. Histology also identified bacterial co-infections in EHP-infected shrimp sampled from slow-growth ponds with low-level mortality. The data confirm that hepatopancreatic microsporidiosis caused by EHP is prevalent in shrimp being cultivated in India. EHP infection control measures thus need to be implemented urgently to limit impacts of slowed shrimp growth. PMID:27503918

  13. Generating a host range-expanded recombinant baculovirus

    PubMed Central

    Wu, Chunfeng; Deng, Zihao; Long, Zhao; Cai, Yi; Ying, Zhongfu; Yin, Hanqi; Yuan, Meijin; Clem, Rollie J.; Yang, Kai; Pang, Yi

    2016-01-01

    As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc∆P35) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc∆P35 or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc∆P35, while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV. PMID:27321273

  14. Generating a host range-expanded recombinant baculovirus.

    PubMed

    Wu, Chunfeng; Deng, Zihao; Long, Zhao; Cai, Yi; Ying, Zhongfu; Yin, Hanqi; Yuan, Meijin; Clem, Rollie J; Yang, Kai; Pang, Yi

    2016-01-01

    As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc(∆P35)) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc(∆P35) or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc(∆P35), while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV. PMID:27321273

  15. Genetic analysis of Black Tiger shrimp (Penaeus monodon) across its natural distribution range reveals more recent colonization of Fiji and other South Pacific islands

    PubMed Central

    Waqairatu, Salote S; Dierens, Leanne; Cowley, Jeff A; Dixon, Tom J; Johnson, Karyn N; Barnes, Andrew C; Li, Yutao

    2012-01-01

    The Black Tiger shrimp (Penaeus monodon) has a natural distribution range from East Africa to the South Pacific Islands. Although previous studies of Indo-Pacific P. monodon have found populations from the Indian Ocean and Australasia to differ genetically, their relatedness to South Pacific shrimp remains unknown. To address this, polymorphisms at eight shared microsatellite loci and haplotypes in a 418-bp mtDNA-CR (control region) sequence were examined across 682 P. monodon from locations spread widely across its natural range, including the South Pacific islands of Fiji, Palau, and Papua New Guinea (PNG). Observed microsatellite heterozygosities of 0.82–0.91, allele richness of 6.85–9.69, and significant mtDNA-CR haplotype variation indicated high levels of genetic diversity among the South Pacific shrimp. Analysis of microsatellite genotypes using a Bayesian STRUCTURE method segregated Indo-Pacific P. monodon into eight distinct clades, with Palau and PNG shrimp clustering among others from Southeast Asia and eastern Australia, respectively, and Fiji shrimp clustering as a distinct group. Phylogenetic analyses of mtDNA-CR haplotypes delineated shrimp into three groupings, with shrimp from Fiji again being distinct by sharing no haplotypes with other populations. Depending on regional location, the genetic structures and substructures identified from the genotyping and mtDNA-CR haplotype phylogeny could be explained by Metapopulation and/or Member–Vagrant type evolutionary processes. Neutrality tests of mutation-drift equilibrium and estimation of the time since population expansion supported a hypothesis that South Pacific P. monodon were colonized from Southeast Asia and eastern Australia during the Pleistocene period over 60,000 years ago when land bridges were more expansive and linked these regions more closely. PMID:22957205

  16. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    PubMed

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  17. Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

  18. THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

    EPA Science Inventory

    Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

  19. MEMBRANOUS LABYRINTH IN BACULOVIRUS-INFECTED CRUSTRACEAN CELLS: POSSIBLE ROLES IN VIRAL REPRODUCTION

    EPA Science Inventory

    The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. t is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and o...

  20. Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) and a wild-type isolate (Sf3) of the same baculovirus. Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioas...

  1. Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

    PubMed Central

    Haase, Santiago; Sciocco-Cap, Alicia; Romanowski, Víctor

    2015-01-01

    Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway. PMID:25941826

  2. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  3. Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus.

    PubMed Central

    Johnson, T M; Mann, W R; Dragland, C J; Anderson, R C; Nemecek, G M; Bell, P A

    1995-01-01

    The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively. Images Figure 1 Figure 2 Figure 4 PMID:7626037

  4. Changes in trace metals in hemolymph of baculovirus infected noctuid larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied how biologically relevant trace metals (i.e., micronutrients) in the plasma of larvae of Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of plas...

  5. Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haema...

  6. DEVELOPMENT OF AN IN SITU TOXICITY ASSAY SYSTEM USING RECOMBINANT BACULOVIRUSES. (R825433)

    EPA Science Inventory

    A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or m...

  7. Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): Baculovirus expression, biochemical properties and organophosphate insensitivity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirm...

  8. Functional characterization of hesp018, a baculovirus-encoded serpin gene.

    PubMed

    Ardisson-Araujo, Daniel M P; Rohrmann, George F; Ribeiro, Bergmann M; Clem, Rollie J

    2015-05-01

    The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects. PMID:25573886

  9. Tissue specificity of a baculovirus expressed, basement membrane-degrading protease in larvae of Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from flesh fly Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm, Heliothis virescens, significantly faste...

  10. Iron levels change in larval Heliothis virescens tissues following baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inductively-coupled plasma mass spectrometry (ICP-MS) and 59Fe radiotracers were used to investigate changes in levels of iron (Fe) in the tissues of Heliothis virescens following baculovirus infection. Fe concentrations were determined by ICP-MS in hemolymph collected from 4th instar larvae infect...

  11. CHARACTERIZATION OF THE DNA OF A NONOCCLUDED BACULOVIRUS, HZ-1V

    EPA Science Inventory

    The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsC1-ethidium bromide density gr...

  12. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    PubMed Central

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd+ baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation. PMID:24362443

  13. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  14. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    PubMed Central

    Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  15. Expression of hemocyanin and digestive enzyme messenger RNAs in the hepatopancreas of the Black Tiger Shrimp Penaeus monodon.

    PubMed

    Lehnert, Sigrid A; Johnson, Samuel E

    2002-10-01

    In order to define the cellular site of synthesis for hemocyanin and digestive enzymes in the decapod hepatopancreas, we studied the expression of messenger ribonucleic acids (RNAs) for these molecules in the epithelium lining hepatopancreas tubules. In situ hybridisation of gene probes for the digestive enzymes amylase, cathepsin-L, cellulase, chitinase-1 and trypsin to tissue sections of the shrimp hepatopancreas confirmed that the F-cells lining tertiary, secondary and primary ducts are the sites of synthesis for digestive enzyme messenger RNA (mRNA). The F-cells also contained mRNA for the hemocyanin gene. This finding raises important questions on the mechanism by which mature hemocyanin accumulates in the shrimp hemolymph. Our in situ hybridisation studies further showed that Penaeus monodon F-cells remain transcriptionally active for digestive enzyme mRNAs during periods of starvation. PMID:12381378

  16. CHARACTERIZATION OF THE GLYCOSYLATED ECDYSTEROIDS IN THE HEMOLYMPH OF BACULOVIRUS-INFECTED GYPSY MOTH LARVAE AND CELLS IN CULTURE

    EPA Science Inventory

    Fourth-instar gypsy moth (Lymantria dispar; Lepidoptera: Lymantriidae) larvae, infected with the gypsy moth baculovirus (LdNPV), show an elevated and prolonged extension of the hemolymph ecdysteroid titer peak associated with molting. The ecdysteroid immunoreactivity associated w...

  17. Use of Bacterial Artificial Chromosomes in Baculovirus Research and Recombinant Protein Expression: Current Trends and Future Perspectives

    PubMed Central

    Roy, Polly; Noad, Rob

    2012-01-01

    The baculovirus expression system is one of the most successful and widely used eukaryotic protein expression methods. This short review will summarise the role of bacterial artificial chromosomes (BACS) as an enabling technology for the modification of the virus genome. For many years baculovirus genomes have been maintained in E. coli as bacterial artificial chromosomes, and foreign genes have been inserted using a transposition-based system. However, with recent advances in molecular biology techniques, particularly targeting reverse engineering of the baculovirus genome by recombineering, new frontiers in protein expression are being addressed. In particular, BACs have facilitated the propagation of disabled virus genomes that allow high throughput protein expression. Furthermore, improvement in the selection of recombinant viral genomes inserted into BACS has enabled the expression of multiprotein complexes by iterative recombineering of the baculovirus genome. PMID:23762754

  18. Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.

    PubMed

    Wu, Qunfeng; Yu, Fulai; Xu, Jinfang; Li, Yang; Chen, Huanchun; Xiao, Shaobo; Fu, Zhen F; Fang, Liurong

    2014-06-25

    Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. PMID:24793501

  19. Bdellovibrio and Like Organisms Enhanced Growth and Survival of Penaeus monodon and Altered Bacterial Community Structures in Its Rearing Water

    PubMed Central

    Li, Huanhuan; Chen, Cheng; Sun, Qiuping; Liu, Renliang

    2014-01-01

    In this study, a 96-h laboratory reduction test was conducted with strain BDHSH06 (GenBank accession no. EF011103) as the test strain for Bdellovibrio and like organisms (BALOs) and 20 susceptible marine bacterial strains forming microcosms as the targets. The results showed that BDHSH06 reduced the levels of approximately 50% of prey bacterial strains within 96 h in the seawater microcosms. An 85-day black tiger shrimp (Penaeus monodon) rearing experiment was performed. The shrimp survival rate, body length, and weight in the test tanks were 48.1% ± 1.2%, 99.8 ± 10.0 mm, and 6.36 ± 1.50 g, respectively, which were values significantly (P < 0.05) higher than those for the control, viz., 31.0% ± 2.1%, 86.0 ± 11.1 mm, and 4.21 ± 1.56 g, respectively. With the addition of BDHSH06, total bacterial and Vibrio numbers were significantly reduced (P < 0.05) by 1.3 to 4.5 log CFU · ml−1 and CFU · g−1 in both water and shrimp intestines, respectively, compared to those in the control. The effect of BDHSH06 on bacterial community structures in the rearing water was also examined using PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of rearing water samples from the control and test tanks revealed that the amounts of 44% of the bacterial species were reduced when BDHSH06 was added to the rearing water over the 85-day rearing period, and among these, approximately 57.1% were nonculturable. The results of this study demonstrated that BDHSH06 can be used as a biocontrol/probiotic agent in P. monodon culture. PMID:25107962

  20. Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Puthawibool, Teeranart; Senapin, Saengchan; Kiatpathomchai, Wansika; Flegel, Timothy W

    2009-03-01

    Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5min hybridization with an FITC-labeled DNA probe and 5min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV). PMID:19022295

  1. Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes.

    PubMed

    Abdul-Aziz, Muslihudeen A; Schöfl, Gerhard; Mrotzek, Grit; Haryanti, Haryanti; Sugama, Ketut; Saluz, Hans Peter

    2015-09-01

    Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo-Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite-based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts). PMID:26380687

  2. Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes

    PubMed Central

    Abdul-Aziz, Muslihudeen A; Schöfl, Gerhard; Mrotzek, Grit; Haryanti, Haryanti; Sugama, Ketut; Saluz, Hans Peter

    2015-01-01

    Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo-Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite-based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts). PMID:26380687

  3. Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus

    PubMed Central

    Nie, Zuo-Ming; Zhang, Zhi-Fang; Wang, Dan; He, Ping-An; Jiang, Cai-Ying; Song, Li; Chen, Fang; Xu, Jie; Yang, Ling; Yu, Lin-Lin; Chen, Jian; Lv, Zheng-Bing; Lu, Jing-Jing; Wu, Xiang-Fu; Zhang, Yao-Zhou

    2007-01-01

    Background The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome. Results The genome of AnpeNPV, which infects Chinese tussah silkworm (Antheraea pernyi), was sequenced and analyzed. The genome was 126,629 bp in size. The G+C content of the genome, 53.4%, was higher than that of most of the sequenced baculoviruses. 147 open reading frames (ORFs) that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. Of the 147 ORFs, 143 appeared to be homologous to other baculovirus genes, and 4 were unique to AnpeNPV. Furthermore, there are still 29 and 33 conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. In addition, the total number of genes common to all lepidopteran NPVs is sill 74, however the 74 genes are somewhat different from the 74 genes identified before because of some new sequenced NPVs. Only 6 genes were found exclusively in all lepidopteran NPVs and 12 genes were found exclusively in all Group I NPVs. AnpeNPV encodes v-trex(Anpe115, a 3' to 5' repair exonuclease), which was observed only in CfMNPV and CfDEFNPV in Group I NPVs. This gene potentially originated by horizontal gene transfer from an ancestral host. In addition, AnpeNPV encodes two conotoxin-like gene homologues (ctls), ctl1 and ctl2, which were observed only in HycuNPV, OpMNPV and LdMNPV. Unlike other baculoviruses, only 3 typical homologous regions (hrs) were identified containing 2~9 repeats of a 30 bp-long palindromic core. However, 24 perfect or imperfect direct repeats (drs) with a high degree of AT content were found within the intergenic spacer regions that

  4. Production of small antibacterial peptides using silkworm-baculovirus protein expression system.

    PubMed

    Fukushima, Mai; Iiyama, Kazuhiro; Yamashita, Jun; Furue, Masutaka; Tsuji, Gaku; Imanishi, Shigeo; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2013-01-01

    The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm-baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus. PMID:23742088

  5. The cellular substrate: a very important requirement for baculovirus in vitro replication.

    PubMed

    Miltenburger, H G; Naser, W L; Harvey, J P; Huber, J; Huger, A M

    1984-01-01

    We established more than 200 primary cell lines of Cydia pomonella (coding moth). 81 of them were selected and screened for replication of two baculoviruses (from two different subgroups): the Choristoneura murinana NPV and the Cydia pomonella GV. Although all these cell lines had been derived from the same insect species, they varied largely in their response to challenge with the NPV. Most of them showed CPE or produced different amounts of polyhedra. Interestingly, we also found a few cell lines that were permissive for GV replication. To our knowledge this is the first time that GV replication in cell lines has been obtained. Our results show that cell line properties are most important for baculovirus in vitro replication. PMID:6516539

  6. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    PubMed

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  7. Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells

    PubMed Central

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul

    2012-01-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  8. Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design.

    PubMed

    Lin, Shih-Yeh; Yeh, Chia-Tsui; Li, Wan-Hua; Yu, Cheng-Ping; Lin, Wen-Chin; Yang, Jyh-Yuan; Wu, Hsueh-Ling; Hu, Yu-Chen

    2015-10-01

    Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease in the Asia-Pacific region. To produce the virus-like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co-express EV71 P1 polypeptide and 3CD protease using the Bac-to-Bac(®) vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v-cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF-P1-C3CD, a recombinant baculovirus constructed using the flashBAC GOLD(TM) system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five(TM) cells with BacF-P1-C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross-protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High Five(TM) culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. PMID:25997678

  9. Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

    PubMed

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul; Ambudkar, Suresh V

    2012-02-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  10. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    PubMed

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  11. Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1

    PubMed Central

    Seshagiri, Somasekar; Miller, Lois K.

    1997-01-01

    We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1. PMID:9391073

  12. Control of programmed cell death by the baculovirus genes p35 and iap.

    PubMed Central

    Clem, R J; Miller, L K

    1994-01-01

    The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells. Images PMID:8035800

  13. The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

    PubMed Central

    Prikhod’ko, Elena A.; Miller, Lois K.

    1999-01-01

    While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection. PMID:10400766

  14. Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

    PubMed

    Wilson, L E; Wilkinson, N; Marlow, S A; Possee, R D; King, L A

    1997-04-01

    A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains. PMID:9105619

  15. Gene organization and transcription of TED, a lepidopteran retrotransposon integrated within the baculovirus genome.

    PubMed Central

    Friesen, P D; Nissen, M S

    1990-01-01

    A single copy of the retrotransposon TED, from the moth Trichoplusia ni (a lepidopteran noctuid), was identified within the DNA genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Determination of the complete nucleotide sequence (7,510 base pairs) of the integrated copy indicated that TED belongs to the family of retrotransposons that includes Drosophila melanogaster elements 17.6 and gypsy and thus represents the first nondipteran member of this invertebrate group to be identified. The internal portion of TED, flanked by long terminal repeats (LTRs), is composed of three long open reading frames comparable in size and location to the gag, pol, and env genes of the vertebrate retroviruses. Sequence similarity with the dipteran elements was the highest within individual domains of TED open reading frame 2 (pol region) that are also conserved among the retroviruses and encode protease, reverse transcriptase, and integrase functions, respectively. Mapping the 5' and 3' termini of TED RNAs indicated that the LTRs have a retroviral U3-R-U5 structural organization that is capable of directing the synthesis of transcripts that represent potential substrates for reverse transcription and intermediates in transposition. Abundant RNAs were also initiated from a site within the 5' LTR that matches the consensus motif for the promoter of late, hyperexpressed baculovirus genes. The presence of this viruslike promoter within TED and its subsequent activation only after integration within the viral genome suggest a possible symbiotic relationship with the baculovirus that could extend transposon host range. Images PMID:1692964

  16. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  17. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    PubMed

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  18. Phagocytic activity, respiratory burst, cytoplasmic free-Ca(2+) concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress.

    PubMed

    Xian, Jian-An; Wang, An-Li; Ye, Chao-Xia; Chen, Xiao-Dan; Wang, Wei-Na

    2010-08-01

    The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24h, 48h, 72h and 96h LC(50) (median lethal concentration) of Cu(2+) on P. monodon (11.63+/-1.14g) were found to be 3.49, 1.54, 0.73 and 0.40mgL(-1), respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca(2+) (cf-Ca(2+)) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu(2+) (0, 0.05, 0.5, 1.5 and 3.5mgL(-1)) for 0, 6, 12, 24 and 48h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05mgL(-1) Cu(2+). THC decreased after Cu-exposure to 0.5mgL(-1) for 48h, 1.5mgL(-1) for 24h and 3.5mgL(-1) for 12h. Phagocytic activity decreased in P. monodon following 48h exposure to 3.5mgL(-1) Cu(2+). RB was induced after 6h exposure to 0.5, 1.5 and 3.5mgL(-1) Cu(2+). cf-Ca(2+) concentration increased after 48h exposure to 0.5mgL(-1) Cu(2+), and 12h exposure to 1.5 and 3.5mgL(-1) Cu(2+). The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48h exposure to 0.5, 1.5 and 3.5mgL(-1) Cu(2+). These results indicate that Cu can induce oxidative stress, elevation of cf-Ca(2+) and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu(2+) to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis. PMID:20398793

  19. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

    PubMed

    Xu, Xin-Gang; Tong, De-Wen; Wang, Zhi Sheng; Zhang, Qi; Li, Zhao-Cai; Zhang, Kuan; Li, Wei; Liu, Hung-Jen

    2011-06-01

    Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections. PMID:21793437

  20. Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Chaillet, Maxime; Berger, Imre; Alves, Paula M

    2016-09-10

    The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts. PMID:27378622

  1. Three-dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block-face scanning electron microscopy.

    PubMed

    Feng, Tianyi; Paterson, Brian D; Webb, Robyn; Johnston, Stephen D

    2016-05-01

    Serial Block-Face Scanning Electron Microscopy (SBF-SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF-SEM provided a large dataset of sequential electron-microscopic-level images that facilitated comprehensive ultrastructural observations and three-dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane-less dense granules. A large electron-lucent vesicle and "arch-like" structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF-SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans. J. Morphol. 277:565-574, 2016. © 2016 Wiley Periodicals, Inc. PMID:26877112

  2. Proteomic analysis of differentially expressed proteins in the lymphoid organ of Vibrio harveyi-infected Penaeus monodon.

    PubMed

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Wang, Hao-Ching; Lo, Chu Fang; Tassanakajon, Anchalee

    2012-05-01

    The protein expression profiles of the lymphoid organ, taken from mock and systemic Vibrio harveyi-infected Penaeus monodon at 6 and 48 h post infection, were revealed. The considerable changes in the expression level of several proteins were observed between the mock and V. harveyi-infected shrimps. From 30 analyzed protein spots with 27 differentially expressed, 21 were known proteins with the most common of these being cytoskeleton proteins (33%) which were all down-regulated upon systemic bacterial infection. Other six proteins including four proteins that are involved in the shrimp immunity (alpha-2-macroglobulin, transglutaminase, heat shock protein 1 and hemocyanin subunit Y), and two proteins that are involved in metabolism (triosephosphate isomerase) and cell signaling (14-3-3 like protein), displayed significantly decreased expression levels. There was, however, an increase in the expression level of the ATP synthase beta subunit, a protein involved in energy balance. Transcription levels of ATP synthase beta subunit and 14-3-3 like protein were up- and down-regulated, respectively, in accord with the observed protein expression levels, but the alpha-2-macroglobulin transcript levels were significantly increased in contrast to the decreased protein expression levels. Interestingly, partial gene silencing of ATP synthase beta subunit revealed a high cumulative mortality of the knockdown shrimps (73.3%) and a dramatic reduction of the total hemocyte numbers in the survival shrimps. These altered proteins are likely to play essential roles in shrimp defense against the pathogenic bacterium V. harveyi. PMID:22302389

  3. Inhibition of Luminescence and Virulence in the Black Tiger Prawn (Penaeus monodon) Pathogen Vibrio harveyi by Intercellular Signal Antagonists

    PubMed Central

    Manefield, Michael; Harris, Lachlan; Rice, Scott A.; de Nys, Rocky; Kjelleberg, Staffan

    2000-01-01

    Expression of luminescence in the Penaeus monodon pathogen Vibrio harveyi is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is N-hydroxy butanoyl-l-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in V. harveyi are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga Delisea pulchra on luminescence and toxin production in a virulent strain of V. harveyi were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in V. harveyi cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from V. harveyi cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of V. harveyi prawn infections. PMID:10788385

  4. In vivo therapeutic potentiality of red seaweed, Asparagopsis (Bonnemaisoniales, Rhodophyta) in the treatment of Vibriosis in Penaeus monodon Fabricius

    PubMed Central

    Manilal, Aseer; Selvin, Joseph; George, Shiney

    2011-01-01

    The crude extract of the red seaweed, Asparagopsis sp. was evaluated for in vivo antibacterial activity against the shrimp vibrio pathogens. The algal extract was rationalized with commercial shrimp feed and orally administered for different duration of time followed by the artificial bacterial challenge experiment. In dose titration experiments, the oral administration of Asparagopsis sp. at a dosage of 850 mg kg–1 of biomass was highly efficacious in the treatment of natural infestations of Vibriosis in Penaeus monodon. The results of the confirmatory dose experiment revealed that the prophylactic treatment with moderate dose of 850 mg kg–1 of biomass day–1 for four weeks followed by 14 days of post infection therapy was highly effective in controlling Vibrio infection in shrimps. Moreover, results of the percent survival index and microbiological analysis clearly show that Asparagopsis extract incorporated medicated feed had broad therapeutic potential for managing shrimp Vibriosis. In addition, in vivo trials and results obtained in this work are based on the crude organic extract sourced from an unidentified Asparagopsis cryptic lineage, therefore further molecular analysis to identify the species will be required. PMID:23961176

  5. Microwave-assisted derivatisation and LC-MS/MS determination of nitrofuran metabolites in farm-raised prawns (Penaeus monodon).

    PubMed

    Palaniyappan, Venkatesh; Nagalingam, Arun Kumar; Ranganathan, Hari Prasad; Kandhikuppam, Krishnamoorthy Bharathi; Kothandam, Hari Prasath; Vasu, Soumya

    2013-01-01

    A new microwave-assisted derivatisation and LC-MS/MS method has been developed for the analysis of nitrofuran metabolites - 3-amino-5-morpholino-methyl-1,3-oxa-zolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) - in farm-raised prawns (Penaeus monodon) from the coastal regions of South India. Analysis was carried out by reverse-phase column (Phenomenex Luna C18) with gradient elution using mobile phase A (0.02% acetic acid in water) and mobile phase B (0.02% acetic acid in acetonitrile), at a flow rate of 200 μl min(-1) and an injection volume of 20 μl. Microwave-assisted derivatisation was achieved in 6 min with good recovery. The results showed that the samples collected from Andhra Pradesh and Karnataka contained residues of nitrofuran metabolites in the range from 5.0 to 40 ng g(-1). This work emphasises the importance of ensuring the safety of seafood and that a new method of derivatisation is applicable for the analysis of nitrofuran metabolites in seafood. PMID:23883190

  6. Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, Penaeus monodon.

    PubMed

    Shi, Jinxuan; Fu, Mingjun; Zhao, Chao; Zhou, Falin; Yang, Qibin; Qiu, Lihua

    2016-03-01

    Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp. PMID:26637414

  7. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon).

    PubMed

    Unajak, Sasimanas; Boonsaeng, Vichai; Jitrapakdee, Sarawut

    2006-10-01

    We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection. PMID:16938476

  8. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    PubMed

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  9. Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection.

    PubMed

    Rattanarojpong, Triwit; Khankaew, Suthiwat; Khunrae, Pongsak; Vanichviriyakit, Rapeepun; Poomputsa, Kanokwan

    2016-07-10

    White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future. PMID:27164257

  10. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  11. Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

    PubMed Central

    Lee, Hee-Jung; Cho, Hansam; Kim, Mi-Gyeong; Heo, Yoon-Ki; Cho, Yeondong; Gwon, Yong-Dae; Park, Ki Hoon; Jin, Hyerim; Kim, Jinyoung; Oh, Yu-Kyoung; Kim, Young Bong

    2015-01-01

    Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×108 copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×109 copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×1010 copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×109 copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses. PMID:25789464

  12. Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus.

    PubMed

    Braconi, Carla Torres; Ardisson-Araújo, Daniel Mendes Pereira; Paes Leme, Adriana Franco; Oliveira, Juliana Velasco de Castro; Pauletti, Bianca Alves; Garcia-Maruniak, Alejandra; Ribeiro, Bergmann Morais; Maruniak, James E; Zanotto, Paolo Marinho de Andrade

    2014-04-01

    Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery. PMID:24443474

  13. Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

    PubMed

    Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

    2016-03-01

    Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides. PMID:26787118

  14. Identification and characterisation of microsatellite DNA markers in order to recognise the WSSV susceptible populations of marine giant black tiger shrimp, Penaeus monodon.

    PubMed

    Chakrabarty, Usri; Dutta, Sourav; Mallik, Ajoy; Mondal, Debabrata; Mandal, Nripendranath

    2015-01-01

    White spot disease (WSD) which is caused by white spot syndrome virus (WSSV) creates severe epizootics in captured and cultured black tiger shrimp, resulting a huge loss in the economic output of the aquaculture industry worldwide. Performing selective breeding using DNA markers would prove to be a potential cost effective strategy for long term disease control in shrimps. In the present investigation, microsatellite DNA fingerprints were compared between naturally occurring WSSV resistant and susceptible populations of Penaeus monodon. After PCR with a set of shrimp specific primers three reproducible DNA fragments of varying sizes were found, among which 442 bp and 236 bp fragments were present in considerably higher frequencies in the WSSV susceptible shrimp population (p ≤ 0.0001). After WSSV challenge experiment the copy no. of WSSV was determined using real-time PCR, where it was found to be almost 4 × 10(3) fold higher in WSSV susceptible shrimps than in the resistant ones. Thus, these microsatellite DNA markers will be useful to distinguish between WSSV susceptible and resistant brood stocks of P. monodon. Sequencing studies revealed that these DNA markers were novel in P. monodon. Highest WSSV resistance using these DNA markers, was observed in the shrimp populations of Andaman Island and Chennai among the different coastal areas of India, suggesting these places as safe for specific pathogen resistant brood stock shrimp collection. This study will be a very effective platform towards understanding the molecular pathogenesis of WSD for generation of disease free shrimp aquaculture industry. PMID:26407974

  15. In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV).

    PubMed

    Tang, Kathy F J; Pantoja, Carlos R; Poulos, Bonnie T; Redman, Rita M; Lightnere, Donald V

    2005-02-28

    Infectious myonecrosis virus (IMNV) was recently found to be the cause of necrosis in the skeletal muscle of farm-reared Litopenaeus vannamei from northeastern Brazil. Nucleic acid extracted from semi-purified IMN virions showed that this virus contains a 7.5 kb RNA genome. A cDNA library was constructed, and a clone, designated as IMNV-317, was labeled with digoxigenin-11-dUTP and used as a gene probe for in situ hybridization (ISH). This probe specifically detected IMNV in infected tissues. To determine the susceptibility of 3 species of penaeid shrimp (L. vannamei, L. stylirostris, Penaeus monodon) to IMNV infection, juveniles were injected with purified virions and observed for clinical signs of infection and mortality over a 4 wk period. All L. vannamei exhibited typical lesions after 6 d, and lesions were visible in all L. stylirostris by Day 13. The clinical signs of opaque muscle were not seen in P. monodon, due to their highly pigmented exoskeleton precluding visual detection of lesions. Moderate mortality (20%) occurred in infected L. vannamei. No mortalities were observed in either L. stylirostris or P. monodon. Histological examination and ISH indicated that all 3 species are susceptible to IMNV infection. Using ISH, IMNV was detected in tissues including the skeletal muscle, lymphoid organ, hindgut, and phagocytic cells within the hepatopancreas and heart. In all 3 species, skeletal muscle cells produced the strongest ISH reactions. Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection. PMID:15819442

  16. Expression of Penaeus monodon ortholog of Niemann-Pick type C-2 in the spermatic tract, and its role in sperm cholesterol removal.

    PubMed

    Chotwiwatthanakun, Charoonroj; Sangatit, Jutharat; Santimanawong, Wanida; Surinlert, Piyaporn; Prommoon, Juthatip; Weerachatyanukul, Wattana; Withyachumnarnkul, Boonsirm; Vanichviriyakit, Rapeepun

    2016-03-01

    Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. © 2016 Wiley Periodicals, Inc. PMID:26822874

  17. Distribution of Pleuroncodes monodon larvae over the continental shelf of south-central Chile: Field and modeling evidence for partial local retention and transport

    NASA Astrophysics Data System (ADS)

    Yannicelli, Beatriz; Castro, Leonardo; Parada, Carolina; Schneider, Wolfgang; Colas, Francois; Donoso, David

    2012-01-01

    In situ and modeled spatial distribution of squat lobster ( Pleuroncodes monodon) larvae over the continental shelf off south central Chile (35-37°S) was analyzed along with currents and hydrography. We aimed to identify the main larval transport/retention characteristics in the study area, which constitutes the southernmost P. monodon fishing grounds embedded in the Humboldt Current System. We hypothesized that the main contribution to population renewal originates in the two persistent adult aggregations close to the nursery ground that occurs over a continental shelf terrace limited by two submarine canyons. Two extensive bio-physical field campaigns were carried out during the main 2001-2002 upwelling season field data indicated that larvae were released from late austral winter to spring from spots to the north and south of the nursery. Zoea I were found mainly below 50 m depth in southward-flowing waters, whereas older zoea dominated in northward flowing layers above 50 m. Larvae were circumscribed between the coast and the shelf break front and pelagic retention areas were identified over the widest shelf area. Megalopa and juveniles during March, were only found over the nursery area. Individual based simulations coupled to the output of a hydrodynamic model (climatological configuration) for the studied area, showed that the release sites close to the nursery made the largest contribution to recruitment. Sites further north could also contribute to recruitment if hatching occurred later in the upwelling season. The contribution of vertical behavior to larval success was also important, as was the former’s interaction with the site and time of larval release. Our results support the relevance of coastal circulation (affected by topography) on the persistence of P. monodon populations off southern Chile, and the modulation of temporal variability. These results might apply to other abundant species in the area.

  18. Ecologically acceptable strategy for the use of genetically engineered baculovirus pesticides. Book chapter

    SciTech Connect

    Wood, H.A.; Hughes, P.R.; van Beek, N.; Hamblin, M.

    1990-01-01

    The basis for the first U.S. Environmental Protection Agency approval for the field release of a genetically-engineered virus has been to take advantage of the biological properties of baculovirus in such a way that an engineered virus could possess enhanced pesticidal properties but, at the same time, would pose no environmental or health hazards. The ultimate goal of these investigations is to reduce the agricultural requirement for synthetic chemical pesticides through the development of viral pesticides with enhanced pesticidal properties.

  19. Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).

    PubMed

    Harder, T C; Osterhaus, A D

    1997-01-20

    A glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids. Phylogenetic analysis gave evidence for a close evolutionary relationship between PhHV-1 and members of the Varicellovirus genus of the alpha-Herpesvirinae and canid herpesvirus in particular. In PhHV-1-infected Crandell feline kidney cells gB is expressed as a 113-kDa glycosylated molecule which is proteolytically cleaved into at least two fragments of 67 and 53-59 kDa apparently forming disulfide-linked heterodimers of 140 kDa. Cell surface expression of PhHV-1 gB was confirmed by FACS analysis. Thus, synthesis and processing of the gB protein of PhHV-1 follows a pattern also observed in other Varicelloviruses. Since the gB protein of herpesviruses, expressed in the baculovirus system, has been shown to be a suitable target for vaccine design, we used this system for expression of PhHV-1 gB. Recombinant (rec) baculovirus-expressed gB was identified as a 105-kDa glycosylated molecule. Proteolytic cleavage into fragments of 62 and 52 kDa was markedly delayed compared to wild-type (wt) gB. Wt and rec gB harbored endoglycosidase H (precursor)- as well as N-glycosidase F-sensitive N-glycans (proteolytic fragments). Baculovirus-expressed gB appeared to be antigenically authentic, since it was recognized in radioimmunoprecipitation and immune peroxidase monolayer assays by PhHV-1-neutralizing seal sera and by gB-specific neutralizing murine monoclonal antibodies. Furthermore, PhHV-1-neutralizing antibodies were induced in mice following immunization with baculovirus-expressed gB, indicating its suitability for incorporation in a candidate vaccine for seals. PMID:9018133

  20. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    PubMed

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases. PMID:26666439

  1. Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus

    PubMed Central

    2012-01-01

    Background West Nile Virus (WNV) is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells. Results In the present study, recombinant baculoviruses expressing WNV premembrane (prM) and envelope (E) proteins under the cytomegalovirus (CMV) promoter with or without vesicular stomatitis virus glycoprotein (VSV/G) were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse) induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ) in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6. Conclusions These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus. PMID:22799608

  2. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  3. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    SciTech Connect

    Hiraki, Toshiki; Shibayama, Naoya; Yoon, Young-Ho; Yun, Kyung-Mook; Hamamoto, Toshiro; Tame, Jeremy R. H.; Park, Sam-Yong

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  4. Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy

    PubMed Central

    Ang, Wei Xia; Zhao, Ying; Kwang, Timothy; Wu, Chunxiao; Chen, Can; Toh, Han Chong; Mahendran, Ratha; Esuvaranathan, Kesavan; Wang, Shu

    2016-01-01

    Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene therapy triggered significantly increased bladder infiltrations of inflammatory monocytes, CD4+, CD8+ and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC. PMID:27273619

  5. [Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus].

    PubMed

    Xu, Peng-Wei; Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Liu, Xiao-Yu; Zeng, Xian-Yin; Zhang, Zhi-Qing

    2012-05-01

    The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines. PMID:22764525

  6. Bioactive baculovirus nanohybrids for stent based rapid vascular re-endothelialization

    NASA Astrophysics Data System (ADS)

    Paul, Arghya; Elias, Cynthia B.; Shum-Tim, Dominique; Prakash, Satya

    2013-08-01

    Present study, for the first time, reports the development of a nanohybridized baculovirus based stent that can locally promote vascular re-endothelialization by efficient delivery of pro-angiogenic vascular endothelial growth factor (Vegf) genes. In vitro data demonstrated rapid expression of functionally active Vegf by the bioactive stent-transduced vascular cells. In vivo site-specific transgene expression was observed at the stented regions of balloon-denuded canine femoral artery, which eventually lead to significant endothelial recovery at the injured sites. A significant reduction in neointima formation (2.23 +/- 0.56 mm2 vs 2.78 +/- 0.49 mm2 and 3.11 +/- 0.23 mm2, p < 0.05; n = 8) and percent stenosis was observed in treated stent group compared to negative control and bare metal stent groups. These findings collectively implicate the potential of this newly developed baculovirus based biotherapeutic stent to ameliorate damaged vascular biology and attenuate re-narrowing of stented artery by inhibiting neointima formation.

  7. Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

    PubMed Central

    Chernomordik, L; Leikina, E; Cho, M S; Zimmerberg, J

    1995-01-01

    We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion. PMID:7707532

  8. Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.

    PubMed

    Tan, Boon-Huan; Brown, Gaie; Sugrue, Richard J

    2007-01-01

    Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains. PMID:17502677

  9. Bioactive baculovirus nanohybrids for stent based rapid vascular re-endothelialization

    PubMed Central

    Paul, Arghya; Elias, Cynthia B.; Shum-Tim, Dominique; Prakash, Satya

    2013-01-01

    Present study, for the first time, reports the development of a nanohybridized baculovirus based stent that can locally promote vascular re-endothelialization by efficient delivery of pro-angiogenic vascular endothelial growth factor (Vegf) genes. In vitro data demonstrated rapid expression of functionally active Vegf by the bioactive stent-transduced vascular cells. In vivo site-specific transgene expression was observed at the stented regions of balloon-denuded canine femoral artery, which eventually lead to significant endothelial recovery at the injured sites. A significant reduction in neointima formation (2.23 ± 0.56 mm2 vs 2.78 ± 0.49 mm2 and 3.11 ± 0.23 mm2, p < 0.05; n = 8) and percent stenosis was observed in treated stent group compared to negative control and bare metal stent groups. These findings collectively implicate the potential of this newly developed baculovirus based biotherapeutic stent to ameliorate damaged vascular biology and attenuate re-narrowing of stented artery by inhibiting neointima formation. PMID:23917680

  10. Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity*

    PubMed Central

    Sugiura, Nobuo; Setoyama, Yuka; Chiba, Mie; Kimata, Koji; Watanabe, Hideto

    2011-01-01

    Chondroitin sulfate is a linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4–9) and temperature (30–60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism. PMID:21715327

  11. Molecular characterization and expression profile of MAP2K1ip1/MP1 gene from tiger shrimp, Penaeus monodon.

    PubMed

    Yang, Lishi; Liu, Xianjun; Huang, Jianhua; Yang, Qibin; Qiu, Lihua; Liu, Wenjing; Jiang, Shigui

    2012-05-01

    MAPK kinase 1 interacting protein 1 (MAP2K1ip1) is an important scaffold proteins of the mitogen-activated protein kinase (MAPK) pathway that form an active signaling module and enhance the specificity and spatiality of MAPK signaling. In the present study, we identified and characterized a MAP2K1ip1 cDNA from tiger shrimp Penaeus monodon (designated as PmMAP2K1ip1). The open reading frame of PmMAP2K1ip1 is 372 bp encoding 123 amino-acid residues with a MAPK interaction domain. The predicted PmMAP2Kip1 protein is 13.6 KDa with the theoretical isoelectric point of 6.3. PmMAP2K1ip1 shared the highest amino acid with Nasonia vitripennis and Strongylocentrotus purpuratus, at 48% and 47.5%, respectively. Phylogenic analysis shows PmMAP2Kip1 is clustering with SpMAP2Kip1, and close to the group of MAP2Kip1s from insect. Furthermore, semiquantitative RT-PCR revealed PmMAP2Kip1 is widely distributed in most examined tissues except nerve, and high expressed in ovary, hemocyte, intestines and hepatopancreas. Meanwhile, PmMAP2k1ip1 is expressed ubiquitously during larval and sex gland development, and keep a high level at the initial development stage. Quantitative real time RT-PCR revealed PmMAP2K1ip1 were up-regulated by lipopolysaccharide and peptidoglycan (PGN) in haemocyte. These data reveal MAP2K1ip1 is a multifunction protein that involved development and immune response. It is benefit to characterize other MAPK signal genes and elucidate the molecular regulation mechanism of MAPK signaling in tiger shrimp. PMID:22209950

  12. Cloning, characterization, and expression of the macrophage migration inhibitory factor gene from the black tiger shrimp (Penaeus monodon).

    PubMed

    Xie, Bobo; Fu, Mingjun; Zhao, Chao; Shi, Jinxuan; Shi, Gongfang; Jiao, Zongyao; Qiu, Lihua

    2016-09-01

    Macrophage migration inhibitory factor (MIF) is an ancient cytokine that engages in innate immune system of vertebrates and invertebrates. In this study, the MIF gene homologue (PmMIF) was cloned from the black tiger shrimp, Penaeus monodon. The full-length cDNA sequence of PmMIF was 838 bp and contained 78 bp 5' untranslated region (UTR) and 397 bp 3' UTR, and an open reading frame (ORF) of 363 bp which coded 120 amino acids (aa). Multiple alignment analysis showed that the deduced amino acid sequence shared 98% identities with MIF from closely related species of Litopenaeus vannamei. Quantitative real-time PCR (qRT-PCR) analysis indicated that PmMIF was highly expression observed in hepatotpancreas and gills. After Vibrio harveyi challenge, PmMIF mRNA level in hepatopancreas and gills were sharply up-regulated at 6 h post-injection, and reached the maximum at 12 h. PmMIF expression level in the hepatopancreas and gills were up-regulated markedly under low (2.3%) and high (4.3%) salinity exposure, respectively. PmMIF expression level in gills increased significantly at 12 h and reached peak values (2.5- fold, 6.4-fold and 1.8-fold compared with the control) at 12 h, 48 h and 12 h after zinc, cadmium and copper exposure, respectively. In the hepatopancreas, the expression of PmMIF reached maximum levels (8.5- fold, 6.2-fold and 2.1-fold compared with the control) at 24 h, 6 h and 48 h after zinc, cadmium and copper exposure, respectively. All the results indicate that PmMIF plays an important role in responding in the innate immune system of shrimps. PMID:27514787

  13. Immune gene expression profile of Penaeus monodon in response to marine yeast glucan application and white spot syndrome virus challenge.

    PubMed

    Wilson, Wilsy; Lowman, Douglas; Antony, Swapna P; Puthumana, Jayesh; Bright Singh, I S; Philip, Rosamma

    2015-04-01

    Immunostimulant potential of eight marine yeast glucans (YG) from Candida parapsilosis R20, Hortaea werneckii R23, Candida spencermartinsiae R28, Candida haemulonii R63, Candida oceani R89, Debaryomyces fabryi R100, Debaryomyces nepalensis R305 and Meyerozyma guilliermondii R340 were tested against WSSV challenge in Penaeus monodon post larvae (PL). Structural characterization of these marine yeast glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. PL were fed 0.2% glucan incorporated diet once in seven days for a period of 45 days and the animals were challenged with white spot syndrome virus (WSSV). The immunostimulatory activity of yeast glucans were assessed pre- and post-challenge WSSV by analysing the expression profile of six antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and 13 immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, caspase, catalase, glutathione peroxidase, glutathione-s-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO), Rab-7, superoxide dismutase and transglutaminase. Expression of seven WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, thymidine kinase and VP28 were also analysed to detect the presence and intensity of viral infection in the experimental animals post-challenge. The study revealed that yeast glucans (YG) do possess immunostimulatory activity against WSSV and also supported higher survival (40-70 %) post-challenge WSSV. Among the various glucans tested, YG23 showed maximum survival (70.27%), followed by YG20 (66.66%), YG28 (60.97%), YG89 (58.53%), YG100 (54.05%), YG63 (48.64%), YG305 (45.7%) and YG340 (43.24%). PMID:25555812

  14. Avian influenza neuraminidase 1 (N1) ELISA using baculovirus expressed antigen and its application on DIVA vaccination strategy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISA was developed using baculovirus express N1 protein from the A/Chicken/Indonesia/11/03 (H5N1) virus. The objective of this study was to evaluate the specificity and sensitivity of the N1-ELISA. The specificity of the ELISA was tested with a chicken anti-sera panel raised against N1 to N9 v...

  15. Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

    PubMed

    Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

    2013-01-01

    Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. PMID:24401775

  16. Induction of an IAP antagonist in Culex quinquefasciatus larvae in response to infection by the baculovirus CuniNPV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CuniNPV is a member of the Dipteran–specific baculoviruses in the genus Deltabaculovirus that specifically infects mosquito larvae within the genus Culex while species of Aedes and Anopheles are refractory. Infections are restricted to the nuclei of larval midgut epithelial cells with transmission...

  17. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

    PubMed

    Ge, Jingping; An, Qi; Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  18. Baculovirus infection of the armyworm (Lepidoptera:Noctuidae) feeding on spiny- or smooth-edged grass (Festuca spp.) leaf blades

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Susceptibility of the armyworm, Mythimna unipuncta (Haworth), to infection by a baculovirus isolated from a Kentucky armyworm population was compared on two suspected progenitors of tall fescue, Festuca mairei and Festuca arundinacea subsp. fenas, with spiny leaf margins intact or removed to test wh...

  19. Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.

    PubMed

    Kim, Yuna; Kim, Jinhyun; Kang, Kyoungsoo; Lyoo, Young S

    2002-03-01

    Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells. PMID:14614268

  20. New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus.

    PubMed

    Regev, Avital; Rivkin, Hadassah; Gurevitz, Michael; Chejanovsky, Nor

    2006-12-22

    Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner. PMID:17141223

  1. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens

    PubMed Central

    Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  2. Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to assess the occurrence of baculovirus infections in mosquitoes and characterize them by using molecular tools. Fortnightly collections were made of mosquito larvae in the city of Caraguatatuba. Six larvae of Culex quinquefasciatus were isolated that had white cysts (nodul...

  3. Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

    PubMed

    Chiou, Tzu-Ting; Lu, Jenn-Kan; Wu, Jen-Leih; Chen, Thomas T; Ko, Chi-Fong; Chen, Jiann-Chu

    2007-01-01

    A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage. PMID:16820207

  4. A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

    PubMed Central

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  5. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    PubMed Central

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  6. Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

    PubMed

    Fleming-Davies, Arietta E; Dwyer, Greg

    2015-12-01

    A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen. PMID:26655986

  7. Transcriptome analyses of insect cells to facilitate baculovirus-insect expression.

    PubMed

    Yu, Kai; Yu, Yang; Tang, Xiaoyan; Chen, Huimin; Xiao, Junyu; Su, Xiao-Dong

    2016-05-01

    The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins. PMID:27017378

  8. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

    PubMed

    Liu, Lan-Jun; Suzuki, Takako; Tsunemitsu, Hiroshi; Kataoka, Michiyo; Ngata, Noriyo; Takeda, Naokazu; Wakita, Takaji; Miyamura, Tatsuo; Li, Tian-Cheng

    2008-01-01

    The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity. PMID:18998045

  9. Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells.

    PubMed

    Laukkanen, M L; Oker-Blom, C; Keinänen, K

    1996-09-24

    Trichoplusia ni (High Five) and Spodoptera frugiperda (Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase of Autographa californica nuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications. PMID:8831686

  10. Identification of a single-nucleocapsid baculovirus isolated from Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

    PubMed

    Wang, Liqun; Yi, Jianping; Zhu, Shanying; Li, Bing; Chen, Yan; Shen, Weide; Wang, Wenbing

    2008-01-01

    A nucleopolyhedrovirus isolated from infected larvae of Clanis bilineata tsingtauica was characterized. Electron microscopical studies on the ultrastructure of C. bilineata nucleopolyhedrovirus (ClbiSNPV) occlusion bodies (OBs) showed several virions (up to 16) with a single nucleocapsid packaged within a single viral envelope. The diameter of the OBs was 0.77-1.7 mum with a mean of 1.13 +/- 0.19 mum. The complete sequence of the ClbiSNPV polyhedrin (polh) gene contained 741 nucleotides, predicting a protein of 246 amino acids. Phylogenetic analyses using the complete sequence of the polh genes indicated that ClbiSNPV clusters with Group II NPVs. This is the first record of a baculovirus from C. bilineata. PMID:18584114

  11. Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

    PubMed

    Grant, Rebecca J; Kelley, Karen L; Maruniak, James E; Garcia-Maruniak, Alejandra; Barrett, Tom; Manire, Charles A; Romero, Carlos H

    2010-07-14

    The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals. PMID:20005643

  12. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    PubMed

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine. PMID:27015979

  13. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  14. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells.

    PubMed

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

    2006-05-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type. PMID:16545777

  15. Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

    PubMed

    Theilmann, D A; Chantler, J K; Stweart, S; Flipsen, H T; Vlak, J M; Crook, N E

    1996-04-01

    A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins. PMID:8615018

  16. Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

    Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection. PMID:9311875

  17. The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells.

    PubMed

    Chen, Yun-Ru; Zhong, Silin; Fei, Zhangjun; Hashimoto, Yoshifumi; Xiang, Jenny Z; Zhang, Shiying; Blissard, Gary W

    2013-06-01

    Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5' capped transcription start sites and 3' polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3' ends showed that 92 (77%) of the 3' PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle. PMID:23536684

  18. Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

    PubMed

    Sugiura, T; Sugita, S; Imagawa, H; Kanaya, T; Ishiyama, S; Saeki, N; Uchiyama, A; Tanigawa, M; Kuwano, A

    2001-10-01

    The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses. PMID:11543878

  19. Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.

    2012-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals. PMID:23035220

  20. Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp.

    PubMed

    Liu, Bo; Gong, Yongchang; Li, Zhen; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2016-06-01

    Grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in China. This study was aimed to develop a novel oral vaccine against grass carp reovirus (GCRV). GCRV vp6 and vp7 genes with β-actin promoter of Megalobrama amblycephala and polyhedrin promoter (Ph10) of baculovirus, respectively, were cloned into plasmid pFast™-Dual to construct a vector pFast-PHVP7-AVP6, which was used to generate a recombinant baculovirus BacFish-vp6/vp7 via Bac-to-Bac system. The VP7 expression was analyzed from freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae by western blotting, and VP6 expression was analyzed from orally vaccinated fish with the freeze-dried powder by RT-PCR. The VP6 expression was also analyzed from both CIK cells transduced with BacFish-vp6/vp7 and tissues of vaccinated fish by immunofluorescence analysis. Recombinant VP7 could be detected from the BacFish-vp6/vp7-infected silkworm pupae. Pathological changes were not observed in CIK cells transduced with BacFish-vp6/vp7, and VP6 expression was found in CIK cells. When the grass carps were orally administrated with the freeze-dried powder, vp6 gene transcription was found in blood of the vaccinated fishes and VP6 protein was observed in liver and kidney of the vaccinated fish by immunofluorescence analysis. These results indicated that vp7 gene was expressed in the BacFish-vp6/vp7-infected silkworm and vp6 gene was expressed in orally vaccinated fish with freeze-dried powder of the BacFish-vp6/vp7-infected silkworm pupae, suggesting the possibility to use the powder as an orally administrated vaccine. PMID:27085857

  1. Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

    PubMed Central

    Rebolledo, Dulce; Guevara, Roger; Murillo, Rosa

    2015-01-01

    Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen. PMID

  2. Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua.

    PubMed

    Rebolledo, Dulce; Lasa, Rodrigo; Guevara, Roger; Murillo, Rosa; Williams, Trevor

    2015-01-01

    Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82-93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen. PMID

  3. Immunogenicity of a Trivalent Human Papillomavirus L1 DNA-Encapsidated, Non-Replicable Baculovirus Nanovaccine

    PubMed Central

    Heo, Yoon-Ki; Cho, Yeondong; Gwon, Yong-Dae; Kim, Mi-Gyeong; Park, Ki Hoon; Oh, Yu-Kyoung; Kim, Young Bong

    2014-01-01

    Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58. PMID:24759938

  4. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    PubMed Central

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  5. Expression of an antiviral protein from Lonomia obliqua hemolymph in baculovirus/insect cell system.

    PubMed

    Carmo, A C V; Giovanni, D N S; Corrêa, T P; Martins, L M; Stocco, R C; Suazo, C A T; Moraes, R H P; Veiga, A B G; Mendonça, R Z

    2012-05-01

    The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress. PMID:22230047

  6. Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores.

    PubMed

    Vuthiphandchai, V; Nimrat, S; Kotcharat, S; Bart, A N

    2007-11-01

    The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days. PMID:17900683

  7. Expression profile of key immune-related genes in Penaeus monodon juveniles after oral administration of recombinant envelope protein VP28 of white spot syndrome virus.

    PubMed

    Thomas, Ancy; Sudheer, Naduvilamuriparampu Saidumuhammed; Kiron, Viswanath; Bright Singh, Issac S; Narayanan, Rangarajan Badri

    2016-07-01

    White spot syndrome virus (WSSV) is the most catastrophic pathogen the shrimp industry has ever encountered. VP28, the abundant envelope protein of WSSV was expressed in bacteria, the purified protein administered orally to Penaeus monodon juveniles and its immune modulatory effects examined. The results indicated significant up-regulation of caspase, penaeidin, crustin, astakine, syntenin, PmRACK, Rab7, STAT and C-type lectin in animals orally administered with this antigen. This revealed the immune modulations in shrimps followed by oral administration of rVP28P which resulted in the reduced transcription of viral gene vp28 and delay in mortality after WSSV challenge. The study suggests the potential of rVP28P to elicit a non-specific immune stimulation in shrimps. PMID:27154537

  8. Enhanced effect of fluorescent whitening agent on peroral infection for recombinant baculovirus in the host Bombyx mori L.

    PubMed

    Wang, Bing; Shang, Jinyan; Liu, Xunli; Cui, Weizheng; Wu, Xiaofeng; Zhao, Na

    2007-01-01

    The low efficiency of the oral infectivity of recombinant polyhedrin-negative baculovirus is a major bottleneck in the application of the baculovirus expression system in the silkworm (Bombyx mori L). In this study, the effects of a fluorescent whitening agent on improving the oral infection for the recombinant Bombyx mori nuclear polyhedrosis virus in silkworm larva and their possible mechanism were investigated. The results showed that the peroral infection can be remarkably enhanced by adding VBL into the larval artificial diet. The maximum infection rate reached as high as 90% with the concentration of VBL (1%), which was then considered as optimal. The total protease activity and pH value of the larval intestinal juice were found to be lower when compared to the control, indicating an abnormal physiological change of the larval digestive system by VBL, which, in turn, resulted in improved peroral infection of recombinant virus. PMID:17160363

  9. Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66

    PubMed Central

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Onishi, Momo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitu

    2012-01-01

    Baculovirus envelope protein ODV-E66 (67–704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67–704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å3 Da−1. PMID:22297996

  10. The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

    PubMed

    Leu, Jiann-Horng; Liu, Kuan-Fu; Chen, Kuan-Yu; Chen, Shu-Hwa; Wang, Yu-Bin; Lin, Chung-Yen; Lo, Chu-Fang

    2015-04-01

    By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp. PMID:25499032

  11. Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure.

    PubMed

    Wang, Yun; Liu, Lihui; Huang, Jianhua; Duan, Yafei; Wang, Jun; Fu, Mingjun; Lin, Heizhao

    2016-01-01

    Glutathione S-transferases (GSTs) are a family of multifunctional phase II enzymes that are involved in the detoxification of exogenous and endogenous compounds. In this study, a full-length cDNA of Mu-class GST (PmMuGST) was isolated from the hepatopancreas of Penaeus monodon using rapid amplification of cDNA ends method. The full length cDNA of PmMuGST is 867 bp, contains an open read frame of 660 bp, and encodes a polypeptide of 219 amino acids with a molecular mass of 25.61 kDa and pI of 6.15. Sequence analysis indicated that the predicted protein sequence of PmMuGST was very similar to (86 %) that of Litopenaeus vannamei. A conserved domain of GST_N_Mu_like (PSSM: cd03075) and GST_C_family_superfamily_like (PSSM: cl02776) was indentified in PmMuGST. Real time quantitative RT-PCR analysis indicated that PmMuGST was present in all of the tested tissues. PmMuGST transcripts both in the hepatopancreas and in the muscle were significantly induced after 14 days of treatment with a low dosage of AFB1 (50 μg/kg) exposure and were significantly inhibited after 42 and 56 days of a high dosage of AFB1 (1000, 2500 μg/kg AFB1) exposure. Taken together, the Mu-class GST from P. monodon was inducible and was involved in the response to AFB1 exposure. PMID:27386274

  12. DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses.

    PubMed

    Nielsen, Cydney B; Cooper, Dawn; Short, Steven M; Myers, Judith H; Suttle, Curtis A

    2002-11-01

    Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests. Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided. DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses. We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity. We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses. McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada. Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity). Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity). Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses. PMID:12507483

  13. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    SciTech Connect

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  14. Display of VP1 on the Surface of Baculovirus and Its Immunogenicity against Heterologous Human Enterovirus 71 Strains in Mice

    PubMed Central

    Kiener, Tanja K.; Chow, Vincent T. K.; Kwang, Jimmy

    2011-01-01

    Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization. Methodology/Principal Finding In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains. Conclusion Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns. PMID:21747954

  15. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.

    PubMed

    Urakawa, T; Ferguson, M; Minor, P D; Cooper, J; Sullivan, M; Almond, J W; Bishop, D H

    1989-06-01

    A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses. PMID:2543785

  16. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    SciTech Connect

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  17. Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

    PubMed Central

    Iatrou, K; Meidinger, R G; Goldsmith, M R

    1989-01-01

    Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required. Images PMID:2556701

  18. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    PubMed

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  19. Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

    PubMed

    Rueda, P; Fominaya, J; Langeveld, J P; Bruschke, C; Vela, C; Casal, J I

    2000-11-22

    We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure. PMID:11115693

  20. Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

    PubMed Central

    2013-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections. Methods Immunoblotting analysis of recombinant baculovirus and immunofluorescent staining of baculovirus-infected cells were followed using anti-ORF2 monoclonal antibodies. The BALB/c mice were immunized intramuscularly with this baculovirus. The titers of antibodies were mensurated with a Cap-protein-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The IFN-γ response in splenocytes harvested from immunized mice was measured by ELISA. Student's t-test was used to compare immune responses of different groups. Results In this study, we successfully constructed a dual-expression-system-based recombinant baculovirus BV-GD-ORF2, which can display the PCV2 capsid (Cap) protein and VSV-G protein on the viral envelope and also expressing Cap protein on transduced mammalian cells, thereby functioning as both a subunit and a DNA vaccine. After infection, the Cap protein was expressed and displayed on the viral surface, as demonstrated with an indirect fluorescence assay and immunoblotting. The vaccination of mice with recombinant baculovirus BV-GD-ORF2 successfully induced robust Cap-protein-specific humoral and cellular immune responses. Conclusions Our findings collectively demonstrate that the recombinant baculovirus BV-GD-ORF2 is a potential vaccine against PCV2 infections. PMID:24161107

  1. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    SciTech Connect

    Vanarsdall, Adam L.; Mikhailov, Victor S.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-08-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout

  2. Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

    PubMed Central

    Vanarsdall, Adam L.; Mikhailov, Victor S.; Rohrmann, George F.

    2009-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using pulsed-field gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 hours post-infection, DBP co-localized with replicated DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp

  3. Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Kim, Ji-Ye; Seul, Hee-Jeong; Lee, Hee-Soo; Kwon, Hyuk-Moo; Sung, Haan-Woo

    2014-03-01

    Avian paramyxovirus 2 (APMV-2) infections are associated with respiratory diseases in poultry worldwide. The hemagglutination inhibition (HI) test is a useful tool for surveillance and monitoring of this virus. In this study, full-length hemagglutinin (HN) gene of APMV-2 was chemically synthesized based on its published sequence, cloned and expressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. The biological, antigenic and immunogenic properties of the expressed protein were evaluated to assess its ability to produce diagnostic reagents for HI testing. Recombinant APMV-2 HN protein showed two distinct bands with molecular masses of 64 and 75kDa, which showed hemagglutination (HA) and neuraminidase activities, respectively. The recombinant HN (rHN) protein extracted from infected cells produced high HA titers (2(13) per 25μL). HA activity of the protein was inhibited by APMV-2 antiserum, although there were weak cross reactions with other APMV serotype antisera. The rHN protein induced high titers of APMV-2-specific antibodies in immunized chickens based on the HI test. These results indicated that recombinant APMV-2 HN protein is a useful alternative to the APMV-2 antigen in HI assays. PMID:24374124

  4. A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats.

    PubMed

    Ebling, Peter M; Holmes, Stephen B

    2002-12-01

    A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (OB) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. OBs, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5 g of lyophilized L, F-H and mineral soil horizons, and 1.0 ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively. PMID:12476994

  5. Baculovirus Induced Transcripts in Hemocytes from the Larvae of Heliothis virescens

    PubMed Central

    Breitenbach, Jonathan E.; Shelby, Kent S.; Popham, Holly J.R.

    2011-01-01

    Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae. PMID:22163334

  6. A baculovirus anti-apoptosis gene homolog of the Trichoplusia ni granulovirus.

    PubMed

    Bideshi, D K; Anwar, A T; Federici, B A

    1999-01-01

    An inhibitor of apoptosis (iap) gene homolog (Tn-iap) of the Trichoplusia ni granulovirus (TnGV) was cloned, sequenced and mapped on the genome of TnGV. Tn-iap encoded a protein (Tn-IAP) of 301 amino acids with a predicted molecular mass of 35 kDa. The Tn-IAP contained the two sequence motifs, BIRs and RING finger, characteristic of IAP proteins, and shared identities of 21-27% and similarities of 28-53% with IAP proteins of Cydia pomonella GV (Cp-IAP), Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (MNPV) (Op-IAP1, 3), Autographa californica MNPV (Ac-IAP1), Bombyx mori NPV (Bm-IAP1), Lymantria dispar MNPV (Ld-IAP3) and Buzura suppressaria single nucleocapsid NPV (Bs-IAP1). However, Tn-IAP shared no significant homology with baculovirus IAP2 proteins. Using an antisense Tn-iap probe, two major transcripts of approximately 800 nt and 1600 nt were detected by Northern blot analysis of RNA extracted from the fat body of T. ni larvae infected with the TnGV. Unlike Cp-IAP and Op-IAP3, however, Tn-IAP did not rescue virion occlusion in SF21 cells infected with a p35-deficient AcMNPV mutant. Tn-IAP's synthesis in vivo but failure to rescue p35-deficient AcMNPV in SF21 cells suggests it is a functional IAP that is only effective in certain cell types. PMID:10541013

  7. Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

    PubMed

    Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

    2016-01-01

    Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171-177, 2016. PMID:26519022

  8. Baculovirus induced transcripts in hemocytes from the larvae of Heliothis virescens.

    PubMed

    Breitenbach, Jonathan E; Shelby, Kent S; Popham, Holly J R

    2011-11-01

    Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae. PMID:22163334

  9. Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

    PubMed

    Katsuma, S; Deng, D X; Zhou, C L; Iwanaga, M; Noguchi, Y; Kobayashi, M; Maeda, S

    2000-10-01

    A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin. PMID:11129641

  10. Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.

    PubMed Central

    Ritchie, P J; Decout, A; Amey, J; Mann, C J; Read, J; Rosseneu, M; Scott, J; Shoulders, C C

    1999-01-01

    The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex. PMID:10036224

  11. Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

    PubMed

    Johnson, T M; Pease, E A; Li, J K; Tien, M

    1992-08-01

    Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification. PMID:1632652

  12. Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

    PubMed

    Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

    2010-11-01

    Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. PMID:20713098

  13. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.

    PubMed

    Felberbaum, Rachael S

    2015-05-01

    The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge. PMID:25800821

  14. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    PubMed

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function. PMID:18713650

  15. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    SciTech Connect

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-03-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.

  16. Adaptive Immune Responses Elicited by Baculovirus and Impacts on Subsequent Transgene Expression In Vivo

    PubMed Central

    Luo, Wen-Yi; Lin, Shih-Yeh; Lo, Kai-Wei; Lu, Chia-Hsin; Hung, Chang-Lin; Chen, Chi-Yuan; Chang, Chien-Chung

    2013-01-01

    Baculovirus (BV) is a promising gene therapy vector and typically requires readministration because BV mediates transient expression. However, how the prime-boost regimen triggers BV-specific adaptive responses and their impacts on BV readministration, transgene expression, and therapeutic/vaccine efficacy remain unknown. Here we unraveled that BV injection into BALB/c mice induced the production of BV-specific antibodies, including IgG1 and IgG2a, which could neutralize BV by antagonizing the envelope protein gp64 and impede BV-mediated transgene expression. Moreover, humans did not possess preexisting anti-BV antibodies. BV injection also elicited BV-specific Th1 and Th2 responses as well as CD4+ and CD8+ T cell responses. gp64 was a primary immunogen to activate the antibody and CD8+ T cell response, with its peptide at positions 457 to 465 (peptide 457-465) being the major histocompatibility complex (MHC) class I epitope to stimulate CD8+ T cell and cytotoxic responses. Nonetheless, a hybrid Sleeping Beauty-based BV enabled long-term expression for >1 year by a single injection, indicating that the T cell responses did not completely eradicate BV-transduced cells and implicating the potential of this hybrid BV vector for gene therapy. These data unveil that BV injection triggers adaptive immunity and benefit rational design of BV administration schemes for gene therapy and vaccination. PMID:23408634

  17. Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

    PubMed Central

    Seong, Baik Lin; Nguyen, Huan Huu; Chang, Jun

    2016-01-01

    The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic. PMID:27023684

  18. Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication

    PubMed Central

    Dong, Zhan-Qi; Hu, Nan; Zhang, Jun; Chen, Ting-Ting; Cao, Ming-Ya; Li, Hai-Qing; Lei, Xue-Jiao; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2015-01-01

    We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42–61 and aa 72–101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication. PMID:26660313

  19. Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication.

    PubMed

    Dong, Zhan-Qi; Hu, Nan; Zhang, Jun; Chen, Ting-Ting; Cao, Ming-Ya; Li, Hai-Qing; Lei, Xue-Jiao; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2015-01-01

    We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42-61 and aa 72-101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication. PMID:26660313

  20. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function.

    PubMed

    Biernat, Magdalena A; Eker, André P M; van Oers, Monique M; Vlak, Just M; van der Horst, Gijsbertus T J; Chaves, Inês

    2012-02-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses. PMID:22306969

  1. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

    PubMed

    Kusano-Arai, Osamu; Fukuda, Rie; Kamiya, Wakana; Iwanari, Hiroko; Hamakubo, Takao

    2016-07-01

    The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface. PMID:27095060

  2. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    SciTech Connect

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M. )

    1990-03-06

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ({sup 3}H)azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the ({sup 3}H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

  3. Hyperactivity and tree-top disease induced by the baculovirus AcMNPV in Spodoptera exigua larvae are governed by independent mechanisms

    NASA Astrophysics Data System (ADS)

    van Houte, Stineke; Ros, Vera I. D.; van Oers, Monique M.

    2014-04-01

    Although many parasites are known to manipulate the behavior of their hosts, the mechanisms underlying such manipulations are largely unknown. Baculoviruses manipulate the behavior of caterpillar hosts by inducing hyperactivity and by inducing climbing behavior leading to death at elevated positions (tree-top disease or Wipfelkrankheit). Whether hyperactivity and tree-top disease are independent manipulative strategies of the virus is unclear. Recently, we demonstrated the involvement of the protein tyrosine phosphatase ( ptp) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity in Spodoptera exigua larvae. Here we show that AcMNPV ptp is not required for tree-top disease, indicating that in S. exigua baculovirus-induced hyperactivity and tree-top disease are independently induced behaviors that are governed by distinct mechanisms.

  4. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    PubMed Central

    2011-01-01

    Background Foot-and-mouth disease (FMD) is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV) under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE), and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA) and indirect sandwich-ELISA (IS-ELISA) showed both recombinant baculoviruses (with or without T-cell epitopes) were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ). Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P < 0.01) and higher neutralizing antibody titer (P < 0.05). Although the inactivated vaccine produced the highest titer of neutralizing antibodies, a lower IFN-γ expression was observed compared to the two recombinant pseudotype baculoviruses. Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection

  5. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

    PubMed Central

    2013-01-01

    Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

  6. Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells.

    PubMed

    Tran, Trinh T B; Dietmair, Stefanie; Chan, Leslie C L; Huynh, Hoai T; Nielsen, Lars K; Reid, Steven

    2012-03-01

    Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus-insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell-baculovirus systems for bioprocess optimization. PMID:22166686

  7. Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

    PubMed Central

    Korth, K L; Levings, C S

    1993-01-01

    The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms. Images Fig. 1 Fig. 2 Fig. 5 Fig. 6 PMID:8475086

  8. Aromatic/heterocyclic amino acids and the simulated sunlight-ultraviolet inactivation of the Heliothis/Helicoverpa baculovirus

    SciTech Connect

    Ignoffo, C.M.; Garcia, C.

    1995-04-01

    Tryptophan, of five aromatic/heterocyclic amino acids (tyrosine, phenylalanine, proline, histidine) provided significant protection of the Heliothis baculovirus (HzSNPV) from inactivation by simulated ultraviolet (SUV). Fifty percent of SUV protection of HzSNPV with tryptophan or tyrosine was obtained at 0.03 mg/ml and 0.5 mg/ml, respectively. Rates as high as 100.0 mg/ml of phenylalanine, histidine, or proline provided <50% protection. The extent of tryptophan protection was correlated with its absorption in the sunlight UV-B spectra. 16 refs., 2 tabs.

  9. Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses.

    PubMed

    Grasela, James J; McIntosh, Arthur H; Ringbauer, Joseph; Goodman, Cynthia L; Carpenter, James E; Popham, Holly J R

    2012-05-01

    The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) in Australia, Hawaii, South Africa, and various Caribbean islands, has posed great concern as to the possible threat to native, endangered species of cactus in the southeastern USA as well as with the potential to cause a major infestation of commercial and agricultural cactus crops in Mexico. A number of control measures have been investigated with varying degrees of success including, field exploration for cactus moth-specific parasitoids, insecticides, fungal, bacterial, and nematode agents. Current tactics used by the USA-Mexico binational program to eradicate cactus moth from Mexico and mitigate its westward movement in the USA include host plant removal, the manual removal and destruction of egg sticks and infected cacti stems, and the Sterile Insect Technique. One other approach not taken until now is the development of a cactus moth cell line as a tool to facilitate the investigation of baculoviruses as an alternative biocontrol method for the cactus moth. Consequently, we established C. cactorum cell lines derived from adult ovarian tissue designated as BCIRL-Cc-AM and BCIRL-Cc-JG. The mean cell population doubling time was 204.3 and 112 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with weekly medium change, while the doubling time was 176.6 and 192.6 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with a daily change of medium. In addition, the daily versus weekly change in medium was reflected in the percentage viability with both cell lines showing higher levels with a daily medium change. Of the three baculoviruses tested, only the recombinant AcMNPV-hsp70Red and GmMNPV at a multiplicity of infection (MOI) of 1.0 were able to demonstrate significant production of extracellular virus (ECV) in each of the cell lines, whereas both cell lines were

  10. Baculovirus p35 increases pancreatic {beta}-cell resistance to apoptosis

    SciTech Connect

    Hollander, Kenneth; Bar-Chen, Michal; Efrat, Shimon . E-mail: sefrat@post.tau.ac.il

    2005-07-01

    {beta}-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in {beta}-cells exposed to cytokines and hIAPP, as well as the effects of p35 on {beta}-cell function. The p35 gene was introduced into {beta}TC-tet cells, a differentiated murine {beta}-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted {beta}-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from {beta}-cell damage caused by hIAPP in type 2 diabetes.

  11. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    PubMed

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins. PMID:26820862

  12. Identification and characterization of vlf-1, a baculovirus gene involved in very late gene expression.

    PubMed Central

    McLachlin, J R; Miller, L K

    1994-01-01

    We have identified a gene required for strong expression of the polyhedrin gene by characterizing a mutant, tsB837, of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which is temperature sensitive (ts) for occluded virus production at the nonpermissive temperature. Marker rescue experiments utilizing an overlapping set of AcMNPV genomic clones revealed that the gene responsible for the ts mutant phenotype mapped to a region between 46 and 48 map units. Fragments (2.2 kb) from both wild-type AcMNPV and tsB837 genomes spanning the mutated region were sequenced, and a single nucleotide difference was observed. This mutation is predicted to substitute a single amino acid within a 44.4-kDa polypeptide. Analysis of protein synthesis in wild-type- and mutant-infected cells at the nonpermissive temperature indicated that polyhedrin synthesis was dramatically reduced in the mutant. Northern (RNA) blot analysis revealed that the mutant had markedly reduced levels of polyhedrin transcripts. Transcripts of another very late gene, p10, were also reduced but to a lesser degree. The transcription of two late genes (603 ORF and vp39) was neither reduced nor temporally delayed. Thus, the gene encoding this very late expression factor, designated vlf-1, regulates the levels of very late gene transcripts, and the tsB837 mutation affects the levels of polyhedrin gene transcripts more strongly than those of p10 transcripts. The product of the newly identified gene has a surprising but significant relationship to a family of integrases and resolvases. Images PMID:7966564

  13. The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication.

    PubMed Central

    Lu, A; Miller, L K

    1995-01-01

    A set of 18 plasmid subclones of the Autographa californica nuclear polyhedrosis virus genome supports expression from a late viral promoter in transient expression assays (J. W. Todd, A. L. Passarelli, and L. K. Miller, J. Virol. 69:968-974, 1995). Using this set of plasmids, we have assigned a role for each of the 18 genes required for optimal late gene expression with respect to its involvement at the levels of transcription, translation, and/or DNA replication. RNase protection analyses demonstrated that all of the known late expression factor genes (lefs) affected the steady-state level of reporter gene RNA. Thus, none of the lefs appeared to be specifically involved in translation. A subset of the lefs supported plasmid replication; ie-1, lef-1, lef-2, lef-3, p143, and p35 were essential for plasmid replication, while ie-n, lef-7, and dnapol had stimulatory effects. The predicted sequence of lef-7 suggests that it is a homolog of herpesvirus single-stranded DNA-binding protein (UL29). The role of p35 in plasmid replication appears to be suppression of apoptosis, because p35 could be functionally replaced in the replication assay by either Cp-iap or Op-iap, two heterologous baculovirus genes which suppress apoptosis by a mechanism which appears to differ from that of p35. Thus, one or more of the replication-related lefs or the process of plasmid replication appears to induce cellular apoptosis. Our results indicate that the remaining lefs, lefs 4 through 11, p47, and 39K (pp31), function either at the level of transcription or at that of mRNA stabilization. PMID:7815565

  14. Baculovirus-mediated miRNA regulation to suppress hepatocellular carcinoma tumorigenicity and metastasis.

    PubMed

    Chen, Chiu-Ling; Wu, Jaw-Ching; Chen, Guan-Yu; Yuan, Pei-Hsiang; Tseng, Yen-Wen; Li, Kuei-Chang; Hwang, Shiaw-Min; Hu, Yu-Chen

    2015-01-01

    MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expressed in HCC cells. MiR-151 is aberrantly overexpressed in HCC cells and promotes HCC metastasis yet its roles on HCC tumorigenicity are unknown. To combat HCC tumorigenicity/metastasis, we developed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors that expressed (i) miR-122 precursors (pre-miR-122), (ii) miR-151 sponges, or (iii) pre-miR-122 and miR-151 sponges. Transduction of aggressive HCC cells (Mahlavu) with the pre-miR-122-expressing BV tremendously enhanced miR-122 levels for >6 weeks, suppressed the levels of downstream effectors (e.g., ADAM10 and Bcl-w), proliferation, anchorage-independent growth, motility and migration/invasion in vitro. Intratumoral injection of the pre-miR-122-expressing BV attenuated the HCC growth/metastasis. The miR-151 sponges-expressing BV diminished the miR-151 levels for 6 weeks, enhanced RhoGDIA expression, suppressed RhoGTPases, as well as motility and migration/invasion of Mahlavu cells. Intratumoral injection of the miR-151 sponge-expressing BV impeded not only HCC metastasis but also cell proliferation, MMP expression and tumor growth in vivo. The BV co-expressing pre-miR-122 and miR-151 sponges also simultaneously enhanced miR-122 expression and inhibited miR-151, and conferred antitumor/anti-metastasis effects albeit lack of synergism. These data implicate the potentials of the SB-based hybrid BV for persistently modulating miRNA and suppressing HCC tumorigenicity/metastasis. PMID:25023326

  15. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

    PubMed Central

    Lebacq-Verheyden, A M; Kasprzyk, P G; Raum, M G; Van Wyke Coelingh, K; Lebacq, J A; Battey, J F

    1988-01-01

    The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands. Images PMID:3211139

  16. Recombinant Baculovirus Associated with Bilosomes as an Oral Vaccine Candidate against HEV71 Infection in Mice

    PubMed Central

    Premanand, Balraj; Prabakaran, Mookkan; Kiener, Tanja K.; Kwang, Jimmy

    2013-01-01

    Background Human enterovirus 71 (HEV71) is one of the major pathogen responsible for hand, foot and mouth disease (HFMD). Currently no effective vaccine or antiviral drugs are available. Like poliovirus, EV71 is transmitted mainly by the feco-oral route. To date the majority of the studied EV71 vaccine candidates are administered parenterally. Injectable vaccines induce good systemic immunity but mucosal responses are often unsatisfactory, whereas mucosal vaccines provide both systemic and mucosal immunity. Therefore, oral immunization appears to be an attractive alternative to parenteral immunization. Methodology/Principal Findings In this report, we studied the efficacy of an orally administered vaccine candidate developed using recombinant baculovirus displaying VP1 (Bac-VP1) in a murine model. Gastrointestinal delivery of Bac-VP1 significantly induced VP1-specific humoral (IgG) and mucosal (IgA) immune responses. Further, we studied the efficacy of the Bac-VP1 associated with bilosomes and observed that the Bac-VP1 associated with bilosomes elicited significantly higher immune responses compared to bilosomes non-associated with Bac-VP1. However, mice immunized subcutaneously with live Bac-VP1 had significantly enhanced VP1 specific serum IgG levels and higher neutralizing antibody titers compared with mice orally immunized with live Bac-VP1 alone or associated with bilosomes. Conclusion Bilosomes have been shown to possess inherent adjuvant properties when associated with antigen. Therefore Bac-VP1 with bilosomes could be a promising oral vaccine candidate against EV71 infections. Thus, Bac-VP1 loaded bilosomes may provide a needle free, painless approach for immunization against EV71, thereby increasing patient compliance and consequently increasing vaccination coverage. PMID:23390538

  17. Lepidopteran Ortholog of Drosophila Breathless Is a Receptor for the Baculovirus Fibroblast Growth Factor

    PubMed Central

    Katsuma, Susumu; Daimon, Takaaki; Mita, Kazuei; Shimada, Toru

    2006-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an ∼110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis. PMID:16699027

  18. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

  19. Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV).

    PubMed

    Balasubramanian, G; Sarathi, M; Venkatesan, C; Thomas, John; Hameed, A S Sahul

    2008-12-01

    The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp. PMID:18834943

  20. Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage.

    PubMed

    Qiu, Lihua; Jiang, Shigui; Zhou, Falin; Zhang, Dianchang; Huang, Jianhua; Guo, Yihui

    2008-09-01

    The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage. PMID:17629788

  1. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution.

    PubMed

    Nagamine, Toshihiro; Sako, Yasushi

    2016-01-01

    Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143), but not BmNPV-P143 (BmP143) or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV. PMID:27244571

  2. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  3. The Effect of MicroRNA bantam on Baculovirus AcMNPV Infection in Vitro and in Vivo

    PubMed Central

    Shi, Xiaojie; Ran, Zihan; Li, Sisi; Yin, Juan; Zhong, Jiang

    2016-01-01

    The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction. PMID:27196923

  4. Optimization of the production of triabin, a novel thrombin inhibitor, in High Five™ insect cells infected with a recombinant baculovirus.

    PubMed

    Vallazza, M; Petri, T

    1999-03-01

    The isolation of a new type of thrombin inhibitor, called triabin, from the saliva of the hematophagous bug Triatoma pallidipennis, has recently been described. In the in vitro platelet aggregation inhibition assay triabin has a similar potency as the thrombin inhibitor hirudin now in phase III clinical trials. However, in another in vitro assay using a low molecular weight substrate for thrombin, triabin does not inhibit thrombin completely even at 6 fold higher molar doses in comparison with hirudin. This means that triabin has a novel mode of action towards thrombin making triabin into an interesting candidate as a therapeutic agent. Recently it has been shown that a recombinant baculovirus can be efficiently used for the triabin production in insect cells and that the yields in adherent cultures of High Five™ cells (approx. 20 mg l-1) were about 7 fold higher than in adherent cultures of Sf9 cells (approx. 3 mg l- 1). To optimize the triabin yield from the baculovirus/insect cell expression system, experiments were performed with suspension adapted cultures of High Five™ cells to investigate the effects of the state of the host cell, of the multiplicity of infection, of the cell density at the time of infection and of supplementation of the medium with nutrients and oxygen. Triabin yields of up to 200 mg l-1, as determined by an activity assay, could finally be obtained here. PMID:22359057

  5. The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae)

    PubMed Central

    Aragão-Silva, C. W.; Andrade, M. S.; Ardisson-Araújo, D. M. P.; Fernandes, J. E. A.; Morgado, F. S.; Báo, S. N.; Moraes, R. H. P.; Wolff, J. L. C.; Melo, F. L.; Ribeiro, B. M.

    2016-01-01

    Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable. PMID:27282807

  6. The Effect of MicroRNA bantam on Baculovirus AcMNPV Infection in Vitro and in Vivo.

    PubMed

    Shi, Xiaojie; Ran, Zihan; Li, Sisi; Yin, Juan; Zhong, Jiang

    2016-01-01

    The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction. PMID:27196923

  7. Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors.

    PubMed

    Mu, Jingfang; van Lent, Jan W M; Smagghe, Guy; Wang, Yun; Chen, Xinwen; Vlak, Just M; van Oers, Monique M

    2014-11-01

    The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process. PMID:25006078

  8. The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae).

    PubMed

    Aragão-Silva, C W; Andrade, M S; Ardisson-Araújo, D M P; Fernandes, J E A; Morgado, F S; Báo, S N; Moraes, R H P; Wolff, J L C; Melo, F L; Ribeiro, B M

    2016-01-01

    Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable. PMID:27282807

  9. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

    PubMed

    Stewart, Meredith E; Bonne, Nicolai; Shearer, Patrick; Khalesi, Bahman; Sharp, Margaret; Raidal, Shane

    2007-05-01

    Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI. PMID:17218022

  10. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment

    SciTech Connect

    Yuan Meijin; Wu Wenbi; Liu Chao; Wang Yanjie; Hu Zhaoyang; Yang Kai Pang Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc{sup P48-KO-PH-GFP} was unable to propagate in cell culture, while a 'repair' Bacmid vAc{sup P48-REP-PH-GFP} was able to replicate in a manner similar to a wild-type Bacmid vAc{sup PH-GFP}. Titration assays and Western blotting confirmed that vAc{sup P48-KO-PH-GFP} was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  11. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution

    PubMed Central

    Nagamine, Toshihiro; Sako, Yasushi

    2016-01-01

    Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143), but not BmNPV-P143 (BmP143) or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV. PMID:27244571

  12. Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

    PubMed Central

    Meulenberg, J J; Bende, R J; Pol, J M; Wensvoort, G; Moormann, R J

    1995-01-01

    The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV. PMID:8574824

  13. DEPENDENCE OF ECDYSTEROID METABOLISM AND DEVELOPMENT IN HOST LARVAE ON THE TIME OF BACULOVIRUS INFECTION AND THE ACTIVITY OF THE UDP-GLUCOSYL TRANSFERASE GENE.

    EPA Science Inventory

    Infection of fourth-instar gypsy moth (Lymantria dispar, Lepidoptera: Lymantriidae) larvae with the wild-type (Wt) gypsy moth baculovirus, LdNPV on the first day post-molt, or infection of fifth instars on the fifth day post-molt, results in elevated ecdysteroid levels in both he...

  14. Development of a Real-Time qPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest

    PubMed Central

    Graham, Robert I.; Tummala, Yamini; Rhodes, Glenn; Cory, Jenny S.; Shirras, Alan; Grzywacz, David; Wilson, Kenneth

    2015-01-01

    Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics. PMID:26463414

  15. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    PubMed

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. PMID:26923062

  16. Early Detection of Baculovirus Expression and Infection in Lepidopteran Larvae Fed Occlusion Bodies of an AcMNPV Recombinant Carrying a Red Fluorescent Protein Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method has been devised utilizing a baculovirus recombinant (AcMNPV hsp70Red) carrying a red fluorescent protein (RFP) gene under the early heat shock promoter (hsp70) to assess potential infectivity of larvae fed occlusion bodies. A time study was employed whereby first and third instars of Trich...

  17. Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?

    PubMed

    Ros, Vera I D; van Houte, Stineke; Hemerik, Lia; van Oers, Monique M

    2015-01-01

    Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour. PMID:25443568

  18. Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism

    PubMed Central

    Martins, Alexandra; Shuman, Stewart

    2003-01-01

    The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded β barrel (the so-called ‘triphosphate tunnel’). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure–activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of γ phosphate hydrolysis. PMID:12595553

  19. Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.

    PubMed

    Chakravarty, Sanchita; Suraj, K; Gupta, Satish Kumar

    2005-05-01

    To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway. PMID:15805145

  20. Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system☆

    PubMed Central

    Lu, Huiyuan; Ma, Jun; Liu, Nian; Wang, Shoulin

    2010-01-01

    Objective CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe3+ or hemin alone. Either co-addition of 5-ALA and Fe3+ or addition of 5-ALA or Fe3+ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). Conclusion 5-ALA and Fe3+ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression. PMID:23554636

  1. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

    PubMed

    Jose, Seena; Mohandas, A; Philip, Rosamma; Bright Singh, I S

    2010-11-01

    Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals. PMID:20807537

  2. A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

    PubMed

    Su, Jianguo; Oanh, Dang T H; Lyons, Russell E; Leeton, Lisa; van Hulten, Marielle C W; Tan, Siok-Hwee; Song, Linsheng; Rajendran, K V; Walker, Peter J

    2008-02-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. PMID:18166489

  3. Detection of a new microsporidium Perezia sp. in shrimps Penaeus monodon and P. indicus by histopathology, in situ hybridization and PCR.

    PubMed

    Han, Jee Eun; Tang, Kathy F J; Pantoja, Carlos R; Lightner, Donald V; Redman, Rita M; Le Groumellec, Marc

    2016-07-01

    Samples of microsporidia-infected shrimps exhibiting clinical signs of cotton shrimp disease were collected from Madagascar, Mozambique, and the Kingdom of Saudi Arabia from 2005 to 2014. The tails of the infected shrimps appeared opaque and whitish; subsequent histological examination revealed the presence of cytoplasmic inclusions and mature spores in tissues of the muscle, hepatopancreas, gills, heart, and lymphoid organ. PCR analysis targeting the small subunit rDNA (SSU rDNA) from infected samples resulted in the amplification of a 1.2 kbp SSU rDNA sequence fragment 94% identical to the corresponding region in the genome of the microsporidian Perezia nelsoni, which infects populations of Penaeus setiferus in the USA. Its SSU rDNA sequence was 100% identical among isolates from Madagascar and Saudi Arabia, indicating that shrimps from the Red Sea and Indian Ocean were infected with the same microsporidium, the novel Perezia sp. A 443 bp fragment of the SSU rDNA sequence was cloned, labeled with digoxigenin and subjected to an in situ hybridization assay with tissue sections of Perezia sp.-infected Penaeus monodon from Madagascar and Mozambique, and P. indicus from Saudi Arabia. The probe hybridized to the mature spores in the hepatopancreas and muscle from which the spores had been obtained for DNA isolation. This assay was specific, showing no reaction to another microsporidium, Enterocytozoon hepatopenaei (EHP), infecting the hepatopancreas of shrimp P. stylirostris cultured in SE Asian countries. We also developed an SSU rDNA-based PCR assay, specific for the novel Perezia sp. This PCR did not react to EHP, nor to genomic DNA of shrimp and other invertebrates. PMID:27409240

  4. Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships.

    PubMed

    Tourtip, Somjintana; Wongtripop, Somjai; Stentiford, Grant D; Bateman, Kelly S; Sriurairatana, Siriporn; Chavadej, Jittipan; Sritunyalucksana, Kallaya; Withyachumnarnkul, Boonsirm

    2009-09-01

    A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon. PMID:19527727

  5. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.

    PubMed

    Zhang, Ziguo; Yang, Jing; Barford, David

    2016-02-15

    The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells. PMID:26454197

  6. Production and purification of VP2 protein of porcine parvovirus expressed in an insect-baculovirus cell system

    PubMed Central

    2010-01-01

    The porcine parvovirus (PPV) VP2 protein was expressed in an insect-baculovirus cell system and was purified using Ni-NTA affinity column chromatography. The recombinant 6-His-tagged VP2 protein with molecular mass (Mr) of about 64 kDa was detected by anti-his antibody and anti-PPV serum. Electron microscopy showed that the purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The expressed VP2 was antigenically similar to the native capsid protein according to HA and a Western blotting assay performed with polyclonal antibodies collected from an outbreak of PPV in one farm. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of PPV or in the vaccination against PPV in the future. PMID:21143963

  7. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    PubMed

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  8. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    PubMed Central

    da Silva, Haroldo Cid; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-01-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  9. Baculovirus display of single chain antibody (scFv) using a novel signal peptide

    PubMed Central

    2010-01-01

    Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV vector resulted in

  10. Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System

    PubMed Central

    Yazdani, Yaghoub; Azari, Shahram; Kalhor, Hamid Reza

    2016-01-01

    Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer’s disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. PMID:27123417

  11. A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system.

    PubMed

    Schlaeppi, Jean-Marc; Henke, Mario; Mahnke, Marion; Hartmann, Steffen; Schmitz, Rita; Pouliquen, Yann; Kerins, Brendan; Weber, Eric; Kolbinger, Frank; Kocher, Hans P

    2006-12-01

    The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%. PMID:16904904

  12. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus

    PubMed Central

    Crava, Cristina M.; Jakubowska, Agata K.; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity. PMID:25993013

  13. Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

    PubMed

    Crava, Cristina M; Jakubowska, Agata K; Escriche, Baltasar; Herrero, Salvador; Bel, Yolanda

    2015-01-01

    Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity. PMID:25993013

  14. Baculovirus DNA Replication-Specific Expression Factors Trigger Apoptosis and Shutoff of Host Protein Synthesis during Infection▿

    PubMed Central

    Schultz, Kimberly L. W.; Friesen, Paul D.

    2009-01-01

    Apoptosis is an important antivirus defense. To define the poorly understood pathways by which invertebrates respond to viruses by inducing apoptosis, we have identified replication events that trigger apoptosis in baculovirus-infected cells. We used RNA silencing to ablate factors required for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transfection with double-stranded RNA (dsRNA) complementary to the AcMNPV late expression factors (lefs) that are designated as replicative lefs (lef-1, lef-2, lef-3, lef-11, p143, dnapol, and ie-1/ie-0) blocked virus DNA synthesis and late gene expression in permissive Spodoptera frugiperda cells. dsRNAs specific to designated nonreplicative lefs (lef-8, lef-9, p47, and pp31) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lefs functioned during infection as defined. Silencing the replicative lefs prevented AcMNPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lefs did not. Thus, the activity of replicative lefs or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, AcMNPV-induced apoptosis was suppressed by silencing the replicative lefs in cells from a divergent species, Drosophila melanogaster. Silencing replicative but not nonreplicative lefs also abrogated AcMNPV-induced shutdown of host protein synthesis, suggesting that virus DNA replication triggers inhibition of host biosynthetic processes and that apoptosis and translational arrest are linked. Our findings suggest that baculovirus DNA replication triggers a host cell response similar to the DNA damage response in vertebrates, which causes translational arrest and apoptosis. Pathways for detecting virus invasion and triggering apoptosis may therefore be conserved between insects and mammals. PMID:19706708

  15. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    PubMed

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects. PMID:26323262

  16. A Model to Study the Phenotypic Changes of Insect Cell Transfection by Copepod Super Green Fluorescent Protein (cop-GFP) in Baculovirus Expression System

    PubMed Central

    Shokrollahi, Narjes; Shahbazzadeh, Delavar; Pooshang-Bagheri, Kamran; Habibi-Anbouhi, Mahdi; Jahanian-Najafabadi, Ali; Behdani, Mahdi

    2016-01-01

    Background: Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells. Methods: The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein (copGFP). In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells. Results: Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis. Conclusion: The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection. PMID:26518237

  17. Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b.

    PubMed

    Liao, Ya-Hsin; Chang, Yu-Han; Sung, Li-Yu; Li, Kuei-Chang; Yeh, Chia-Lin; Yen, Tzu-Chen; Hwang, Shiaw-Min; Lin, Kun-Ju; Hu, Yu-Chen

    2014-06-01

    Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adipose-derived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling ≈94% of defect area and ≈89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing. PMID:24674465

  18. Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

    PubMed

    Meneses-Ruiz, D M; Laclette, J P; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, J C

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. PMID:22110386

  19. Protective Efficacy of a Single Dose of Baculovirus Hemagglutinin-Based Vaccine in Chickens and Ducks Against Homologous and Heterologous H5N1 Virus Infections

    PubMed Central

    Park, Eun Hye; Song, Byung Min; Yum, Jung; Kim, Ji An; Oh, Seung Kyoo; Kim, Hyun Soo; Cho, Gil Jae

    2014-01-01

    Abstract Outbreaks of the highly pathogenic H5N1 virus in poultry and humans are ongoing. Vaccination is an efficient method for prevention of H5N1 infection. Using chickens and ducks, we assessed the efficacy of a vaccine comprising H5N1 hemagglutinin (HA) protein produced in a baculovirus expression system. The immunized chickens and ducks were protected against lethal infection by H5N1 in an antigen dose-dependent manner. Complete protection against homologous challenge and partial protection against heterologous challenge were achieved in chickens immunized with 5 μg HA protein and in ducks immunized with 10 μg HA protein. The IgG antibody subtype was mainly detected in the sera and tissues, including the lungs. The neuraminidase (NA) inhibition assay was negative in immunized chickens and ducks. Our results indicated that the expressed HA protein by baculovirus was immunogenic to both chickens and ducks, and the immunized chickens and ducks were protected from the lethal infections of highly pathogenic H5N1 influenza virus, though ducks required more HA protein than chickens to be protected. Also, baculovirus HA-vaccinated poultry can be differentiated from infected poultry by NA inhibition assay. PMID:25211640

  20. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    PubMed Central

    Meneses-Ruiz, DM; Laclette, JP; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, JC

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. PMID:22110386

  1. Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers

    PubMed Central

    Fung, King Leung; Kapoor, Khyati; Pixley, Jessica N.; Talbert, Darrell J.; Kwit, Alexandra D.T.; Ambudkar, Suresh V.

    2016-01-01

    The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [3H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp– or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system PMID:26622052

  2. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    PubMed Central

    Suzuki, Hitoshi; Matsumoto, Norihiko; Suzuki, Tomoyuki; Chang, Myint OO; Takaku, Hiroshi

    2009-01-01

    Background Hepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing

  3. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties

    PubMed Central

    2013-01-01

    Background Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. Methods A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3’-5’-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman’s assay in microplates. Results A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for

  4. Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming.

    PubMed

    Shikano, Ikkei; Hua, Kevin Ngoc; Cory, Jenny S

    2016-05-01

    Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to

  5. Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

    PubMed

    Yee, C J; DeFrances, M C; Bell, A; Bowen, W; Petersen, B; Michalopoulos, G K; Zarnegar, R

    1993-08-10

    A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by

  6. Virus-like particles of porcine bocavirus generated by recombinant baculoviruses can be applied to sero-epidemic studies.

    PubMed

    Zhang, Wenjing; Sano, Natsuha; Kataoka, Michiyo; Ami, Yasushi; Suzaki, Yuriko; Wakita, Takaji; Ikeda, Hidetoshi; Li, Tian-Cheng

    2016-06-01

    Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm(3), respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV. PMID:26959654

  7. Incorporation of partial polyhedrin homology sequences (PPHS) enhances the production of cloned foreign genes in a baculovirus expression system.

    PubMed

    Gong, Zhaohui; Jin, Yongfeng; Zhang, Yaozhou

    2006-03-01

    Baculovirus expression vector systems (BEVSs) have been used extensively for high-level expression of cloned foreign genes. In many instances, the levels of recombinant protein(s) produced in insect cells and larvae are insufficient for experimental purposes. Thus new techniques and methods are needed to increase significantly the protein expression levels in BEVS. In the present paper, we describe the incorporation of a 15 bp element derived from the 5'-end partial sequence of the polyhedrin gene, which contains the non-coding sequence ATAAAT and the coding sequence ATGCCGAAT, into the 5'-end of the CTB (cholera toxin B subunit)-INS (insulin) fusion gene. With the addition of the PPHS (partial polyhedrin homology sequences), two extra amino acids (Pro-Asn) were added to the N-terminus of the mCTB-INS (modified CTB-INS) fusion protein. This new fusion protein was expressed in both insect cells and larvae using BEVSs. We found that the addition of PPHS enhanced 4-fold the expression of CTB-INS in both insect cells and larvae. Further analysis revealed that the additional two amino acids in mCTB-INS did not significantly affect binding affinity for G(M1) ganglioside. Therefore the PPHS can be used as a constitutive element immediately downstream of the polyhedrin promoter to induce significant increases in the expression levels of cloned foreign genes. PMID:16313236

  8. Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

    PubMed

    Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

    2015-12-01

    Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. PMID:26160190

  9. Baculovirus-Vectored Multistage Plasmodium vivax Vaccine Induces Both Protective and Transmission-Blocking Immunities against Transgenic Rodent Malaria Parasites

    PubMed Central

    Mizutani, Masanori; Iyori, Mitsuhiro; Blagborough, Andrew M.; Fukumoto, Shinya; Funatsu, Tomohiro; Sinden, Robert E.

    2014-01-01

    A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen. PMID:25092912

  10. Densovirus Is a Mutualistic Symbiont of a Global Crop Pest (Helicoverpa armigera) and Protects against a Baculovirus and Bt Biopesticide

    PubMed Central

    Xu, Pengjun; Liu, Yongqiang; Graham, Robert I.; Wilson, Kenneth; Wu, Kongming

    2014-01-01

    Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus. PMID:25357125

  11. Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response.

    PubMed

    Liu, Ming-Kun; Lin, Jhe-Jhih; Chen, Chung-Yung; Kuo, Szu-Cheng; Wang, Yu-Ming; Chan, Hong-Lin; Wu, Tzong Yuan

    2016-01-01

    BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy. PMID:27314325

  12. Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

    PubMed Central

    Greenfield, C; Patel, G; Clark, S; Jones, N; Waterfield, M D

    1988-01-01

    The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production. Images PMID:2834199

  13. Pathogen persistence in the environment and insect-baculovirus interactions: disease-density thresholds, epidemic burnout, and insect outbreaks.

    PubMed

    Fuller, Emma; Elderd, Bret D; Dwyer, Greg

    2012-03-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when larvae consume foliage contaminated with particles released from infectious cadavers. Using field-transmission experiments, we are able to estimate persistence time under natural conditions, and inserting our estimates into a standard epidemic model suggests that epidemics are often terminated by a combination of pupation and burnout rather than by burnout alone, as predicted by theory. Extending our models to allow for multiple generations, and including environmental transmission over the winter, suggests that the virus may survive over the long term even in the absence of complex persistence mechanisms, such as environmental reservoirs or covert infections. Our work suggests that estimates of persistence times can lead to a deeper understanding of environmentally transmitted pathogens and illustrates the usefulness of experiments that are closely tied to mathematical models. PMID:22322229

  14. Pathogen Persistence in the Environment and Insect-Baculovirus Interactions: Disease-Density Thresholds, Epidemic Burnout and Insect Outbreaks

    PubMed Central

    Fuller, Emma; Elderd, Bret D.

    2013-01-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when larvae consume foliage contaminated with particles released from infectious cadavers. Using field-transmission experiments, we are able to estimate persistence time under natural conditions, and inserting our estimates into a standard epidemic model suggests that epidemics are often terminated by a combination of pupation and burnout, rather than by burnout alone as predicted by theory. Extending our models to allow for multiple generations, and including environmental transmission over the winter, suggests that the virus may survive over the long term even in the absence of complex persistence mechanisms, such as environmental reservoirs or covert infections. Our work suggests that estimates of persistence times can lead to a deeper understanding of environmentally transmitted pathogens, and illustrates the usefulness of experiments that are closely tied to mathematical models. PMID:22322229

  15. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    PubMed

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  16. In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells.

    PubMed

    Thoene, Jess; Goss, Thomas; Witcher, Marc; Mullet, Jodi; N'Kuli, Francisca; Van Der Smissen, Patrick; Courtoy, Pierre; Hahn, Si Houn

    2013-05-01

    Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~1.05 g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T1/2 ~48 h), saturable (down to ~40% of initial after 4 days) and stable (>one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy. PMID:23465695

  17. Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

    PubMed

    Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gravisaco, María José; Lucero, María Soledad; Richetta, Matías; Gómez, Evangelina; Berinstein, Analía

    2016-07-01

    Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases. PMID:27063861

  18. Delivery of glutamine synthetase gene by baculovirus vectors: a proof of concept for the treatment of acute hyperammonemia.

    PubMed

    Torres-Vega, M A; Vargas-Jerónimo, R Y; Montiel-Martínez, A G; Muñoz-Fuentes, R M; Zamorano-Carrillo, A; Pastor, A R; Palomares, L A

    2015-01-01

    Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver diseases, can cause neuropsychiatric complications, which in the worst cases result in brain damage, coma or death. Diverse treatments exist for the treatment of hyperammonemia, but they have limited efficacy, adverse effects and elevated cost. Gene therapy is a promising alternative that is explored here. A baculovirus, termed Bac-GS, containing the glutamine synthetase (GS) gene was constructed for the in vitro and in vivo treatment of hyperammonemia. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a high expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia. When Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of ammonia in blood decreased 351 μM, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles from the rats transduced with Bac-GS. These results show that gene delivery for overexpressing GS in muscle tissue is a promising alternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy or UCD. PMID:25338921

  19. Studies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

    PubMed

    Zerka, Agata; Rydzak, Joanna; Lass, Anna; Szostakowska, Beata; Nahorski, Wacław; Wroczyńska, Agnieszka; Myjak, Przemyslaw; Krotkiewski, Hubert; Jaskiewicz, Ewa

    2016-04-01

    The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines. PMID:26439848

  20. Production of hepatitis E virus-like particles presenting multiple foreign epitopes by co-infection of recombinant baculoviruses

    PubMed Central

    Shima, Ryoichi; Li, Tian Cheng; Sendai, Yutaka; Kataoka, Chikako; Mori, Yoshio; Abe, Takayuki; Takeda, Naokazu; Okamoto, Toru; Matsuura, Yoshiharu

    2016-01-01

    Hepatitis E virus (HEV) causes not only endemics via a fecal-oral route but also sporadic cases via zoonotic transmission or blood transfusion. HEV-like particles (HEV-LP) produced by using a baculovirus expression system are considered a candidate for mucosal vaccines for HEV infection. In this study, we attempted to produce a chimeric HEV-LP presenting various foreign epitopes on its surface. Expression of the recombinant capsid proteins carrying a myc- or FLAG-tag inserted between amino acid residues 488 and 489, which are located in the exterior loop on the protruding domain of the HEV capsid, resulted in the production of recombinant HEV-LP. Although expression of the recombinant capsid protein carrying the HA-tag inserted at the same site failed to produce any particles, co-expression with the myc-tagged capsid protein successfully yielded a chimeric HEV-LP consisting of both recombinant capsid proteins. Immunoprecipitation analyses confirmed that the chimeric particles present these foreign epitopes on the surface. Similar results were obtained for the expression of the recombinant capsid proteins carrying neutralizing epitopes of Japanese encephalitis virus. These results suggest the chimeric HEV-LP system provides a novel vaccine carrier that can accommodate multiple neutralizing epitopes on its surface. PMID:26905478

  1. Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response

    PubMed Central

    Liu, Ming-Kun; Lin, Jhe-Jhih; Chen, Chung-Yung; Kuo, Szu-Cheng; Wang, Yu-Ming; Chan, Hong-Lin; Wu, Tzong Yuan

    2016-01-01

    BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy. PMID:27314325

  2. Ligand binding properties of muscarinic acetylcholine receptor subtypes (m1-m5) expressed in baculovirus-infected insect cells.

    PubMed

    Dong, G Z; Kameyama, K; Rinken, A; Haga, T

    1995-07-01

    Five subtypes of muscarinic acetylcholine receptors (m1-m5) have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system. Up to 6 nmol of muscarinic acetylcholine receptors were produced by 1 liter culture; 0.3 to 0.6 (human m1), 3 to 6 (human m2), 2 to 4 (rat m3), 1 to 2 (rat m4) and 0.5 to 1 (human m5) nmol. Pirenzepine, AF-DX116 and hexahidrosiladifenidol showed the highest affinity for the m1, m2 and m3 subtype, respectively, indicating that these receptors expressed in Sf9 cells retain the same substrate specificity as those in mammalian tissues or cultured cells. Among 32 kinds of muscarinic ligands examined in the present studies, prifinium was found to have the highest affinity for the m4 subtype, and pilocarpine, oxotremorine, McN-A343 and promethazine the highest affinity for the m5 subtype, although the differences in the affinities among the five subtypes were less than 10-fold. Alcuronium increased the binding of [3H]N-methylscopalamine to the m2 subtype, but not the m1, m4 and m5 subtypes and only slightly to the m3 subtype. Similar but smaller effects of fangchinoline and tetrandrine were found for [3H]N-methylscopalamine binding to only the m3 subtype. These effects may also be useful for the discrimination of individual subtypes. PMID:7616422

  3. Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

    PubMed

    Lundkvist, A; Vapalahti, O; Plyusnin, A; Sjölander, K B; Niklasson, B; Vaheri, A

    1996-11-01

    Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family. PMID:8896239

  4. Validation of a Commercial Insulated Isothermal PCR-based POCKIT Test for Rapid and Easy Detection of White Spot Syndrome Virus Infection in Litopenaeus vannamei

    PubMed Central

    Tsai, Yun-Long; Wang, Han-Ching; Lo, Chu-Fang; Tang-Nelson, Kathy; Lightner, Donald; Ou, Bor-Rung; Hour, Ai-Ling; Tsai, Chuan-Fu; Yen, Cheng-Chi; Chang, Hsiao-Fen Grace; Teng, Ping-Hua; Lee, Pei-Yu

    2014-01-01

    Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61–95.56%) and specificity 97% (95% CI: 94.31–98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei. PMID:24625894

  5. N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

    PubMed

    Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

    2016-05-01

    The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. PMID:27030553

  6. Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions.

    PubMed

    Nguyen, Quan; Palfreyman, Robin W; Chan, Leslie C L; Reid, Steven; Nielsen, Lars K

    2012-01-01

    The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from cultured H. zea cells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection of H. zea cells with H. Armigera single-capsid nucleopolyhedrovirus (HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base for H. zea insect-HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of

  7. Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system.

    PubMed

    Ring, M; Meredith, J; Wiens, C; Macins, A; Brock, H W; Phillips, J E; Theilmann, D A

    1998-01-01

    We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl- transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2-3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270-fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses. PMID:9612936

  8. Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent, cross-clade protection against H5N1 influenza virus in mice.

    PubMed

    Zhang, Jie; Fan, Hui-Ying; Zhang, Zhen; Zhang, Juan; Zhang, Jiao; Huang, Jian-Ni; Ye, Yu; Liao, Ming

    2016-01-27

    H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8(+) T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity. PMID:26724200

  9. Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

    PubMed

    Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto; Corva, Santiago Gerardo; Mórtola, Eduardo Carlos; Larsen, Alejandra; Jarvis, Donald L; Echeverría, María Gabriela

    2013-07-01

    Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine. PMID:23631926

  10. Improvement in the UV resistance of baculoviruses by displaying nano-zinc oxide-binding peptides on the surfaces of their occlusion bodies.

    PubMed

    Li, Jin; Zhou, Yin; Lei, Chengfeng; Fang, Wei; Sun, Xiulian

    2015-08-01

    The sensitivity of baculoviruses to UV radiation severely limits their large-scale application as biological insecticides. The polyhedron envelope of a baculovirus, which is composed of carbohydrate and polyhedron envelope protein (PEP), is a significant structure for the stability and persistence of occlusion bodies (OBs) under environmental conditions. The results of this study revealed that the rough pitted surface phenotype of a pep-null Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could not be rescued by any of its homologues, such as Helicoverpa armigera nucleopolyhedrovirus pep or Cydia pomonella granulovirus putative peps. In contrast, the N-terminal and middle flexible region (NM region, 1-167 aa) of AcMNPV PEP were able to form an intact OB envelope. Furthermore, this region was capable of carrying eGFP to the surfaces of the OBs. To improve the UV resistance of AcMNPV OBs, two peptides capable of specifically binding to nano-ZnO were separately fused to the NM region of PEP. Under laboratory conditions, infectivity of the recombinant viruses binding to nano-ZnO particles was about ninefold higher than that without the nano-ZnO particles after UV-B irradiation. Pot experiments revealed that the half-life of the recombinant baculovirus binding nano-ZnO particles was 3.3 ± 0.15 days, which was significantly longer than that of the control virus (0.49 ± 0.06 days). These results therefore represent a new approach for the protection the baculoviral insecticides against UV irradiation in the field. PMID:25895092

  11. The Baculovirus Core Gene ac83 Is Required for Nucleocapsid Assembly and Per Os Infectivity of Autographa californica Nucleopolyhedrovirus

    PubMed Central

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection. PMID:23864639

  12. Safety and immunogenicity of H5N1 influenza vaccine based on baculovirus surface display system of Bombyx mori.

    PubMed

    Jin, Rongzhong; Lv, Zhengbing; Chen, Qin; Quan, Yanping; Zhang, Haihua; Li, Si; Chen, Guogang; Zheng, Qingliang; Jin, Lairong; Wu, Xiangfu; Chen, Jianguo; Zhang, Yaozhou

    2008-01-01

    Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans. PMID:19079592

  13. Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system

    USGS Publications Warehouse

    Shivappa, R.B.; McAllister, P.E.; Edwards, G.H.; Santi, N.; Evensen, O.; Vakharia, V.N.

    2005-01-01

    Various attempts to develop a vaccine against infectious pancreatic necrosis virus (IPNV) have not yielded consistent results. Thus, at present, no commercial vaccine is available that can be used with confidence to immunize fry of salmon and trout. We generated a cDNA clone of the large genome segment A of an IPNV Sp strain and expressed all structural protein genes in insect cells and larvae using a baculovirus expression system. Green fluorescent protein was also co-expressed as a reporter molecule. High yields of IPNV proteins were obtained and the structural proteins self assembled to form virus-like particles (VLPs). We tested the immunogenicity of the putative VLP antigen in immersion vaccine experiments (two concentrations) in rainbow trout (Oncorhynchus mykiss) fry, and by intraperitoneal immunisation of Atlantic salmon (Salmo salar) pre-smolts using an oil adjuvant formulation. Rainbow trout were challenged by immersion using either the Sp or the VR-299 strain of IPNV two or three weeks post-vaccination, while Atlantic salmon were bath challenged with Sp strain after two months, after parr-smolt transformation. In the rainbow trout fry challenged two weeks post-immunization, cumulative mortality rates three weeks post challenge were 14 % in the fry that had received the highest dose versus 8 % in the control groups. No indication of protection was seen in repeated trials using a lower dose of antigen and challenge three weeks post-immunisation. The cumulative mortality rate of intraperitoneally immunised Atlantic salmon post-smolts four weeks post challenge was lower (56 %) than in the control fish (77 %), showing a dose-response pattern.

  14. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.

    PubMed Central

    Guarino, L A; Summers, M D

    1986-01-01

    The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1. Images PMID:3944847

  15. Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.

    PubMed

    Pajot-Augy, E; Bozon, V; Remy, J J; Couture, L; Salesse, R

    1999-03-01

    The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors. PMID:10102991

  16. The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

    PubMed

    Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

    2016-06-01

    A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future. PMID:27177910

  17. Cell Susceptibility to Baculovirus Transduction and Echovirus Infection Is Modified by Protein Kinase C Phosphorylation and Vimentin Organization

    PubMed Central

    Turkki, Paula; Makkonen, Kaisa-Emilia; Huttunen, Moona; Laakkonen, Johanna P.; Ylä-Herttuala, Seppo; Airenne, Kari J.

    2013-01-01

    Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively. PMID:23824807

  18. Single Chain Variable Fragment Against Aβ Expressed in Baculovirus Inhibits Abeta Fibril Elongation and Promotes its Disaggregation

    PubMed Central

    Fang, Fang; Song, Lin-Lin; Jiao, Yue-Ying; Wang, He; Peng, Xiang-Lei; Zheng, Yan-Peng; Wang, Jun; He, Jin-Sheng; Hung, Tao

    2015-01-01

    Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD. PMID:25919299

  19. Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells.

    PubMed

    Nakayama, Tomofumi; Yuasa, Eri; Kanemaru, Ayumi; Saito, Masayuki; Saitoh, Hisato

    2014-01-01

    Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1. PMID:24637489

  20. Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene▿

    PubMed Central

    Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, WonKyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

    2010-01-01

    Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph. PMID:20219904

  1. Enhancing yield of infectious Bursal disease virus structural proteins in baculovirus expression systems: focus on media, protease inhibitors, and dissolved oxygen.

    PubMed

    Hu, Y C; Bentley, W E

    1999-01-01

    Structural proteins of the poultry pathogen, infectious bursal disease virus (IBDV), were expressed in the baculovirus/insect cell expression system. To date, several reports have indicated that animal virus structural proteins are expressed only at low yield in this system. In this article, several factors were examined to enhance yield. These include medium, dissolved oxygen level, and the addition (in vivo and in vitro) of protease inhibitors. Specifically, two media were compared, and SF-900 II was superior to Ex-Cell 401 for cell growth and IBDV protein expression. A cocktail of protease inhibitors including phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and ethylenediamine tetraacetic acid (EDTA) minimized proteolysis in vitro. Also, aprotinin and pepstatin A deterred product degradation in vivo and increased the product yield nearly 2-fold. Finally, in 3 L bioreactors, a dissolved oxygen tension of 50% DO (air saturation) was optimal. Results demonstrated that several relatively simple adjustments to the baculovirus system significantly improved the yield of IBD virus structural proteins. PMID:10585191

  2. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    PubMed Central

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. PMID:26090442

  3. Transient and Stable Expression of the Neurotensin Receptor NTS1: A Comparison of the Baculovirus-Insect Cell and the T-REx-293 Expression Systems

    PubMed Central

    Xiao, Su; White, Jim F.; Betenbaugh, Michael J.; Grisshammer, Reinhard; Shiloach, Joseph

    2013-01-01

    Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [3H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs. PMID:23696845

  4. Parasitic Manipulation of Host Behaviour: Baculovirus SeMNPV EGT Facilitates Tree-Top Disease in Spodoptera exigua Larvae by Extending the Time to Death

    PubMed Central

    Han, Yue; van Houte, Stineke; Drees, Gerben F.; van Oers, Monique M.; Ros, Vera I. D.

    2015-01-01

    Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e., climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease. PMID:26463412

  5. Parasitic Manipulation of Host Behaviour: Baculovirus SeMNPV EGT Facilitates Tree-Top Disease in Spodoptera exigua Larvae by Extending the Time to Death.

    PubMed

    Han, Yue; van Houte, Stineke; Drees, Gerben F; van Oers, Monique M; Ros, Vera I D

    2015-01-01

    Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e., climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease. PMID:26463412

  6. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

    PubMed

    Premanand, Balraj; Kiener, Tanja K; Meng, Tao; Tan, Yun Rui; Jia, Qiang; Chow, Vincent T K; Kwang, Jimmy

    2012-09-01

    EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice. PMID:22691220

  7. Protection of mice against pandemic H1N1 influenza virus challenge after immunization with baculovirus-expressed stabilizing peptide fusion hemagglutinin protein.

    PubMed

    Yang, Eunji; Cho, Yonggeun; Choi, Jung-Ah; Choi, YoungJoo; Park, Pil-Gu; Park, Eunsun; Lee, Choong Hwan; Lee, Hyeja; Kim, Jongsun; Lee, Jae Myun; Song, Manki

    2015-02-01

    Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein (2 μg/mouse) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility. PMID:25394603

  8. Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

    PubMed Central

    Prud'homme, I; Zhou, E M; Traykova, M; Trotter, H; Chan, M; Afshar, A; Harding, M J

    1997-01-01

    The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV. Images Figure 2. Figure 3. Figure 4. PMID:9342453

  9. Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes.

    PubMed

    Deepika, A; Sreedharan, K; Paria, Anutosh; Makesh, M; Rajendran, K V

    2014-12-01

    The Toll-pathway plays key roles in regulating the innate immune response in invertebrates. Myeloid differentiation factor 88 (MyD88) and Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) are key molecules in this signalling pathway. To investigate the role of Toll-pathway in innate immune response of shrimp, Penaeus monodon, MyD88 (PmMyD88) and TRAF6 (PmTRAF6) were identified and characterised. PmMyD88 cDNA is 1716 bp long with an open reading frame (ORF) of 1449 bp encoding a putative protein of 482 amino acids, with a death domain, a TIR domain and C-terminal extension domain. PmTRAF6 cDNA is 2563 bp long with an ORF of 1785 bp (594 amino acids) with an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled region and a MATH domain. In healthy shrimp, PmMyD88, PmTRAF6 and PmToll were detected in 15 tissues with the highest expression in midgut, eyestalk and lymphoid organ, respectively. Responses of these genes to WSSV in experimentally-infected P. monodon as well as in cultured haemocytes and also effect of poly I:C on the gene expression in vitro was investigated at six time-points in seven tissues. PmToll showed significant up-regulation at all time-points of infection in six tissues and until 24 h post-infection in vitro. However, poly I:C-induced haemocytes showed up-regulation of the gene until 48 h post-exposure. WSSV caused significant up-regulation of PmMyD88 in most of the tissues tested. The virus challenge as well as poly I:C induction in vitro also resulted in significant up-regulation of the gene. Up-regulated expression of PmTRAF6 was detected in haemocytes and lymphoid organ at late stage of infection. In vitro virus challenge showed significant up-regulation of PmTRAF6 at almost all time-points whereas no significant change in the expression was observed on poly I:C induction. The responses of these key genes, observed in the present study, suggest that Toll-pathway as a whole may play a crucial

  10. The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

    PubMed

    Hattori, Tomomi; Nakanishi, Kohei; Mori, Takaaki; Tomita, Masahiro; Tsumoto, Kanta

    2016-01-01

    Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells. PMID:26498840

  11. Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

    PubMed Central

    Yamada, Hayato; Kitaguchi, Koji; Hamajima, Rina; Kobayashi, Michihiro

    2013-01-01

    We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells. PMID:24067961

  12. Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine.

    PubMed

    Huynh, Hoai T; Chan, Leslie C L; Tran, Trinh T B; Nielsen, Lars K; Reid, Steven

    2012-01-01

    A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in-house low-cost serum-free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex. However, VPM3 is composed of a significant number of undefined components, including five different protein hydrolysates, which introduce a challenging lot-to-lot variability to the production process. In this study, an intensive statistical optimization routine was employed to reduce the number of protein hydrolysates in VPM3 medium. Nearly 300 runs (including replicates) were conducted with great efficiency by using 50 mL TubeSpin® bioreactors to propagate insect cell suspension cultures. Fractional factorial experiments were first used to determine the most important of the five default protein hydrolysates, and to screen for seven potential substitutes for the default meat peptone, Primatone RL. Validation studies informed by the screening tests showed that promising alternative media could be formulated based on just two protein hydrolysates, in particular the YST-AMP (Yeast Extract and Amyl Meat Peptone) and YST-POT (Yeast Extract and Lucratone Potato Peptone) combinations. The YST-AMP (meat-based) and YST-POT (meat-free) variants of VPM3 were optimized using response surface methodology, and were shown to be just as good as the default VPM3 and the commercial Sf-900 II media in supporting baculovirus yields, hence providing a means toward a more reproducible and scalable production process for HaSNPV biopesticides. PMID:22323401

  13. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    PubMed

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  14. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    SciTech Connect

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  15. An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha - An example of adaptation to a new host.

    PubMed

    Rabalski, Lukasz; Krejmer-Rabalska, Martyna; Skrzecz, Iwona; Wasag, Bartosz; Szewczyk, Boguslaw

    2016-09-01

    A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocapsids of LdMNPV-BNP are enveloped together in 2-26 virions embedded in occluded bodies (OBs) very similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae in a relatively short time (LT50 of approximately 9days for a dose of 2×10(7)OBs/ml), highlighting the possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene content (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is 157,270 base pairs long and has a notably lower G+C content in comparison to other LdMNPVs (50.3% G+C content compared to an average of 57.4% among other LdMNPVs). According to our phylogenetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneficial as an active component of biopesticides that can be used during forest integrated pest management. PMID:27451947

  16. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    PubMed

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. PMID:26643933

  17. TRANSFECTION WITH BACULOVIRUS DNA

    EPA Science Inventory

    Purified DNA from the nuclear polyhedrosis viruses of Autographa californica (AcM NPV) and Rachiplusia ou (RoM NPV) were found to be infectious in TN-368 cells employing the calcium phosphate precipitation technique (F.L. Graham and A.J. van der Eb, Virology, 52, 456-467, 1973). ...

  18. Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

    PubMed Central

    Li, John J.; Cao, Chune; Fixsen, Sarah M.; Young, Janet M.; Bando, Hisanori; Elde, Nels C.; Katsuma, Susumu; Dever, Thomas E.; Sicheri, Frank

    2015-01-01

    Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase. PMID:26216977

  19. Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase.

    PubMed Central

    Emery-Goodman, A; Hirling, H; Scarpellino, L; Henderson, B; Kühn, L C

    1993-01-01

    Iron regulatory factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in iron metabolism. Binding of IRF to iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after iron deprivation, but is inactive, yet present, under conditions of high cellular iron supply. Recently, IRF was also shown to have aconitase activity indicating the existence of an Fe-S cluster in the protein. In the current study we expressed human IRF in insect cells from recombinant baculovirus and analysed IRE-binding and aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of iron, was fully active in IRE-binding, but showed no aconitase activity. In contrast, IRF made by cells grown in high iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster. Images PMID:8464737

  20. Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation▿ †

    PubMed Central

    Wang, Manli; Tuladhar, Era; Shen, Shu; Wang, Hualin; van Oers, Monique M.; Vlak, Just M.; Westenberg, Marcel

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly. PMID:20519380

  1. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays. PMID:23820164

  2. Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system.

    PubMed

    Khoo, Keng Meng; Chang, Chan Fong; Schubert, Jeffrey; Wondrak, Ewald; Chng, Hiok Hee

    2005-04-01

    CD38 is a type II transmembrane glycoprotein found in myriad mammalian tissues and cell types. It is known for its involvement in the metabolism of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. CD38 itself has been shown to have clinical significance in certain diseases with possible utilization in diagnostic and prognostic applications. Previous studies on several autoimmune diseases have shown the usefulness of recombinant CD38 protein expressed from Escherichia coli and Pichia pastoris in the detection of autoantibodies to CD38 via Western blot and ELISA. In this study, we produced a 6 x His-tagged GST-CD38 fusion protein using a recombinant baculovirus/insect cell expression technique that was purified as a soluble protein. The fusion protein was purified to homogeneity by affinity and gel filtration chromatography steps. It has an apparent molecular mass of 56 kDa on SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by antibodies against human CD38 as well as the polyhistidine tag. Peptide mass fingerprinting analysis confirmed the identity of human CD38 in the fusion protein. PMID:15766882

  3. Self-Assembly and Release of Peste des Petits Ruminants Virus-Like Particles in an Insect Cell-Baculovirus System and Their Immunogenicity in Mice and Goats

    PubMed Central

    Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

    2014-01-01

    Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR. PMID:25117931

  4. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used. PMID:19737754

  5. Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System

    PubMed Central

    Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

    2015-01-01

    Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. Objectives The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. Materials and Methods To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Results Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Conclusions Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV. PMID:26862379

  6. Assembly-defective point mutants of the human immunodeficiency virus type 1 Gag precursor phenotypically expressed in recombinant baculovirus-infected cells.

    PubMed Central

    Hong, S S; Boulanger, P

    1993-01-01

    Two substitution mutants of the human immunodeficiency virus type 1 gag gene product were isolated after nitrous acid mutagenesis of a recombinant baculovirus expressing a non-N-myristylated, p6-deleted Gag precursor (Pr49). Both mutants failed to assemble intracellular Gag virus-like particles, as does the parental recombinant, and therefore expressed a self-assembly defective (Sad) phenotype in insect cells. The mutations consisted of nonconservative changes involving highly conserved hydrophobic residues in the p24 domain, Leu to Pro at position 268 (L268P) and Leu to Ser at amino acid 322 (L322S). Experimental data suggested that the two mutated residues belonged to functionally different regions of the Gag precursor. (i) A partial complementation effect between the two mutants for Gag precursor assembly was observed in coinfection experiments. (ii) The two mutations showed different phenotypes when placed in the N-myristylated context, of which only the L268P mutation abolished extracellular budding and release of Gag particles at the plasma membrane. Both L268P and L322S mutants had a trans-dominant negative effect on the intracellular assembly of a non-N-myristylated, full-length (Pr55) Gag precursor expressed by a coinfecting recombinant. None of the mutants, however, showed any detectable effect in trans on membrane targeting and budding of the coexpressed N-myristylated wild-type Gag precursor. Images PMID:8474175

  7. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system.

    PubMed

    Teng, Chao-Yi; van Oers, Monique M; Wu, Tzong-Yuan

    2013-10-01

    The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or β-synuclein (β-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, β-syn + CALR, or β-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three "helper" genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three "helper" genes, up to 10 dpi was observed. Utilization of this "triple-supporters" containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products. PMID:23900798

  8. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen. PMID:17998551

  9. Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

    PubMed

    Martínez-Torrecuadrada, J L; Díaz-Laviada, M; Roy, P; Sánchez, C; Vela, C; Sánchez-Vizcaíno, J M; Casal, J I

    1996-06-01

    African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors. PMID:8683209

  10. Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

    PubMed

    George, Steve; Jauhar, Altamash M; Mackenzie, Jennifer; Kieβlich, Sascha; Aucoin, Marc G

    2015-09-01

    The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system. PMID:25850946

  11. Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator IE1.

    PubMed

    Rodems, S M; Friesen, P D

    1995-09-01

    The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nuclear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 activity, the effects of site-directed mutations on transcriptional enhancement by the 28-mer and binding of the AcMNPV transregulator IE1 were investigated. In transfection assays and infections with AcMNPV recombinants, activation of a basal viral promoter required sequences within both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays indicated that hr5 probes containing a single 28-mer were bound by in vitro-synthesized IE1. Competition assays using DNA fragments that contained mutated 28-mers demonstrated that both half sites were required for optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMNPV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding and antibody supershift assays, it was demonstrated that IE1 binds to the 28-mer as a dimer. Collectively, these findings support a model in which symmetrical IE1 binding and simultaneous interaction with each half site are required for IE1-mediated transcriptional enhancement by hr5. Thus, sequence-specific binding may be one of the mechanisms by which IE1 directly or indirectly transregulates baculovirus gene expression. PMID:7636981

  12. An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

    PubMed Central

    2014-01-01

    Background Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan. Results An immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody. Conclusion A relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP. PMID:25123112

  13. Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94.

    PubMed Central

    Clem, R J; Robson, M; Miller, L K

    1994-01-01

    The infectivity of Autographa californica nuclear polyhedrosis virus mutants lacking the apoptosis-inhibiting gene p35 is decreased 1,000-fold or more in larvae of the insect Spodoptera frugiperda if the budded form of the virus is administered by hemocoelic injection; this decrease is correlated with the antiviral effects of apoptosis (R. J. Clem and L. K. Miller, J. Virol. 67:3730-3738, 1993). We have extended this correlation by showing that the infectivity of p35 mutant budded virus is restored to wild-type levels by expression of an unrelated baculovirus apoptosis-inhibiting gene, Cp-iap. We have also examined the oral infectivity of the occluded form of mutants lacking p35, the neighboring p94 gene, or both genes by feeding insects occluded virus. The oral infectivity of the p35 mutant was significantly reduced in S. frugiperda larvae, but this reduction (25-fold) was less than that observed for the hemocoelic route of infection (1,000-fold). The disruption of p94 alone had no apparent effect on infectivity by either route. Unexpectedly, however, the disruption of both p35 and p94 restored oral infectivity to nearly wild-type levels but did not exert this compensatory effect on infectivity by hemocoelic injection. Thus, the infectivity of the double p35/p94 mutant is affected in a route-specific manner in S. frugiperda larvae, suggesting a tissue-specific response to p35 and/or p94. Infectivity in a different host, Trichoplusia ni, was unaffected by all the mutants tested, consistent with previous studies indicating a lack of sensitivity to apoptosis in this species. However, T. ni and S. frugiperda larvae infected with p35 mutants failed to exhibit the symptom of morphological disintegration ("melting") typical of a wild-type infection, suggesting that p35 is required for the infection of some tissues in both species. PMID:8084009

  14. Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein.

    PubMed

    Sun, Jianhui; Huang, Liping; Wei, Yanwu; Wang, Yiping; Chen, Dongjie; Du, Wenjuan; Wu, Hongli; Feng, Li; Liu, Changming

    2015-11-01

    Porcine parvovirus type 1 (PPV1) is a major causative agent of embryonic and fetal death in swine. The PPV1 VP2 protein is closely associated with viral immunogenicity for eliciting neutralizing antibodies, but its antigenic structures have been largely unknown. We generated three monoclonal antibodies (MAbs) against baculovirus-expressed recombinant PPV1 VP2 protein. A PEPSCAN analysis identified the minimal B cell linear epitopes of PPV1 VP2 based on these MAbs. Three core epitopes, (228)QQITDA(233), (284)RSLGLPPK(291), and (344)FEYSNGGPFLTPI(356), were defined and mapped onto three-dimensional models of the PPV1 virion and VP2 monomer. The epitope (228)QQITDA(233) is exposed on the virion surface, and the other two are located inside the protein. An alignment of the PPV1 VP2 amino acid sequences showed that (284)RSLGLPPK(291) and (344)FEYSNGGPFLTPI(356) are absolutely conserved, whereas (228)QQITDA(233) has a single substitution at residue 233 in some (S → A or T). We developed a VP2 epitope-based indirect enzyme-linked immunosorbent assay (iELISA) to test for anti-PPV1 antibodies. In a comparative analysis with an immunoperoxidase monolayer assay using 135 guinea pig sera, the VP2-epitope-based iELISA had a concordance rate of 85.19 %, sensitivity of 83.33 %, and specificity of 85.47 %. MAb 8H6 was used to monitor VP2 during the PPV1 replication cycle in vitro with an indirect immunofluorescence assay, which indicated that newly encapsulated virions are released from the nucleus at 24 h postinfection and the PPV1 replication cycle takes less than 24 h. This study provides valuable information clarifying the antigenic structure of PPV1 VP2 and lays the foundations for PPV1 serodiagnosis and antigen detection. PMID:26153140

  15. Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

    PubMed

    Mahiou, J; Abastado, J P; Cabanie, L; Godeau, F

    1998-03-01

    We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart. PMID:9480929

  16. Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

    PubMed

    Gotoh, Takeshi; Awa, Hirono; Kikuchi, Ken-Ichi; Nirasawa, Satoru; Takahashi, Saori

    2010-01-01

    In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells. PMID:20139610

  17. Interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analogue.

    PubMed

    Lacourciere, G M; Vakharia, V N; Tan, C P; Morris, D I; Edwards, G H; Moos, M; Armstrong, R N

    1993-03-16

    A recombinant baculovirus (vEHX) encoding rat hepatic microsomal epoxide hydrolase has been constructed. Infection of Spodoptera frugiperda (Sf9) cells with the recombinant virus results in the expression of the enzyme at a level estimated to be between 5% and 10% of the cellular protein. The enzyme, which can be purified in 15% yield by a simple three-step procedure involving detergent extraction, DEAE-cellulose chromatography, and removal of the detergent on hydroxylapatite, has physical and kinetic properties very close to those of the enzyme obtained from rat liver microsomes. The interaction of the enzyme with two nitrogen-containing analogues of the substrate phenanthrene 9,10-oxide (1) was investigated in order to delineate the contributions of the oxirane group and the hydrophobic surface of the substrate to substrate recognition. The enzyme exhibits altered kinetic properties toward 1,10-phenanthroline 5,6-oxide (2) in which the biphenyl group of 1 is replaced with a bipyridyl group, suggesting that hydrophobic interaction between the complementary surfaces of the substrate and active site has an influence on catalysis. The conjugate acid of the aziridine analogue of 1, phenanthrene 9,10-imine (3), in which the oxirane oxygen is replaced with NH, has a pKa of 6.1, which allows the characterization of both the neutral and protonated aziridine (3H+) as substrate analogues for the enzyme. The pH dependence of the solvolysis reveals that 3H+ rearranges to a 65/35 mixture of 9-aminophenanthrene and 9-amino-10-hydroxy-9,10-dihydrophenanthrene 10(3)-fold faster than does 3. The neutral aziridine is a competitive inhibitor (Ki = 26 microM) of the enzyme at pH 8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8383521

  18. Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens.

    PubMed

    Choi, Jun-Gu; Kim, Min-Chul; Kang, Hyun-Mi; Kim, Kwang-Il; Lee, Kyu-Jun; Park, Choi-Kyu; Kwon, Jun-Hun; Kim, Jae-Hong; Lee, Youn-Jeong

    2013-03-23

    Since 2003, the highly pathogenic avian influenza (HPAI) H5N1 has become a serious problem in animals and an increasing threat to public health. To develop effective vaccines for H5 HPAI in chickens, virus-like particles (VLP) were produced using a baculovirus expression system. The particles comprised hemagglutinin (HA) alone (HA-VLP) or HA in combination with a matrix protein (M1; HAM-VLP) derived from a recent clade 2.3.2.1 H5N1 HPAI virus. To compare the immunogenicity and protective efficacy of these VLPs, 10 μg HAM-VLP, the equivalent amounts of HA incorporated HA-VLP or whole inactivated virus (WIV), were emulsified with mineral oil and used to immunize chickens. The serum hemagglutination inhibition antibody levels induced by HA-VLP and HAM-VLP were comparable to WIV. Antibodies to nucleoprotein were detected only in the WIV group. Immunized chickens in each group survived and were protected against a lethal homologous virus challenge, showing no clinical signs of infection. The challenge virus was detected intermittently in some oropharyngeal swabs, but not in cloacal swabs or various organs, which means that VLPs and WIV provide protection against systemic but not local virus replication in chickens. After the challenge, the HA-VLP group showed significantly increased serum antibody levels compared to the HAM-VLP and WIV groups, and some chickens in the HA-VLP group seroconverted with respect to nucleoprotein. Taken together, these results suggest that VLPs may be an effective method for controlling HPAI in chickens. They could be applied to a differentiating infected from vaccinated animals (DIVA) strategy. In addition, it is likely that HAM-VLP is more efficacious than HA-VLP in chickens. PMID:23265240

  19. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    PubMed Central

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

  20. Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein.

    PubMed

    Zhu, Xuejiao; Liu, Jie; Bai, Juan; Liu, Panrao; Zhang, Tingjie; Jiang, Ping; Wang, Xianwei

    2016-04-01

    Porcine circovirus type 2 (PCV2) causes significant economic losses to the swine industry worldwide. Heat shock proteins (Hsps) can be used as modulators to enhance both innate and adaptive immune responses. In the present study, recombinant baculoviruses expressing the PCV2Cap protein and the N-terminal 22-370 amino acids of porcine Gp96 (Gp96N), Hsp90, and Hsp70 (rBac-cap/Gp96N, rBac-cap/Hsp90 and rBac-cap/Hsp70, respectively) were constructed and the immune responses were examined in mice and piglets. The mouse experiments showed that rBac-cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies, proliferative responses of peripheral blood mononuclear cells (PBMCs) and IFN-γ levels compared to rBac-cap/Hsp90, rBac-cap/Hsp70, or rBac-cap. The pig experiments showed that the levels of anti-PCV2 antibody, proliferative responses of PBMCs, and IFN-γ in the rBac-cap/Gp96N groups were increased compared to those in rBac-cap group. There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-cap/Gp96N and rBac-cap, and the relative daily weight gains were higher than those in the challenge control (CC) group. The pathological lesions, extent of viremia, and viral loads of the vaccinated groups were milder than those in the CC group. Meanwhile, the extent of viremia and viral load present in the rBac-cap/Gp96N group were significantly lower than those in the rBac-cap group. These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2Cap. Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine. PMID:26826323

  1. Intranasal Immunization of Baculovirus Displayed Hemagglutinin Confers Complete Protection against Mouse Adapted Highly Pathogenic H7N7 Reassortant Influenza Virus

    PubMed Central

    Rajesh Kumar, Subaschandrabose; Syed Khader, Syed Musthaq; Kiener, Tanja K.; Szyporta, Milene; Kwang, Jimmy

    2013-01-01

    Background Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus. Methodology/Principal Findings In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA). The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n.) or subcutaneously (s.c.) with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs. Conclusion Our results indicated that protection from high pathogenic H7N7 (NL/219/03) virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections. PMID:23762234

  2. Protective efficacy of a human endogenous retrovirus envelope-coated, nonreplicable, baculovirus-based hemagglutin vaccine against pandemic influenza H1N1 2009.

    PubMed

    Choi, Jae-Yoo; Gwon, Yong-Dae; Kim, Jeong-Ki; Cho, Yeon-Dong; Heo, Yoon-Ki; Cho, Han-Sam; Choi, Tae-Jin; Poo, Ha-Ryoung; Oh, Yu-Kyoung; Kim, Young Bong

    2013-01-01

    Despite the advantages of DNA vaccines, overcoming their lower efficacy relative to that of conventional vaccines remains a challenge. Here, we constructed a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus-based HA vaccine against swine influenza A/California/04/2009(H1N1) hemagglutin (HA) (AcHERV-sH1N1-HA) as an alternative to conventional vaccines and evaluated its efficacy in two strains of mice, BALB/c and C57BL/6. A commercially available, killed virus vaccine was used as a positive control. Mice were intramuscularly administered AcHERV-sH1N1-HA or the commercial vaccine and subsequently given two booster injections. Compared with the commercial vaccine, AcHERV-sH1N1-HA induced significantly higher levels of cellular immune responses in both BALB/c and C57BL/6 mice. Unlike cellular immune responses, humoral immune responses depended on the strain of mice. Following immunization with AcHERV-sH1N1-HA, C57BL/6 mice showed HA-specific IgG titers 10- to 100-fold lower than those of BALB/c mice. In line with the different levels of humoral immune responses, the survival of immunized mice after intranasal challenge with sH1N1 virus (A/California/04/2009) depended on the strain. After challenge with 10-times the median lethal dose (MLD50) of sH1N1 virus, 100% of BALB/c mice immunized with the commercial vaccine or AcHERV-sH1N1-HA survived. In contrast, C57BL/6 mice immunized with AcHERV-sH1N1-HA or the commercial vaccine showed 60% and 70% survival respectively, after challenge with sH1N1 virus. In all mice, virus titers and results of histological analyses of lung tissues were consistent with the survival data. Our results indicate the importance of humoral immune response as a major defense system against influenza viral infection. Moreover, the complete survival of BALB/c mice immunized with AcHERV-sH1N1-HA after challenge with sH1N1 virus suggests the potential of baculoviral vector-based vaccines to achieve an efficacy comparable to

  3. Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

    PubMed

    Kost, T A; Ignar, D M; Clay, W C; Andrews, J; Leray, J D; Overton, L; Hoffman, C R; Kilpatrick, K E; Ellis, B; Emerson, D L

    1997-04-29

    Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA. PMID:9185859

  4. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV).

    PubMed

    de Freitas, Daniele Scandiucci; Coelho, Marly C Felipe; Souza, Manoel T; Marques, Abi; Ribeiro, E Bergmann Morais

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presence of the p35 gene by PCR and/or dot blot hybridization. Transcriptional analysis of regenerated plants showed the presence of specific p35 transcripts in 9 of them. Regenerated plants containing the p35 gene were inoculated with the cowpea aphid-borne mosaic virus (CABMV), the bacterium Xanthomonas axonopodis pv passiflorae, and the herbicide, glufosinate, (Syngenta). None of the plants showed resistance to CABMV. Regenerated plants (p35+) showed less than half of local lesions showed by non-transgenic plants when inoculated with X. axonopodis and some p35+ plants showed increased tolerance to the glufosinate herbicide when compared to non-transgenic plants. PMID:17016672

  5. Silencing of the Baculovirus Op-iap3 Gene by RNA Interference Reveals that It Is Required for Prevention of Apoptosis during Orgyia pseudotsugata M Nucleopolyhedrovirus Infection of Ld652Y Cells†

    PubMed Central

    Means, John C.; Muro, Israel; Clem, Rollie J.

    2003-01-01

    The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function. PMID:12663755

  6. Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

    PubMed

    Swamy, N; Ghosh, S; Schneider, G B; Ray, R

    2001-01-01

    Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal

  7. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    PubMed

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants. PMID:11513247

  8. Co-expression of four baculovirus proteins, IE1, LEF3, P143, and PP31, elicits a cellular chromatin-containing reticulate structure in the nuclei of uninfected cells

    SciTech Connect

    Nagamine, Toshihiro; Abe, Atsushi; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo

    2011-08-15

    Baculovirus DNA replication, transcription, and nucleocapsid assembly occur within a subnuclear structure called the virogenic stroma (VS) that consists of two subcompartments. Specific components of the VS sub-compartments have not been identified except for PP31, a DNA-binding protein that localizes specifically to the electron-dense region of VS. Here, we investigate the dynamic structure of VS using a GFP-tagged PP31 molecule (GFP-PP31). GFP-PP31 localizes to the VS throughout the course of infection. At later times post-infection, a PP31 reticulum distributed within VS was also apparent, indicating that VS sub-compartments compose a reticulate structure. Transient expression of PP31 with the viral proteins, IE1, LEF3, and P143, in uninfected cells resulted in the formation of a reticulate structure containing cellular chromatin and the spatial arrangements of the four proteins within the induced reticulum were the same as those within VS reticulum, suggesting that the two reticula are formed by a similar mechanism.

  9. Interaction of Autographa californica Multiple Nucleopolyhedrovirus Cathepsin Protease Progenitor (proV-CATH) with Insect Baculovirus Chitinase as a Mechanism for proV-CATH Cellular Retention▿†

    PubMed Central

    Hodgson, Jeffrey J.; Arif, Basil M.; Krell, Peter J.

    2011-01-01

    The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA–proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA–green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA–proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA–proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH. PMID:21289117

  10. DEVELOPMENT OF GENETICALLY ENHANCED BACULOVIRUS PESTICIDES

    EPA Science Inventory

    The assessment of potential environmental impacts of genetically improved viral pesticides will include an evaluation of the properties of the foreign gene product(s) as well as the biological properties of altered virus itself. It is anticipated that in the near future several t...

  11. Immunological-based assays for specific detection of shrimp viruses

    PubMed Central

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-01-01

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  12. Immunological-based assays for specific detection of shrimp viruses.

    PubMed

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-02-12

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  13. Development of genetically enhanced baculovirus pesticides (Chapter 5). Book chapter

    SciTech Connect

    Wood, H.A.

    1991-01-01

    The report describes the assessment of the potential environmental impacts of genetically improved viral pesticides, including an evaluation of the properties of the foreign gene product(s) and the biological properties of the altered virus itself. The current field release studies are collecting much of the information which will be needed to assess the environmental safety of these new pesticides. Of primary concern will be the cost-to-benefit ratios as determined by production costs, stability, application, technology, and field efficacy. Despite the improvements afforded through biotechnology, it is clear that viral and other microbial pesticides will only reduce, not eliminate, the agricultural requirements for synthetic pesticides. Even so, biological pesticides are among the best solutions to reducing crop losses in the absence of ecological disturbances and potential health hazards.

  14. Baculovirus induced transcripts in hemocytes from Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using RNA-sequencing digital difference expression profiling methods we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcri...

  15. Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

    PubMed Central

    Guarino, L A; Dong, W; Xu, B; Broussard, D R; Davis, R W; Jarvis, D L

    1992-01-01

    The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle. Images PMID:1433508

  16. Regulation of expression of a baculovirus ecdysteroid UDPglucosyltransferase gene.

    PubMed Central

    O'Reilly, D R; Miller, L K

    1990-01-01

    The Autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid UDPglucosyltransferase which catalyzes the transfer of glucose from UDPglucose to ecdysteroid insect molting hormones. Expression of this gene allows the virus to block molting and pupation of infected insect larvae. In this study, we present the nucleotide sequence of the A. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt was transcribed as two 5'-coterminal mRNAs early in infection. Transferase activity was detected in infected cells and in the extracellular fluid by 3 h after infection. The majority of the activity accumulated in the extracellular fluid. We show that the egt gene product is a 60-kilodalton protein which is secreted from the infected cell. The egt gene is located in a region of the A. californica nuclear polyhedrosis virus genome which exhibited hypervariability in serially passaged virus stocks. Nucleotide sequence analysis revealed that the most common deletion occurring in these serially passaged virus isolates is located in the egt gene. Images PMID:2106039

  17. Monitoring of Water Quality and Microalgae Species Composition of Penaeus monodon Ponds in Pulau Pinang, Malaysia

    PubMed Central

    Shaari, Asma Liyana; Surif, Misni; Latiff, Faazaz Abd.; Omar, Wan Maznah Wan; Ahmad, Mohd Noor

    2011-01-01

    Many reports have revealed that the abundance of microalgae in shrimp ponds vary with changes in environmental factors such as light, temperature, pH, salinity and nutrient level throughout a shrimp culture period. In this study, shrimp cultivation period was divided into three stages (initial = week 0–5, mid = week 6–10 and final = week 11–15). Physical and chemical parameters throughout the cultivation period were studied and species composition of microalgae was monitored. Physical parameters were found to fluctuate widely with light intensity ranging between 182.23–1278 μmol photon m−2s−1, temperature between 29.56°C −31.59°C, dissolved oxygen (DO) between 4.56–8.21 mg/l, pH between 7.65–8.49 and salinity between 20‰–30‰. Ammonium (NH4+-N), nitrite (NO2−-N), nitrate (NO3−-N), and orthophosphate (PO43−-P) concentrations in the pond at all cultivation stages ranged from 0.017 to 0.38 mg/l, 0.24 to 2.12 mg/l, 0.06 to 0.98 mg/l and 0.16 to 1.93 mg/l respectively. Statistical test (ANOVA) showed that there were no significant difference (p<0.05) in nutrients concentrations among the cultivation stages. All nutrients concentrations however were still in the tolerable level and safe for shrimp culture. The chlorophyll a contents were found to range from 5.03±2.17 to 32.61±0.35 μg/l throughout the cultivation period. A total of 19 microalgae species were found in the shrimp pond, with diatoms contributing up to 72% of the species followed by Chlorophyta (11%) and Cyanophyta (11%). However, weekly species abundance varied through the study period. At the initial stage, when there were no shrimps in the pond, Anabaena spp. and Oscillatoria spp. (Cyanophyta) were the dominant species, followed by Chlorella sp. and Dunaliella sp. (Chlorophyta). When shrimps were introduced into the pond, Amphora sp., Navicula sp. Gyrosigma sp. and Nitzschia sp. (diatoms) started to exist. At the middle and towards the final stage of the shrimp culture period diatoms were the dominant species. The Chlorophyta (Chlorella sp.) domination took place only twice, which was at week 2 and 13. The absence of some of the coastal water microalgae species in the shrimp pond was most likely due to the fact that they could not tolerate the physicochemical factors of harsh environment. In this study, Cylindrotheca closterium was regarded as the most tolerant species among the microalgae due to its ability to exist for 6 weeks out of the 15 weeks of cultivation. PMID:24575209

  18. Studies on luminous, Vibrio harveyi associated with shrimp culture system rearing Penaeus monodon.

    PubMed

    Kannapiran, E; Ravindran, J; Chandrasekar, R; Kalaiarasi, A

    2009-09-01

    Microbiological studies in a modified extensive shrimp culture system at Nambuthalai, southeast coast of India were carried out fora period of 120 days. Population dynamics and distribution profile of luminous bacteria and total heterotrophic bacteria in the water sediment and animal samples were monitored. Luminous bacteria associated with exoskeleton, gills and gut were isolated and quantified. The total heterotrophic bacterial counts ranged from 1.3 x 10(4) to 25.3 x 10(4) CFU ml(-1) in water and 1.5 x 10(6) to 26.2 x 10(6) CFU g(-1) in sediment. The V. harveyi population density varied between 0.6 x 10(4) and 8.8 x 10(4) LCFU ml(-1) in water and from 1.2 x 10(6) to 10.4 x 10(6) LCFU g(-1) sediment respectively. The gut of the animal was found to harbor high density of V. harveyi than gills and exoskeleton. The total heterotrophic bacteria and V. harveyi population density showed increasing trend during the culture period. The high V harveyi density observed in this study at the end of the culture period correlated with the outbreak of white spot disease. PMID:20143707

  19. The narwhal (Monodon monoceros) cementum-dentin junction: a functionally graded biointerphase.

    PubMed

    Grandfield, Kathryn; Chattah, Netta Lev-Tov; Djomehri, Sabra; Eidelmann, Naomi; Eichmiller, Frederick C; Webb, Samuel; Schuck, P James; Nweeia, Martin; Ho, Sunita P

    2014-08-01

    In nature, an interface between dissimilar tissues is often bridged by a graded zone, and provides functional properties at a whole organ level. A perfect example is a "biological interphase" between stratified cementum and dentin of a narwhal tooth. This study highlights the graded structural, mechanical, and chemical natural characteristics of a biological interphase known as the cementum-dentin junction layer and their effect in resisting mechanical loads. From a structural perspective, light and electron microscopy techniques illustrated the layer as a wide 1000-2000 μm graded zone consisting of higher density continuous collagen fiber bundles from the surface of cementum to dentin, that parallels hygroscopic 50-100 μm wide collagenous region in human teeth. The role of collagen fibers was evident under compression testing during which the layer deformed more compared to cementum and dentin. This behavior is reflected through site-specific nanoindentation indicating a lower elastic modulus of 2.2 ± 0.5 GPa for collagen fiber bundle compared to 3 ± 0.4 GPa for mineralized regions in the layer. Similarly, microindentation technique illustrated lower hardness values of 0.36 ± 0.05 GPa, 0.33 ± 0.03 GPa, and 0.3 ± 0.07 GPa for cementum, dentin, and cementum-dentin layer, respectively. Biochemical analyses including Raman spectroscopy and synchrotron-source microprobe X-ray fluorescence demonstrated a graded composition across the interface, including a decrease in mineral-to-matrix and phosphate-to-carbonate ratios, as well as the presence of tidemark-like bands with Zn. Understanding the structure-function relationships of wider tissue interfaces can provide insights into natural tissue and organ function. PMID:25205746

  20. Genetic Correlations Between Carcass Traits And Molecular Breeding Values In Angus Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research elucidated genetic relationships between carcass traits, ultrasound indicator traits, and their respective molecular breeding values (MBV). Animals whose MBV data were used to estimate (co)variance components were not previously used in development of the MBV. Results are presented fo...

  1. The Development of a High Density Linkage Map for Black Tiger Shrimp (Penaeus monodon) Based on cSNPs

    PubMed Central

    Baranski, Matthew; Gopikrishna, Gopalapillay; Robinson, Nicholas A.; Katneni, Vinaya Kumar; Shekhar, Mudagandur S.; Shanmugakarthik, Jayakani; Jothivel, Sarangapani; Gopal, Chavali; Ravichandran, Pitchaiyappan; Kent, Matthew; Arnyasi, Mariann; Ponniah, Alphis G.

    2014-01-01

    Transcriptome sequencing using Illumina RNA-seq was performed on populations of black tiger shrimp from India. Samples were collected from (i) four landing centres around the east coastline (EC) of India, (ii) survivors of a severe WSSV infection during pond culture (SUR) and (iii) the Andaman Islands (AI) in the Bay of Bengal. Equal quantities of purified total RNA from homogenates of hepatopancreas, muscle, nervous tissue, intestinal tract, heart, gonad, gills, pleopod and lymphoid organs were combined to create AI, EC and SUR pools for RNA sequencing. De novo transcriptome assembly resulted in 136,223 contigs (minimum size 100 base pairs, bp) with a total length 61 Mb, an average length of 446 bp and an average coverage of 163× across all pools. Approximately 16% of contigs were annotated with BLAST hit information and gene ontology annotations. A total of 473,620 putative SNPs/indels were identified. An Illumina iSelect genotyping array containing 6,000 SNPs was developed and used to genotype 1024 offspring belonging to seven full-sibling families. A total of 3959 SNPs were mapped to 44 linkage groups. The linkage groups consisted of between 16–129 and 13–130 markers, of length between 139–10.8 and 109.1–10.5 cM and with intervals averaging between 1.2 and 0.9 cM for the female and male maps respectively. The female map was 28% longer than the male map (4060 and 2917 cM respectively) with a 1.6 higher recombination rate observed for female compared to male meioses. This approach has substantially increased expressed sequence and DNA marker resources for tiger shrimp and is a useful resource for QTL mapping and association studies for evolutionarily and commercially important traits. PMID:24465553

  2. A multi-biomarker approach to assess the impact of farming systems on black tiger shrimp (Penaeus monodon).

    PubMed

    Tu, Huynh Thi; Silvestre, Frederic; Wang, Neil; Thome, Jean-Pierre; Phuong, Nguyen Thanh; Kestemont, Patrick

    2010-11-01

    This study examined the advantages of the use of biomarkers as an early warning system by applying it to different shrimp farming systems in Soctrang and Camau provinces, main shrimp producers in Mekong River Delta, Vietnam. Shrimp were collected at 15 different farms divided into four different farming systems: three farms were converted from originally rice paddies into intensive shrimp farming systems (IS1, IS2, IS3); three farms were rice-shrimp integrated farming systems (RS4, RS5, RS6); three farms were intensive farming systems (IS7, IS8, IS9); six farms were extensive shrimp farming systems (From ES1 to ES6). Lipid peroxidation (LPO) and total glutathione (GSH) were measured as well as catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and acetylcholinesterase activities (ACHE). Organ specificity was observed between gills and hepatopancreas with generally higher activity of GST in gills (GSTG) whereas the contrary was observed for LPO level in gills (LPOG). Hierarchical clustering and principal component analysis clearly indicated that shrimp reared in extensive culture system formed a distinct group from those reared in intensive or rice-shrimp integrated systems. CAT in gills (CATG), GPX in gills (GPXG) and hepatopancreas (GPXHP) and ACHE in muscle (ACHEM) of shrimp collected in extensive farms showed a general higher level than those in intensively farmed shrimp. On the contrary, we observed clear high levels of GSTG and GST in hepatopancreas (GSTHP) and LPOG and hepatopancreas (LPOHP) of shrimp sampled in intensive and rice-shrimp integrated systems. Thus, we propose that LPO and CAT, GPX, GST and ACHE can be used as a set of biomarkers for the assessment of health condition and can discriminate between shrimp cultivated in different farming systems. These findings provide the usefulness of integrating a set of biomarkers to define the health status of shrimp in different shrimp culture systems. PMID:20943254

  3. The Genome of the Nucleopolyhedrosis-Causing Virus from Tipula oleracea Sheds New Light on the Nudiviridae Family

    PubMed Central

    Thézé, Julien; Gavory, Frederick; Gaillard, Julien; Poulain, Julie; Drezen, Jean-Michel; Herniou, Elisabeth A.

    2014-01-01

    ABSTRACT A large double-stranded DNA (dsDNA) virus that produces occlusion bodies, typical of baculoviruses, has been described to infect crane fly larvae of the genus Tipula (Diptera, Tipulidae). Because of a lack of genomic data, this virus has remained unclassified. Electron microscopy of an archival virus isolated from Tipula oleracea, T. oleracea nudivirus (ToNV), showed irregularly shaped occlusion bodies measuring from 2 to 5 μm in length and 2 μm in middiameter, filled with rod-shape virions containing single nucleocapsids within a bilayer envelope. Whole-genome amplification and Roche 454 sequencing revealed a complete circular genome sequence of 145.7 kb, containing five direct repeat regions. We predicted 131 open reading frames, including a homolog of the polyhedrin gene encoding the major occlusion body protein of T. paludosa nucleopolyhedrovirus (NPV). BLAST searches demonstrated that ToNV had 21 of the 37 baculovirus core genes but shared 52 genes with nudiviruses (NVs). Phylogenomic analyses indicated that ToNV clearly belongs to the Nudiviridae family but should probably be assigned to a new genus. Among nudiviruses, ToNV was most closely related to the Penaeus monodon NV and Heliothis zea NV clade but distantly related to Drosophila innubia NV, the other nudivirus infecting a Diptera. Lastly, ToNV was found to be most closely related to the nuvidirus ancestor of bracoviruses. This was also reflected in terms of gene content, as ToNV was the only known exogenous virus harboring homologs of the Cc50C22.6 and 27b (Cc50C22.7) genes found in the nudiviral genomic cluster involved in bracovirus particle production. IMPORTANCE The Nudiviridae is a family of arthropod dsDNA viruses from which striking cases of endogenization have been reported (i.e., symbiotic bracoviruses deriving from a nudivirus and the endogenous nudivirus of the brown planthopper). Although related to baculoviruses, relatively little is known about the genomic diversity of

  4. Inactivation of nuclear polyhedrosis virus (Baculovirus subgroup A) by monochromatic UV radiation

    SciTech Connect

    Griego, V.M.; Martignoni, M.E.; Claycomb, A.E.

    1985-03-01

    Monochromatic radiation at wavelengths of 290, 300, 310, and 320 nm inactivated occluded nuclear polyhedrosis virus of the Douglas-fir tussock moth, Orgyia pseudotsugata. Data indicate that all of the wavelengths are capable of causing virus inactivation; much greater fluences are needed for virus inactivation as the wavelength increases.

  5. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  6. EXPRESSION EFFICIENCY OF A SCORPION NEUROTOXIN, AAHIT, USING BACULOVIRUS IN INSECT CELLS. (R825433)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  7. Interactions between an injected polydnavirus and per os baculovirus in gypsy moth larvae.

    PubMed

    D'Amico, V; Podgwaite, J D; Zerillo, R; Taylor, P; Fuester, R

    2013-10-01

    Larval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. dispar nucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus (CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or in the form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificial diet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitism nor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concurrent challenges with LdMNPV. PMID:23933012

  8. p13 from group II baculoviruses is a killing-associated gene

    PubMed Central

    Lu, Nan; Du, Enqi; Liu, Yangkun; Qiao, Hong; Yao, Lunguang; Pan, Zishu; Lu, Songya; Qi, Yipeng

    2012-01-01

    p13 gene was first described in Leucania separata multinuclear polyhedrosis virus (Ls-p13) several years ago, but the function of P13 protein has not been experimentally investigated to date. In this article, we indicated that the expression of p13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (Ha-p13) was regulated by both early and late promoter. Luciferase assay demonstrated that the activity of Ha-p13 promoter with hr4 enhancer was more than 100 times in heterologous Sf9 cells than that in nature host Hz-AM1 cells. Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocal microscopic analysis showed that both mainly located in the cytoplasm membrane at 48 h. Results of RNA interference indicated that Ha-p13 was a killing-associated gene for host insects H. armigera. The AcMNPV acquired the mentioned killing activity and markedly accelerate the killing rate when expressing Ls-p13. In conclusion, p13 is a killing associated gene in both homologous and heterologous nucleopolyhedrovirus. [BMB Reports 2012; 45(12): 730-735] PMID:23261060

  9. Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field

    PubMed Central

    Slavicek, James; Podgwaite, John D.; Webb, Ralph; Fuester, Roger; Peiffer, Randall A.

    2013-01-01

    Nucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by the v-chiA genes. We used recombinant and naturally occurring strains of the Lymantria dispar NPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functional v-chiA genes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and the v-chiA gene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings. PMID:23624474

  10. EVALUATION OF A RECOMBINANT DIAMONDBACK MOTH BACULOVIRUS IN SELECTED LEPIDOPTERAN CELL LINES AND LARVAE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diamondback moth (DBM), Plutella xylostella (L.) is one of the most important pests of the cabbage family, as well as other vegetable crops throughout the world. Its control by chemical insecticides as well as by the biopesticide Bacillus thuringiensis has become more difficult due to the devel...

  11. Gut transcription in Helicoverpa zea is dynamically altered in response to baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Helicoverpa zea transcriptome was analyzed 24 hours after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (P<0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detect...

  12. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    PubMed

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  13. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35

    PubMed Central

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its ‘reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  14. APPLICATION OF A NOVEL RADIOIMMUNOASSAY TO IDENTIFY BACULOVIRUS STRUCTURAL PROTEINS THAT SHARE INTERSPECIES ANTIGENIC DETERMINANTS

    EPA Science Inventory

    Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125 I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five bacul...

  15. Production of human beta interferon in insect cells infected with a Baculovirus expression vector

    SciTech Connect

    Smith, G.E.; Summers, M.D.; Fraser, M.J.

    1983-12-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 x 10/sup 6/ U of interferon activity was produced by 10/sup 6/ infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

  16. Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication.

    PubMed

    Asser-Kaiser, Sabine; Radtke, Pit; El-Salamouny, Said; Winstanley, Doreen; Jehle, Johannes A

    2011-02-20

    An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGV(hsp-eGFP)) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae. PMID:21190707

  17. Deletion of v-chiA from a baculovirus reduces horizontal transmission in the field.

    PubMed

    D'Amico, Vincent; Slavicek, James; Podgwaite, John D; Webb, Ralph; Fuester, Roger; Peiffer, Randall A

    2013-07-01

    Nucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by the v-chiA genes. We used recombinant and naturally occurring strains of the Lymantria dispar NPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functional v-chiA genes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and the v-chiA gene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings. PMID:23624474

  18. Isolation and Characterization of a Baculovirus Associated with the Insect Parasite Cotesia marginiventris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiple nucleopolyhedrovirus was found associated with the parasitoid Cotesia marginiventris and was isolated from stung Trichoplusia ni larvae which were used to rear the parasite. The wild type virus was plaque purified by infecting a Heliothis subflexa (BCIRL-HsAM1) cell line and isolating se...

  19. Gut Transcription in Helicoverpa zea is Dynamically Altered in Response to Baculovirus Infection

    PubMed Central

    Noland, Jeffrey E.; Breitenbach, Jonathan E.; Popham, Holly J. R.; Hum-Musser, Sue M.; Vogel, Heiko; Musser, Richard O.

    2013-01-01

    The Helicoverpa zea transcriptome was analyzed 24 h after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (p < 0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detected in the gut epithelial tissue; where 63% of these genes were down-regulated and 37% of genes were up-regulated compared to the mock-infected control. Genes that play important roles in digestive physiology were noted as being generally down-regulated. Among these were aminopeptidases, trypsin-like serine proteases, lipases, esterases and serine proteases. Genes related to the immune response reacted in a complex nature having peptidoglycan binding and viral antigen recognition proteins and antiviral pathway systems down-regulated, whereas antimicrobial peptides and prophenoloxidase were up-regulated. In general, detoxification genes, specifically cytochrome P450 and glutathione S-transferase were down-regulated as a result of infection. This report offers the first comparative transcriptomic study of H. zea compared to HzSNPV infected H. zea and provides further groundwork that will lead to a larger understanding of transcriptional perturbations associated with viral infection and the host response to the viral insult in what is likely the most heavily infected tissue in the insect. PMID:26462433

  20. Characterization of a new megabirnavirus that confers hypovirulence with the aid of a co-infecting partitivirus to the host fungus, Rosellinia necatrix.

    PubMed

    Sasaki, Atsuko; Nakamura, Hitoshi; Suzuki, Nobuhiro; Kanematsu, Satoko

    2016-07-01

    A new virus termed Rosellinia necatrix megabirnavirus 2 (RnMBV2) was molecularly and biologically characterized. RnMBV2 was originally harbored in isolate W8 of R. necatrix co-infected with the previously reported virus Rosellinia necatrix partitivirus 1 (RnPV1). RnMBV2 has molecular features similar and different from precedent megabirnaviruses, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1). The two genomic segments of RnMBV2 (9.0-kbp dsRNA1 and 8.0-kbp dsRNA2) each possess two open reading frames (ORF1 and 2 on dsRNA1 and ORF3 and 4 on dsRNA2), with a well conserved 5'-long untranslated region (UTR) of 1.7-1.8kb between the segments, and relatively short 3'-UTR. The RnMBV2 dsRNA1-coded capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) show higher sequence identity to those of SsMBV1 than to those of RnMBV1, whereas the RnMBV2 ORF3-coded protein is more closely related to the counterpart of RnMBV1. No significant amino acid sequence similarity was detected among ORF4-coded sequences of the three megabirnaviruses. Virion transfection and co-culturing allowed for single and double infection of mycelial incompatible isolates W37 and W97 by RnMBV2 and/or RnPV1. Their comparative analyses showed RnMBV2 to be able to confer hypovirulence with the aid of a co-infecting RnPV1, while the individual viruses exhibited asymptomatic infections. Interestingly, RnPV1 accumulation appeared to be increased in co-infected fungal strain with two segments of RnMBV2 relative to singly infected fungal strains. Furthermore, the dispensability of RnMBV2 dsRNA2 was demonstrated to be similar to that of the other two megabirnaviruses. PMID:26718720

  1. Identification and ultrastructural characterization of a novel virus from fish.

    PubMed

    Granzow, H; Weiland, F; Fichtner, D; Schütze, H; Karger, A; Mundt, E; Dresenkamp, B; Martin, P; Mettenleiter, T C

    2001-12-01

    During routine investigations on fish, a virus (isolate DF 24/00) with novel morphological features and hitherto undescribed morphogenesis was isolated from a white bream (Blicca bjoerkna L.; Teleostei, order Cypriniformes). Cell-free virions consist of a rod-shaped nucleocapsid (120-150x19-22 nm) similar to that seen in baculoviruses. The virion has a bacilliform shape (170-200x75-88 nm) reminiscent of rhabdoviruses with an envelope containing coronavirus-like spikes (20-25 nm). DF 24/00 replicated well in various fish cell lines. Inhibitor studies with 5-iodo-2'-deoxyuridine indicated that the viral genome consists of RNA and chloroform sensitivity correlated with ultrastructural demonstration of enveloped virions. The buoyant density of the virus determined in sucrose was 1.17-1.19 g/ml. Preliminary biochemical characterization revealed the presence of six antigenic glycoproteins, three of which contain sugars with concanavalin-A specificity. Ultrastructurally, morphogenesis of virus progeny was detected only in the cytoplasm. Nucleocapsids were observed to bud through membranes of the endoplasmic reticulum and/or Golgi apparatus into dilated vesicles. Egress of mature virions occurs primarily by exocytosis and, only very rarely, by budding directly at the plasma membrane. Morphologically similar viruses had previously been isolated from grass carp (Ctenopharyngodon idella), blue crab (Callinectis sapidus), European shore crab (Carcinus maenas) and shrimp (Penaeus monodon). To date, none of them has been classified. In summary, the first characterization of a new virus that might represent a member of a novel virus family that has morphological features resembling those found in rhabdo-, corona- and baculoviruses is presented. PMID:11714959

  2. The accuracies of DNA-based estimates of genetic merit derived from Angus or multibreed beef cattle training populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several organizations have developed prediction models for molecular breeding values (MBV) for quantitative growth and carcass traits in beef cattle using BovineSNP50 genotypes and phenotypic or EBV data. MBV for Angus cattle have been developed by IGENITY, Pfizer Animal Genetics, and a collaboratio...

  3. Genetic evaluation of Angus cattle for carcass marbling using ultrasound and genomic indicators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to estimate genetic parameters needed to elucidate the relationships of a molecular breeding value for marbling (MBV), intramuscular fat of yearling bulls measured with ultrasound (IMF) and marbling score of harvested steers (MRB), and to assess the utility of MBV and IMF in predicti...

  4. Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus

    PubMed Central

    Wang, Minghong; Wang, Yong; Sun, Xiangzhong; Cheng, Jiasen; Fu, Yanping; Liu, Huiquan; Jiang, Daohong; Ghabrial, Said A.

    2015-01-01

    ABSTRACT Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5′-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. IMPORTANCE Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized

  5. Effects of buffer additives and thermal processing methods on the solubility of shrimp (Penaeus monodon) proteins and the immunoreactivity of its major allergen.

    PubMed

    Lasekan, Adeseye O; Nayak, Balunkeswar

    2016-06-01

    This study examines the potential of two buffer additives (Tween 20 and DTT) to improve the solubility of proteins from shrimp subjected to different heat treatments and the allergenicity of tropomyosin in the extracts. The concentration of soluble proteins extracted by all the buffers from processed shrimp was significantly reduced compared with untreated samples. The concentration of total soluble proteins from heat treated shrimp increased significantly when phosphate buffer containing both surfactant and reducing agent was used as the extraction buffer. However, the concentrations of heat-stable proteins in the buffers were mostly similar. The electrophoretic profile of extracted proteins showed that tropomyosin is very stable under the different heat treatment methods used in this study except for high pressure steaming where the intensity of tropomyosin band was reduced. Competitive inhibition ELISA showed that high pressure steaming reduced the allergenicity of tropomyosin compared with other heat treatments methods. PMID:26830572

  6. Trace metals in the giant tiger prawn Penaeus monodon and mangrove sediments of the Tanzania coast: Is there a risk to marine fauna and public health?

    PubMed

    Rumisha, Cyrus; Mdegela, Robinson H; Kochzius, Marc; Leermakers, Martine; Elskens, Marc

    2016-10-01

    Mangroves ecosystems support livelihood and economic activities of coastal communities in the tropics and subtropics. Previous reports have documented the inefficiency of waste treatment facilities in Tanzania to contain trace metals. Therefore, the rapidly expanding coastal population and industrial sector is likely to threaten mangrove ecosystems with metal pollution. This study analysed trace metals in 60 sediment samples and 160 giant tiger prawns from the Tanzanian coast in order to document the distribution of trace metals and to establish if measured levels present a threat to mangrove fauna and are of public health importance. High levels of Cr, Co, Cu, Fe, Mn, Ni, and V was observed in mangroves of river Pangani, Wami, and Rufiji. Multivariate analysis showed that they originate mainly from weathering and erosion in the river catchments. Extreme enrichment of Cd was observed in a mangrove affected by municipal sewage. The distribution of Hg, Pb, and Zn was related with urbanisation and industrial activities along the coast. The metal pollution index was high at Pangani, Saadani, and Rufiji, suggesting that these estuarine mangroves are also affected by human activities in the catchment. Moderate to considerable ecological risks were observed in all sampled mangroves, except for Kilwa Masoko. It was revealed that As, Cd, and Hg present moderate risks to fauna. High levels of Cu, Fe and Zn were observed in prawns but the level of the non-essential Cd, Hg, and Pb did not exceed the maximum allowed levels for human consumption. However, based on the trends of fish consumption in the country, weekly intake of Hg is likely to exceed provisional tolerable weekly intake level, especially in fishing communities. This calls for measures to control Hg emissions and to strengthen sewage and waste treatment in coastal cities and urban centres in the basin of major rivers. PMID:27281719

  7. Angiotensin AT1 and AT2 Receptors Regulate Basal Skeletal Muscle Microvascular Volume and Glucose Utilization

    PubMed Central

    Chai, Weidong; Wang, Wenhui; Liu, Jia; Barrett, Eugene J.; Carey, Robert M.; Cao, Wenhong; Liu, Zhenqi

    2010-01-01

    Angiotensin II causes vasoconstriction via the type 1 receptor (AT1R) and vasodilatation through the type 2 receptor (AT2R). Both are expressed in muscle microvasculature where substrate exchanges occur. Whether they modulate basal muscle microvascular perfusion and substrate metabolism is not known. We measured microvascular blood volume (MBV), a measure of microvascular surface area and perfusion, in rats during systemic infusion of angiotensin II at either 1 or 100 ng/kg/min. Each caused a significant increase in muscle MBV. Likewise, administration of AT1R blocker losartan increased muscle MBV by >3-fold (p<0.001). Hindleg glucose extraction and muscle interstitial oxygen saturation simultaneously increased by 2–3-fold. By contrast, infusing AT2R antagonist PD123319 significantly decreased muscle MBV by up to 80% (p<0.001). This was associated with a significant decrease in hindleg glucose extraction and muscle oxygen saturation. AT2R antagonism and inhibition of nitric oxide synthase each blocked the losartan-induced increase in muscle MBV and glucose uptake. In conclusion, angiotensin II acts on both AT1R and AT2R to regulate basal muscle microvascular perfusion. Basal AT1R tone restricts muscle MBV and glucose extraction while basal AT2R activity increases muscle MBV and glucose uptake. Pharmacologic manipulation of the balance of AT1R and AT2R activity affords the potential to improve glucose metabolism. PMID:19996061

  8. Effect of the peak cell density of recombinant AcMNPV-infected Hi5 cells on baculovirus yields.

    PubMed

    Huynh, Hoai T; Tran, Trinh T B; Chan, Leslie C L; Nielsen, Lars K; Reid, Steven

    2015-02-01

    The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and β-galactosidase (β-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the β-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.5 and 6 × 10(6) cells/mL and a multiplicity of infection of 10 plaque-forming units (PFU)/cell either in the original or fresh medium conditions. There were negative correlations between the three key virus infection indicators (vDNA, mRNA and β-Gal) and the peak cell density (PCD). However, unlike infected Sf9 cells, the yield decline started at the lowest PCD investigated (0.6 × 10(6) cells/mL). Generally, the yield decline with increasing PCD was most pronounced for β-Gal followed by mRNA and was more moderate for vDNA. The decline was significantly reduced but not totally arrested when fresh medium replacement was used. The results suggest that the reduction in recombinant protein-specific yields at high PCDs is associated with limitations during the up-stream processes of replication and transcription rather than entirely caused by limitations during translation. In addition, low production rates at late infection stages of moderate to high ICDs are a probable cause of the cell density effect. PMID:25472440

  9. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion

    PubMed Central

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  10. Pivotal Role of the Non-hr Origin of DNA Replication in the Genesis of Defective Interfering Baculoviruses

    PubMed Central

    Pijlman, Gorben P.; Dortmans, Jos C. F. M.; Vermeesch, Angela M. G.; Yang, Kai; Martens, Dirk E.; Goldbach, Rob W.; Vlak, Just M.

    2002-01-01

    The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogenous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells. PMID:11991989

  11. Defensive reaper - Induction of mx and Apoptosis in mosquito midgut cells as an innate immune response to baculovirus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many vertebrate and insect viruses posses anti-apoptotic genes that are required for their infectivity. This has led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect anti-viral defense ...

  12. A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper.

    PubMed Central

    Thiem, S M; Miller, L K

    1989-01-01

    An Autographa californica nuclear polyhedrosis virus gene encoding a 30-kilodalton polypeptide with two different sequence motifs characteristic of DNA-binding proteins was identified immediately downstream of the major capsid protein gene (vp39). The gene, CG30, was characterized by sequencing, transcriptional mapping, in vitro translation of hybrid-selected RNA, and comparison of the derived polypeptide sequence with published data bases. The initial ATG of the 792-base-pair CG30 open reading frame is two nucleotides downstream of the vp39 terminal TAA codon. Early transcripts of CG30 initiate within the vp39 coding sequence. At late times, bicistronic transcripts initiate from the vp39 promoter, continue through CG30, and terminate at the same site as the early transcripts. In vitro translation of hybrid-selected early CG30 RNA yields a polypeptide of 30 kilodaltons. The predicted CG30 polypeptide sequence has characteristics of a eucaryotic transcriptional activator and is novel in having two potential DNA-binding domains. A stretch of acidic residues bridges a zinc finger at the amino terminus and a leucine zipper with a flanking basic region at the carboxyl terminus. Images PMID:2507791

  13. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    PubMed

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  14. Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) as a possible threat to native, endangered species of cactus in the southeastern United States as we...

  15. Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells.

    PubMed

    Bel Haj Rhouma, Rahima; Cérutti-Duonor, Martine; Benkhadir, Khadija; Goudey-Perrière, Françoise; El Ayeb, Mohamed; Lopez-Ferber, Miguel; Karoui, Habib

    2005-12-01

    BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV. PMID:16216259

  16. Acetylcholinesterase of the Sand Fly Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression and Biochemical Properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduc...

  17. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

    SciTech Connect

    Wang Shengpeng; Guo Tingqing; Guo Xiuyang; Huang Junting; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-24

    In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

  18. Rheological profile of diets produced using agro-industrial wastes for rearing codling moth larvae for baculovirus biopesticides.

    PubMed

    Gnepe, J R; Tyagi, R D; Brar, S K; Valero, J R

    2011-01-01

    A rheological study of diets using the agro-industrial wastes (brewery wastewater and pomace waste) was carried out in order to obtain a diet most adapted to supply nutrients for growth of codling moth (CM) larvae. Nutritive capacity (g/L) of brewery wastewater (BWW) (25.5 ± 5.5 carbohydrates; 16.9 ± 2.1 proteins; 6 ± 1.6 lipids) and pomace waste (POM) (22.0 ± 0.03 carbohydrates; 11.3 ± 1.3 proteins; 2 ± 0.2 lipids) were essential and important as replacement or in association with other ingredients [soya flour (SF), wheat germ (WG), yeast extract (YE)] of the standard diet for the breeding of codling moth larvae. These diet additives also contributed to the preservation of texture and nutritive content of larvae diet. The eggs and CM larvae were grown on alternate diets under industrial conditions (16:8 h photoperiod; 25 ± 1 °C and 50 ± 0.5 % of humidity). The higher assimilation of nutrients of the diets in BWW and control diet was observed by calculating the rate of hatching of eggs (0.48 to 0.71); larvae growth (0.23 to 0.4) and fertility (1.33 to 3 for control diet). The excellent growth and fertility rates of codling moth larvae were attributed to variations in viscosity (varying from 50 to 266 mPa.s⁻¹), particle size (varying 24.3 μm in 88.05 μm with regard to 110 μm the control diet) and total solids (145.88 g/L POM + YE; 162.08 g/L BWW + YE; 162.2 g/L POM + WG; 173 g/L control; 174.3 g/L BWW + WG) diets. Lower viscosity favored improved diet due to ease of assimilation of nutrients. Thus, rheology is an important parameter during preparation of diets for growth of codling moth larvae as it will dictate the nutrient assimilation which is an important parameter of larvae growth. PMID:21442538

  19. Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.

    PubMed

    Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

    2000-07-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

  20. Engineering a Recombinant Baculovirus with a Peptide Hormone Gene and its Effect on the Corn Earworm, Helicoverpa zea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The helicokinins are peptides identified from Helicoverpa zea that when injected into the larvae were found to cause excessive diuresis and loss of feeding activity. Of the three peptides, helicokinin II (HezK-II) was found to be most potent. A synthetic gene encoding HezK-II was constructed based o...

  1. On the local behaviour of the multidimensional {Lambda}-variation

    SciTech Connect

    Bakhvalov, Alexandr N

    2011-01-14

    Let two classes ({Lambda}{sup 1},...,{Lambda}{sup m})BV and (M{sup 1},...,M{sup m})BV on an interval {Delta} be given. In the paper, we find necessary and sufficient conditions for the {Lambda}-variation of any function in the M-class, over a neighbourhood of every regular point, to tend to zero as the neighbourhood decreases. Bibliography: 10 titles.

  2. Megabirnavirus structure reveals a putative 120-subunit capsid formed by asymmetrical dimers with distinctive large protrusions.

    PubMed

    Miyazaki, Naoyuki; Salaipeth, Lakha; Kanematsu, Satoko; Iwasaki, Kenji; Suzuki, Nobuhiro

    2015-08-01

    Rosellinia necatrix megabirnavirus 1 (RnMBV1) W779 is a bi-segmented dsRNA virus and a strain of the type species Rosellinia necatrix megabirnavirus 1 of the family Megabirnaviridae. RnMBV1 causes severe reduction of both mycelial growth of Rosellinia necatrix in synthetic medium and fungal virulence to plant hosts, and thus has strong potential for virocontrol (biological control using viruses) of white rot. The structure of RnMBV1 was examined by cryo-electron microscopy and three-dimensional reconstruction at 15.7 Å resolution. The diameter of the RnMBV1 capsid was 520 Å, and the capsid was composed of 60 asymmetrical dimers in the T = 1 (so-called T = 2) lattice that is well conserved among dsRNA viruses. However, RnMBV1 has putatively 120 large protrusions with a width of ∼ 45 Å and a height of ∼ 50 Å on the virus surface, making it distinguishable from the other dsRNA viruses. PMID:25968130

  3. Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

    PubMed Central

    Tuffereau, Christine; Benejean, Jacqueline; Alfonso, Anne-Marie Roque; Flamand, Anne; Fishman, Mark C.

    1998-01-01

    The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo. PMID:9445003

  4. Baculovirus replication: characterization of DNA and proteins synthesized by a nuclear polyhedrosis virus of Lymantria dispar, the gypsy moth, in a homologous cell line

    SciTech Connect

    McClintock, J.T.

    1985-01-01

    A multiple-embedded nuclear polyhedrosis virus (NPV) of the gypsy moth, Lymantria dispar (LdMNPV), is used for biological control. However, LdMNPV has low natural virulence and a long infection cycle in relation to other NPVs. Therefore, the replicative cycle of LdMNPV was investigated using a homologous cell line, IPLB-LD-652Y. Based on analyses of virus growth curves LdMNPV nonoccluded virus and polyhedral inclusion bodies appeared approximately 20 and 50 hr postinfection (p.i.), respectively. LdMNPV polypeptides, identified by autoradiography of (/sup 35/S)-methionine labeled fractions in SDS-PAGE, were synthesized in sequential phases: (1) an early ..cap alpha.. phase of replication (4 polypeptides from 4 to 12 hr p.i.), (2) an intermediate ..beta.. phase (20 polypeptides from 12 to 24 hr p.i.), and a late ..gamma.. phase (4 polypeptides from 24 to 28 hr p.i.). In infected cells at least four polypeptides were post-translational cleaved and/or modified. Pulse-labeling with (/sup 3/H)-mannose, (/sup 3/H)-N-acetyl-glucosamine or (/sup 32/P)-monosodium phosphate revealed several viral polypeptides which were glycosylated and/or phosphorylated. DNA:DNA dot hybridization experiments suggested that LdMNPV DNA synthesis was initiated between 12 to 16 hr p.i., increasing significantly thereafter.

  5. Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A.

    PubMed Central

    Andersons, D; Engström, A; Josephson, S; Hansson, L; Steiner, H

    1991-01-01

    A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1720614

  6. Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

    PubMed

    An, K; Gillock, E T; Sweat, J A; Reeves, W M; Consigli, R A

    1999-04-01

    The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells. PMID:10211971

  7. Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

    PubMed Central

    Erdmann, Dirk; Zimmermann, Catherine; Fontana, Patrizia; Hau, Jean-Christophe; De Pover, Alain; Chène, Patrick

    2010-01-01

    Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies. PMID:20357977

  8. A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice

    PubMed Central

    2011-01-01

    Background Avian influenza viruses of H9N2 subtype have become highly prevalent in avian species. Although these viruses generally cause only mild to moderate disease, they can infect a wide variety of species, including chickens, quail, turkeys, ducks, geese, pheasant, partridge, and pigeon, even transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. Results The results showed that stronger immune responses were induced in a mouse model immunized with BV-Dual-HA than in those vaccinated with a DNA vaccine encoding the same antigen. Moreover, complete protection against lethal challenge with H9N2 virus was observed in mice. Conclusion BV-Dual-HA could be utilized as a vaccine candidate against H9N2 virus infection. PMID:21639929

  9. Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus.

    PubMed

    Lee, Jong Seok; Kwon, Young-Man; Hwang, Hye Suk; Lee, Yu-Na; Ko, Eun-Ju; Yoo, Si-Eun; Kim, Min-Chul; Kim, Ki-Hye; Cho, Min Kyoung; Lee, Young-Tae; Lee, You Ri; Quan, Fu-Shi; Kang, Sang-Moo

    2014-10-01

    Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no licensed vaccine against RSV and it is a high priority to develop a safe RSV vaccine. We determined the immunogenicity and protective efficacy of combined virus-like particle and DNA vaccines presenting RSV glycoproteins (Fd.VLP) in comparison with formalin inactivated RSV (FI-RSV). Immunization of mice with Fd.VLP induced higher ratios of IgG2a/IgG1 antibody responses compared to those with FI-RSV. Upon live RSV challenge, Fd.VLP and FI-RSV vaccines were similarly effective in clearing lung viral loads. However, FI-RSV immunized mice showed a substantial weight loss and high levels of T helper type 2 (Th2) cytokines as well as extensive lung histopathology and eosinophil infiltration. In contrast, Fd.VLP immunized mice did not exhibit Th2 type cytokines locally and systemically, which might contribute to preventing vaccine-associated RSV lung disease. These results indicate that virus-like particles in combination with DNA vaccines represent a potential approach for developing a safe and effective RSV vaccine. PMID:25173478

  10. Molecular probes for identification of pathogenic viruses in mosquitoes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral pathogens that cause disease in mosquitoes belong to three major groups: baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus); iridoviruses (MIVs) (Iridoviridae: Chloriridovirus); and cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus). Baculoviruses and iridoviruses are DNA vir...

  11. Estimation of the Proportion of Variation Accounted for by DNA Tests. I: Genetic Variance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The proportion of genetic variation accounted for (Rg2) is an important characteristic of a DNA test. For each of 3 levels of narrow sense heritability of the observed trait (h2gy) and 4 levels of Rg2, 500 independent replicates of an observed trait and a molecular breeding value (MBV) for 1000 offs...

  12. Understanding the Mechanisms of Immunopathogenesis of Human and Bovine Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extensive investigations have revealed that zoonotic pathogens in the Mycobacterium tuberculosis complex (MTBC) evolved from a common ancestor. Although all the members can cause disease in one or more species of mammals, Mycobacterium tuberculosis (Mtb) and M. bovis (Mbv) are the major pathogens ...

  13. Using software security analysis to verify the secure socket layer (SSL) protocol

    NASA Technical Reports Server (NTRS)

    Powell, John D.

    2004-01-01

    nal Aeronautics and Space Administration (NASA) have tens of thousands of networked computer systems and applications. Software Security vulnerabilities present risks such as lost or corrupted data, information the3, and unavailability of critical systems. These risks represent potentially enormous costs to NASA. The NASA Code Q research initiative 'Reducing Software Security Risk (RSSR) Trough an Integrated Approach '' offers, among its capabilities, formal verification of software security properties, through the use of model based verification (MBV) to address software security risks. [1,2,3,4,5,6] MBV is a formal approach to software assurance that combines analysis of software, via abstract models, with technology, such as model checkers, that provide automation of the mechanical portions of the analysis process. This paper will discuss: The need for formal analysis to assure software systems with respect to software and why testing alone cannot provide it. The means by which MBV with a Flexible Modeling Framework (FMF) accomplishes the necessary analysis task. An example of FMF style MBV in the verification of properties over the Secure Socket Layer (SSL) communication protocol as a demonstration.

  14. Overview of genomic selection in small populations of beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efficiency and reproduction are important to beef production and are enhanced by using breeds adapted to specific management strategies and environments and by crossbreeding. Thus, dozens of breeds are currently used in the U.S. Genomic prediction of breeding value (MBV) needs large trait and geno...

  15. Yield and quality grade outcomes as affected by molecular breeding values for commercial beef cattle.

    PubMed

    Thompson, N M; DeVuyst, E A; Brorsen, B W; Lusk, J L

    2015-05-01

    Although genetic tests for many economically important beef cattle traits are commercially available, additional information is needed to help the industry better understand how the results from these tests translate into phenotypic outcomes. This information has important implications for marker-assisted management. The objective of this research was to determine the relationship between molecular breeding values (MBV) characterizing yield grade and marbling and distributions of phenotypic outcomes for yield grade and quality grade. Using data collected from commercially-fed cattle, mixed-model regression equations were estimated for yield grade and quality grade outcomes for both the full sample of commercial cattle (n= 8,995) and a subsample of black-hided steers (n = 4,790). Significant yield grade (P < 0.01) and marbling (P < 0.01) MBV effects were found. However, the yield grade MBV held up better at predicting phenotypic outcomes than the marbling MBV. Estimated conditional probability mass functions of yield and quality grade outcomes for the general population and black-hided steers were similar. Since distributions for black-hided steers were expected to be more applicable from a management perspective, we focused our analysis on these animals. For example, black-hided steers with "low" genetic potential for yield grade and marbling had about a 29% chance of achieving the base price or better on a price grid (yield grade 3 or lower and quality grade Low Choice or better). Increasing genetic potential for marbling increased the likelihood of achieving this same outcome. However, increasing genetic potential for yield grade was unlikely to increase overall carcass quality given its large deleterious effect on quality grade outcomes. Instead, simultaneous improvements in genetic potential for yield grade and marbling offered much more reliable improvements in overall carcass quality. For example, an animal with "moderate" genetic potential for both yield

  16. 76 FR 28422 - Marine Mammals; File No. 16053

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-17

    ...), rough- toothed dolphin (Steno bredanensis), beluga whale (Delphinapterus leucas), narwhal (Monodon monoceros), Pacific white-sided dolphin (Lagenorhynchus obliquidens), Atlantic white-sided dolphin (L. acutus), white-beaked dolphin (L. albirostris), hourglass dolphin (L. cruciger), dusky dolphin...

  17. Integrating Genomics with Nutrition Models to Improve the Prediction of Cattle Performance and Carcass Composition under Feedlot Conditions

    PubMed Central

    Tedeschi, Luis O.

    2015-01-01

    Cattle body composition is difficult to model because several factors affect the composition of the average daily gain (ADG) of growing animals. The objective of this study was to identify commercial single nucleotide polymorphism (SNP) panels that could improve the predictability of days on feed (DOF) to reach a target United States Department of Agriculture (USDA) grade given animal, diet, and environmental information under feedyard conditions. The data for this study was comprised of crossbred heifers (n = 681) and steers (n = 836) from commercial feedyards. Eleven molecular breeding value (MBV) scores derived from SNP panels of candidate gene polymorphisms and two-leptin gene SNP (UASMS2 and E2FB) were evaluated. The empty body fat (EBF) and the shrunk body weight (SBW) at 28% EBF (AFSBW) were computed by the Cattle Value Discovery System (CVDS) model using hip height (EBFHH and AFSBWHH) or carcass traits (EBFCT and AFSBWCT) of the animals. The DOFHH was calculated when AFSBWHH and ADGHH were used and DOFCT was calculated when AFSBWCT and ADGCT were used. The CVDS estimates dry matter required (DMR) by individuals fed in groups when observed ADG and AFSBW are provided. The AFSBWCT was assumed more accurate than the AFSBWHH because it was computed using carcass traits. The difference between AFSBWCT and AFSBWHH, DOFCT and DOFHH, and DMR and dry matter intake (DMI) were regressed on the MBV scores and leptin gene SNP to explain the variation. Our results indicate quite a large range of correlations among MBV scores and model input and output variables, but MBV ribeye area was the most strongly correlated with the differences in DOF, AFSBW, and DMI by explaining 8, 13.2 and 6.5%, respectively, of the variation. This suggests that specific MBV scores might explain additional variation of input and output variables used by nutritional models in predicting individual animal performance. PMID:26599759

  18. Development of a new Sonovue™ contrast-enhanced ultrasound approach reveals temporal and age-related features of muscle microvascular responses to feeding

    PubMed Central

    Mitchell, William Kyle; Phillips, Bethan E; Williams, John P; Rankin, Debbie; Smith, Kenneth; Lund, Jonathan N; Atherton, Philip J

    2013-01-01

    Compromised limb blood flow in aging may contribute to the development of sarcopenia, frailty, and the metabolic syndrome. We developed a novel contrast-enhanced ultrasound technique using Sonovue™ to characterize muscle microvasculature responses to an oral feeding stimulus (15 g essential amino acids) in young (∼20 years) and older (∼70 years) men. Intensity-time replenishment curves were made via an ultrasound probe “fixed” over the quadriceps, with intermittent high mechanical index destruction of microbubbles within muscle vasculature. This permitted real-time measures of microvascular blood volume (MBV), microvascular flow velocity (MFV) and their product, microvascular blood flow (MBF). Leg blood flow (LBF) was measured by Doppler and insulin by enzyme-linked immunosorbent assay. Steady-state contrast concentrations needed for comparison between different physiological states were achieved <150 sec from commencing Sonovue™ infusion, and MFV and MBV measurements were completed <120 sec thereafter. Interindividual coefficients of variation in MBV and MFV were 35–40%, (N = 36). Younger men (N = 6) exhibited biphasic vascular responses to feeding with early increases in MBV (+36%, P < 0.008 45 min post feed) reflecting capillary recruitment, and late increases in MFV (+77%, P < 0.008) and MBF (+130%, P < 0.007 195 min post feed) reflecting more proximal vessel dilatation. Early MBV responses were synchronized with peak insulin but not increased LBF, while later changes in MFV and MBF occurred with insulin at post absorptive values but alongside increased LBF. All circulatory responses were absent in old men (N = 7). Thus, impaired postprandial circulation could impact age-related declines in muscle glucose disposal, protein anabolism, and muscle mass. PMID:24303186

  19. Integrating Genomics with Nutrition Models to Improve the Prediction of Cattle Performance and Carcass Composition under Feedlot Conditions.

    PubMed

    Tedeschi, Luis O

    2015-01-01

    Cattle body composition is difficult to model because several factors affect the composition of the average daily gain (ADG) of growing animals. The objective of this study was to identify commercial single nucleotide polymorphism (SNP) panels that could improve the predictability of days on feed (DOF) to reach a target United States Department of Agriculture (USDA) grade given animal, diet, and environmental information under feedyard conditions. The data for this study was comprised of crossbred heifers (n = 681) and steers (n = 836) from commercial feedyards. Eleven molecular breeding value (MBV) scores derived from SNP panels of candidate gene polymorphisms and two-leptin gene SNP (UASMS2 and E2FB) were evaluated. The empty body fat (EBF) and the shrunk body weight (SBW) at 28% EBF (AFSBW) were computed by the Cattle Value Discovery System (CVDS) model using hip height (EBFHH and AFSBWHH) or carcass traits (EBFCT and AFSBWCT) of the animals. The DOFHH was calculated when AFSBWHH and ADGHH were used and DOFCT was calculated when AFSBWCT and ADGCT were used. The CVDS estimates dry matter required (DMR) by individuals fed in groups when observed ADG and AFSBW are provided. The AFSBWCT was assumed more accurate than the AFSBWHH because it was computed using carcass traits. The difference between AFSBWCT and AFSBWHH, DOFCT and DOFHH, and DMR and dry matter intake (DMI) were regressed on the MBV scores and leptin gene SNP to explain the variation. Our results indicate quite a large range of correlations among MBV scores and model input and output variables, but MBV ribeye area was the most strongly correlated with the differences in DOF, AFSBW, and DMI by explaining 8, 13.2 and 6.5%, respectively, of the variation. This suggests that specific MBV scores might explain additional variation of input and output variables used by nutritional models in predicting individual animal performance. PMID:26599759

  20. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3,were previously identified as presumptively encoding acetylcholinesterases, but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant c...

  1. Glucagon-like peptide 1 recruits muscle microvasculature and improves insulin's metabolic action in the presence of insulin resistance.

    PubMed

    Chai, Weidong; Zhang, Xingxing; Barrett, Eugene J; Liu, Zhenqi

    2014-08-01

    Glucagon-like peptide 1 (GLP-1) acutely recruits muscle microvasculature, increases muscle delivery of insulin, and enhances muscle use of glucose, independent of its effect on insulin secretion. To examine whether GLP-1 modulates muscle microvascular and metabolic insulin responses in the setting of insulin resistance, we assessed muscle microvascular blood volume (MBV), flow velocity, and blood flow in control insulin-sensitive rats and rats made insulin-resistant acutely (systemic lipid infusion) or chronically (high-fat diet [HFD]) before and after a euglycemic-hyperinsulinemic clamp (3 mU/kg/min) with or without superimposed systemic GLP-1 infusion. Insulin significantly recruited muscle microvasculature and addition of GLP-1 further expanded muscle MBV and increased insulin-mediated glucose disposal. GLP-1 infusion potently recruited muscle microvasculature in the presence of either acute or chronic insulin resistance by increasing muscle MBV. This was associated with an increased muscle delivery of insulin and muscle interstitial oxygen saturation. Muscle insulin sensitivity was completely restored in the presence of systemic lipid infusion and significantly improved in rats fed an HFD. We conclude that GLP-1 infusion potently expands muscle microvascular surface area and improves insulin's metabolic action in the insulin-resistant states. This may contribute to improved glycemic control seen in diabetic patients receiving incretin-based therapy. PMID:24658303

  2. Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis

    PubMed Central

    Lane, V. Michael; Ball, Louise M.; Hess, John F.

    2010-01-01

    A randomized, blinded, controlled field trial was conducted during summer 2006 in a northern California, USA, herd of beef cattle to evaluate the efficacy of a recombinant Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye). A convenience sample comprised of 127 steers were administered a subcutaneous dose of either adjuvant alone (ISCOM matrices; control group) or recombinant M. bovoculi cytotoxin carboxy terminus adjuvanted with ISCOM matrices (MbvA group) and were boostered 21 days later. The steers were examined once weekly for 15 weeks for evidence of IBK. No significant difference in the cumulative proportion of corneal ulcerations was detected between groups. Compared to the control calves, the MbvA vaccinates had significantly higher increases in serum neutralizing titers to M. bovoculi hemolysin between week 0 and week 6. The prevalence of M. bovis isolations was higher from ulcerated eyes of calves vaccinated with MbvA as compared to control calves. Vaccination of calves against the carboxy terminus of M. bovoculi RTX toxin resulted in significant increases in serum hemolysin neutralizing titers and may modulate organism type cultured from ulcerated eyes of calves in herds where both M. bovis and M. bovoculi exist. Use of M. bovoculi antigens alone in vaccines to prevent IBK may not be beneficial in herds where IBK is associated with both M. bovoculi and M. bovis. PMID:20217228

  3. Identification and expression profile of multiple genes in response to magnesium exposure in Culex quinquefasciatus larvae.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Magnesium is crucial for baculovirus transmission in Culex nigripalpus (Theobald) and Cx. quinquefasciatus (Say) larvae, both in the field and in the laboratory. However, the mechanistic role of magnesium in baculovirus transmission is unknown. To investigate the possible role of a host response fac...

  4. The effects of resistance exercise training on macro- and micro-circulatory responses to feeding and skeletal muscle protein anabolism in older men

    PubMed Central

    Phillips, Bethan E; Atherton, Philip J; Varadhan, Krishna; Limb, Marie C; Wilkinson, Daniel J; Sjøberg, Kim A; Smith, Kenneth; Williams, John P

    2015-01-01

    The anabolic effects of dietary protein on skeletal muscle depend on adequate skeletal muscle perfusion, which is impaired in older people. This study explores fed state muscle microvascular blood flow, protein metabolism and exercise training status in older men. We measured leg blood flow (LBF), muscle microvascular blood volume (MBV) and muscle protein turnover under post-absorptive and fed state (i.v. Glamin to double amino acids, dextrose to sustain glucose ∼7–7.5 mmol l−1) conditions in two groups: 10 untrained men (72.3 ± 1.4 years; body mass index (BMI) 26.5 ± 1.15 kg m2) and 10 men who had undertaken 20 weeks of fully supervised, whole-body resistance exercise training (RET) (72.8 ± 1.4 years; BMI 26.3 ± 1.2 kg m2). We measured LBF by Doppler ultrasound and muscle MBV by contrast-enhanced ultrasound. Muscle protein synthesis (MPS) was measured using [1, 2-13C2] leucine with breakdown (MPB) and net protein balance (NPB) by ring-[D5] phenylalanine tracers. Plasma insulin was measured via ELISA and indices of anabolic signalling (e.g. Akt/mTORC1) by immunoblotting from muscle biopsies. Whereas older untrained men did not exhibit fed-state increases in LBF or MBV, the RET group exhibited increases in both LBF and MBV. Despite our hypothesis that enhanced fed-state circulatory responses would improve anabolic responses to nutrition, fed-state increases in MPS (∼50–75%; P < 0.001) were identical in both groups. Finally, whereas only the RET group exhibited fed-state suppression of MPB (∼–38%; P < 0.05), positive NPB achieved was similar in both groups. We conclude that RET enhances fed-state LBF and MBV and restores nutrient-dependent attenuation of MPB without robustly enhancing MPS or NPB. PMID:25867865

  5. Sequence Analysis of the Genome of the Neodiprion sertifer Nucleopolyhedrovirus†

    PubMed Central

    Garcia-Maruniak, Alejandra; Maruniak, James E.; Zanotto, Paolo M. A.; Doumbouya, Aissa E.; Liu, Jaw-Ching; Merritt, Thomas M.; Lanoie, Jennifer S.

    2004-01-01

    The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses. PMID:15194780

  6. Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

    PubMed

    Abhiman, P B; Shankar, K M; Patil, Rajreddy; Suresh Babu, P P; Sahoo, A K; Shamasundar, B A

    2014-05-01

    Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes. PMID:24803705

  7. Chitin purification from shrimp wastes by microbial deproteination and decalcification.

    PubMed

    Xu, Y; Gallert, C; Winter, J

    2008-06-01

    Chitin was purified from Penaeus monodon and Crangon crangon shells using a two-stage fermentation process with anaerobic deproteination followed by decalcification through homofermentative lactic acid fermentation. Deproteinating enrichment cultures from sewage sludge and ground meat (GM) were used with a proteolytic activity of 59 and 61 mg N l(-1) h(-1) with dried and 26 and 35 mg N l(-1) h(-1) with wet P. monodon shells. With 100 g wet cells of proteolytic bacteria per liter, protein removal was obtained in 42 h. An anaerobic spore-forming bacterium HP1 was isolated from enrichment GM. Its proteolytic activity was 76 U ml(-1) compared to 44 U ml(-1) of the consortium. Glucose was fermented with Lactobacillus casei MRS1 to lactic acid. At a pH of 3.6, calcium carbonate of the shells was solubilised. After deproteination and decalcification of P. monodon or C. crangon shells, the protein content was 5.8% or 6.7%, and the calcium content was 0.3% or 0.4%, respectively. The viscosity of the chitin from P. monodon and C. crangon was 45 and 135 mPa s, respectively, whereas purchased crab shell chitin (practical grade) had a viscosity of 21 mPa s, indicating a higher quality of biologically purified chitin. PMID:18418590

  8. Effects of hypoxia on cerebral and muscle haemodynamics during knee extensions in healthy subjects.

    PubMed

    Gomes, Paulo Sergio Chagas; Matsuura, Cristiane; Bhambhani, Yagesh N

    2013-01-01

    A hypoxic model was used to investigate changes in localized cerebral and muscle haemodynamics during knee extension (KE) in healthy individuals. Thirty-one young healthy volunteers performed one set of KE until failure under hypoxia (14 % O(2)) or normoxia (21 % O(2)) at 50, 75 or 100 % of 1 repetition maximum, in random order, on three occasions. Prefrontal cerebral and vastus lateralis muscle oxygenation and blood volume (Cox, Mox, Cbv and Mbv, respectively) were recorded simultaneously by near-infrared spectroscopy. Hypoxia induced significant declines in Cox [-0.017 ± 0.016 optical density (OD) units] and Mox (-0.014 ± 0.026 OD units) and increases in Cbv (0.017 ± 0.027 OD units) and Mbv (0.016 ± 0.023 OD units) at rest. Hypoxia significantly reduced total work (TW) performed during KE at each exercise intensity. Cox, Cbv, Mox, and Mbv changes during KE did not differ between normoxia and hypoxia. Correlations between TW done and Cox changes under normoxia (r = 0.04, p = 0.182) and hypoxia (r = 0.05, p = 0.122) were not significant. However, TW was significantly correlated with Mox under both normoxia (R (2) = 0.24, p = 0.000) and hypoxia (R (2) = 0.15, p = 0.004). Since changes in Cox and Mox reflect alterations in the balance between oxygen delivery and extraction in these tissues, which, in the brain, is an index of neuronal activation, we conclude that: (1) limitation of KE performance was mediated peripherally under both normoxia and hypoxia, with no additional effect of hypoxia, and (2) because of the low common variance with Mox additional intramuscular factors likely play a role in limiting KE performance. PMID:22544440

  9. A genome-wide association study of meat and carcass traits in Australian cattle.

    PubMed

    Bolormaa, S; Neto, L R Porto; Zhang, Y D; Bunch, R J; Harrison, B E; Goddard, M E; Barendse, W

    2011-08-01

    Chromosomal regions containing DNA variation affecting the traits intramuscular fat percentage (IMF), meat tenderness measured as peak force to shear the LM (LLPF), and rump fat measured at the sacro-iliac crest in the chiller (CHILLP8) were identified using a set of 53,798 SNP genotyped on 940 taurine and indicine cattle sampled from a large progeny test experiment. Of these SNP, 87, 64, and 63 were significantly (P < 0.001) associated with the traits IMF, LLPF, and CHILLP8, respectively. A second, nonoverlapping sample of 1,338 taurine and indicine cattle from the same large progeny test experiment genotyped for 335 SNP, including as a positive control the calpastatin (CAST) c.2832A > G SNP, was used to confirm these locations. In total, 37 SNP were significantly (P < 0.05) associated with the same trait and with the same favorable homozygote in both data sets, representing 27 chromosomal regions. For the trait IMF, the effect of SNP in the confirmation data set was predicted from the discovery set by multiplying the estimated allele effect of each SNP in the discovery set by the number of copies of the reference allele of each SNP in the confirmation set. These weighted effects were then summed over all SNP to generate a molecular breeding value (MBV) for each animal in the confirmation data set. Using a bivariate analysis of MBV and IMF phenotypes of animals in the confirmation set, a panel of 14 SNP explained 5.6 and 15.6% of the phenotypic and genetic variance of IMF, respectively, in the confirmation data set. The amount of variation did not increase as more SNP were added to the MBV and instead decreased to 1.2 and 3.8% of the phenotypic and genetic variance of IMF, respectively, when 329 SNP were included in the analysis. PMID:21421834

  10. Acute cocoa flavanol supplementation improves muscle macro- and microvascular but not anabolic responses to amino acids in older men.

    PubMed

    Phillips, Bethan E; Atherton, Philip J; Varadhan, Krishna; Limb, Marie C; Williams, John P; Smith, Kenneth

    2016-05-01

    The anabolic effects of nutrition on skeletal muscle may depend on adequate skeletal muscle perfusion, which is impaired in older people. Cocoa flavanols have been shown to improve flow-mediated dilation, an established measure of endothelial function. However, their effect on muscle microvascular blood flow is currently unknown. Therefore, the objective of this study was to explore links between the consumption of cocoa flavanols, muscle microvascular blood flow, and muscle protein synthesis (MPS) in response to nutrition in older men. To achieve this objective, leg blood flow (LBF), muscle microvascular blood volume (MBV), and MPS were measured under postabsorptive and postprandial (intravenous Glamin (Fresenius Kabi, Germany), dextrose to sustain glucose ∼7.5 mmol·L(-1)) conditions in 20 older men. Ten of these men were studied with no cocoa flavanol intervention and a further 10 were studied with the addition of 350 mg of cocoa flavanols at the same time that nutrition began. Leg (femoral artery) blood flow was measured by Doppler ultrasound, muscle MBV by contrast-enhanced ultrasound using Definity (Lantheus Medical Imaging, Mass., USA) perflutren contrast agent and MPS using [1, 2-(13)C2]leucine tracer techniques. Our results show that although older individuals do not show an increase in LBF or MBV in response to feeding, these absent responses are apparent when cocoa flavanols are given acutely with nutrition. However, this restoration in vascular responsiveness is not associated with improved MPS responses to nutrition. We conclude that acute cocoa flavanol supplementation improves muscle macro- and microvascular responses to nutrition, independently of modifying muscle protein anabolism. PMID:27120341

  11. Genomic prediction of breeding values in the New Zealand sheep industry using a 50K SNP chip.

    PubMed

    Auvray, B; McEwan, J C; Newman, S-A N; Lee, M; Dodds, K G

    2014-10-01

    The aim of genomic prediction is to predict breeding value from genomic data. We describe the development of genomic prediction equations and accuracies for molecular breeding values (MBV) for industry use, focusing on the methodology used to deal with predictions for the New Zealand sheep population structure. This is made up of a mixture of pure and crossbred animals, but principally Romney based. In particular, we used pedigree-based EBV for 8 traits (weaning weight as a direct effect, weaning weight as a maternal effect, live weight at 8 mo, live weight at 12 mo, greasy fleece weight at 12 mo, lamb fleece weight, adult fleece weight, and number of lambs born) and Illumina OvineSNP50 BeadChip genotypes from 13,420 animals to investigate BLUP with different genomic relationship matrices (GRM) based on SNP markers and to investigate varying sets of older animals (training sets) to predict the MBV of younger animals (validation sets). The GRM tested included modifications to account for allele frequency differences between breeds, rescaling so that the mean GRM is equal to the mean of the traditional pedigree numerator relationship matrix A: , and combining of the GRM with A: using a convex combination with a weight estimated by maximizing a conditional restricted likelihood. We found that these modifications were beneficial and recommend using a breed-adjusted GRM combined with A: . Training data sets with Romney, Coopworth, and Perendale animals all together usually predicted better than using just a pure breed training data set for all traits. But predictions for the breed Perendale were more accurate with a Perendale training set for 3 of the 8 traits. We concluded that using a mixed-breed training set for all combinations of traits and breeds was best but advise that increasing the number of Perendale animals genotyped should be a priority to increase the MBV accuracies obtained for that breed. PMID:25149326

  12. SIMPLE SYSTEM FOR THE PRELIMINARY EVALUATION OF INFECTIVITY AND PATHOGENESIS OF INSECT VIRUS IN A NONTARGET ESTUARINE SHRIMP

    EPA Science Inventory

    Biological control agents (biorationals) are increasingly important in pest control concepts. Certain insect viruses, particularly the baculoviruses (nuclear polyhedrosis viruses), are considered to have potential as biological pesticides, and could be used widely in the environm...

  13. VIRAL PESTICIDES: PRESENT KNOWLEDGE AND POTENTIAL EFFECTS ON PUBLIC AND ENVIRONMENTAL HEALTH (SYMPOSIUM PROCEEDINGS)

    EPA Science Inventory

    Baculoviruses appear to be effective alternatives to chemical pest control. To date deleterious effects on other components of the ecosystem have not been demonstrated. However, safety testing recommended for registration utilize protocols developed for chemical pesticides. Safet...

  14. Complete Genome Sequences of Seven Helicoverpa armigera SNPV-AC53-Derived Strains

    PubMed Central

    2016-01-01

    Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from Helicoverpa armigera SNPV-AC53 (KJ909666). PMID:27151787

  15. Bacterial, but not Baculoviral Infections Stimulate Hemolin Expression in Noctuid Moths

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis vires...

  16. How to Choose In Vitro Systems to Predict In Vivo Drug Clearance: A System Pharmacology Perspective

    PubMed Central

    Wang, Lei; Chiang, ChienWei; Liang, Hong; Wu, Hengyi; Feng, Weixing; Quinney, Sara K.; Li, Jin; Li, Lang

    2015-01-01

    The use of in vitro metabolism data to predict human clearance has become more significant in the current prediction of large scale drug clearance for all the drugs. The relevant information (in vitro metabolism data and in vivo human clearance values) of thirty-five drugs that satisfied the entry criteria of probe drugs was collated from the literature. Then the performance of different in vitro systems including Escherichia coli system, yeast system, lymphoblastoid system and baculovirus system is compared after in vitro-in vivo extrapolation. Baculovirus system, which can provide most of the data, has almost equal accuracy as the other systems in predicting clearance. And in most cases, baculovirus system has the smaller CV in scaling factors. Therefore, the baculovirus system can be recognized as the suitable system for the large scale drug clearance prediction. PMID:26539530

  17. Bacterial but not Baculoviral Infections Stimulate Hemolin Expression in Helicoverpa zea and Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and budworm (Heliothis virescens), whi...

  18. 78 FR 42527 - Government-Owned Inventions; Availability for Licensing

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-16

    ... baculovirus production (allowing high- throughput production with limited screening) Higher efficiency cloning...--Combinatorial Cloning Platform. Licensing Contact: Susan Ano, Ph.D.; 301-435-5515; anos@mail.nih.gov ....

  19. Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridovirus...

  20. UMTRA ground water sampling techniques: Comparison of the traditional and low flow methods

    SciTech Connect

    1995-07-01

    This report describes the potential changes in water quality data that may occur with the conversion from MBV (multiple bore volume) to LF (low flow) sampling and provides two examples of how such a change might impact Project decisions. The existing scientific literature on LF sampling is reviewed and the new LF data from three UMTRA Uranium Mill Tailings Remedial Action Project sites are evaluated seeking answers to the questions posed above. Several possible approaches, that the UMTRA Project may take to address issues unanswered by the literature are presented and compared, and a recommendation is offered for the future direction of the LF conversion effort.