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Sample records for monodon baculovirus mbv

  1. Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon.

    PubMed

    Tang, Kathy F J; Lightner, Donald V

    2011-02-22

    We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

  2. Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.

    PubMed

    Ramesh Kumar, D; Sanjuktha, M; Rajan, J J S; Ananda Bharathi, R; Santiago, T C; Alavandi, S V; Poornima, M

    2014-09-01

    Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Improving baculovirus recombination

    PubMed Central

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation. PMID:12527795

  9. 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.

  10. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  11. Thiamin requirement of juvenile shrimp (Penaeus monodon).

    PubMed

    Chen, H Y; Wu, F C; Tang, S Y

    1991-12-01

    In a 9-wk feeding trial, juvenile shrimp (Penaeus monodon) were fed semipurified diets containing seven levels (0, 20, 40, 60, 80, 160 and 320 mg/kg diet) of supplemental thiamin hydrochloride. The dietary thiamin level required for optimal growth in P. monodon was found to be approximately 14 mg/kg diet based on hemolymph (blood) thiamin analysis. The minimum dietary thiamin level that produced substantial shrimp growth was approximately 13 mg/kg diet. Shrimp fed unsupplemented diets (thiamin content of 0.12 mg/kg diet) did not demonstrate specific deficiency signs, except those universal signs such as retarded growth, poor food conversion and low survival rates.

  12. Baculovirus Transfer Vectors.

    PubMed

    Possee, Robert D; King, Linda A

    2016-01-01

    The production of a recombinant baculovirus expression vector normally involves mixing infectious virus DNA with a plasmid-based transfer vector and then co-transfecting insect cells to initiate virus infection. The aim of this chapter is to provide an update on the range of baculovirus transfer vectors currently available. Some of the original transfer vectors developed are now difficult to obtain but generally have been replaced by superior reagents. We focus on those that are available commercially and should be easy to locate. These vectors permit the insertion of single or multiple genes for expression, or the production of proteins with specific peptide tags that aid subsequent protein purification. Others have signal peptide coding regions permitting protein secretion or plasma membrane localization. A table listing the transfer vectors also includes information on the parental virus that should be used with each one. Methods are described for the direct insertion of a recombinant gene into the virus genome without the requirement for a transfer vector. The information provided should enable new users of the system to choose those reagents most suitable for their purposes.

  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. Recombinant baculovirus displayed vaccine

    PubMed Central

    Prabakaran, Mookkan; Kwang, Jimmy

    2014-01-01

    The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness. PMID:23941989

  15. Recombinant baculoviruses for insect control.

    PubMed

    Inceoglu, A B; Kamita, S G; Hinton, A C; Huang, Q; Severson, T F; Kang, K; Hammock, B D

    2001-10-01

    Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

  16. 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.

  17. 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...

  18. Baculovirus-insect cell expression systems.

    PubMed

    Jarvis, Donald L

    2009-01-01

    In the early 1980s, the first-published reports of baculovirus-mediated foreign gene expression stimulated great interest in the use of baculovirus-insect cell systems for recombinant protein production. Initially, this system appeared to be the first that would be able to provide the high production levels associated with bacterial systems and the eukaryotic protein processing capabilities associated with mammalian systems. Experience and an increased understanding of basic insect cell biology have shown that these early expectations were not completely realistic. Nevertheless, baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. Furthermore, important technological advances over the past 20 years have improved upon the original methods developed for the isolation of baculovirus expression vectors, which were inefficient, required at least some specialized expertise and, therefore, induced some frustration among those who used the original baculovirus-insect cell expression system. Today, virtually any investigator with basic molecular biology training can relatively quickly and efficiently isolate a recombinant baculovirus vector and use it to produce their favorite protein in an insect cell culture. This chapter will begin with background information on the basic baculovirus-insect cell expression system and will then focus on recent developments that have greatly facilitated the ability of an average investigator to take advantage of its attributes.

  19. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  20. Cloning of penaeidin gene promoter in tiger shrimp (Penaeus monodon).

    PubMed

    Ho, Shih-Hu; Song, Yen-Ling

    2009-07-01

    Penaeidins belong to a family of antimicrobial peptides that are expressed in the hemocytes of penaeid shrimps. Using an extender PCR method and a nested PCR, we cloned two types of genomic fragment flanking the 5' end of penaeidin gene in tiger shrimp (Penaeus monodon): Type536 and Type411 sequences. Both fragments contained TATA box, GATA, dorsal and AP-1 motifs and were ligated to an expression vector with a luciferase reporter gene. The constructs were then delivered into Drosophila S2 cell line. The promoter functions of the two fragments were determined using a luciferase expression assay. The study demonstrated that Type411 sequence performed higher transcriptional activity than Type536. Alignment of the upstream sequences of penaeidin genes in P. monodon and Litopenaeus vannamei showed that the promoter regions were obviously more diverse than the 5'UTRs. Phylogenetic analysis indicated the presence of two types of promoters that are not species-specific in the two shrimps.

  1. Dietary pantothenic acid requirement of juvenile grass shrimp, Penaeus monodon.

    PubMed

    Shiau, S Y; Hsu, C W

    1999-03-01

    A feeding trial was conducted to estimate the minimal dietary pantothenic acid (PA) requirement for juvenile grass shrimp, Penaeus monodon. Purified diets with seven levels (0, 20, 40, 60, 120, 240, and 480 mg/kg) of supplemental PA were fed to P. monodon (mean weight 0.88 +/- 0.01 g) for 8 wk. The level of PA detected in the unsupplemented diet was 0.02 mg/kg. Each diet was fed to three replicate groups of shrimp. Feed efficiencies (FE) and protein efficiency ratios were highest in shrimp fed the diets supplemented with 120, 240, and 480 mg PA/kg diet, followed by the groups fed 60 mg/kg, then 40 mg/kg, and finally the unsupplemented control group (P < 0.05). Shrimp fed diets supplemented with PA had significantly higher survival percentages and lower hepatopancreatic lipid concentration than those fed the unsupplemented, control diets. Broken-line regression analyses of weight gain percentage and hepatopancreatic CoA and PA concentrations of the shrimp indicated that the adequate dietary PA concentration in growing P. monodon is 101-139 mg/kg.

  2. Purification of baculovirus vectors using heparin affinity chromatography

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; van der Loo, Johannes CM; Malik, Punam

    2016-01-01

    Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications. PMID:27933303

  3. In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

    PubMed Central

    Tani, Hideki; Limn, Chang Kwang; Yap, Chan Choo; Onishi, Masayoshi; Nozaki, Masami; Nishimune, Yoshitake; Okahashi, Nobuo; Kitagawa, Yoshinori; Watanabe, Rie; Mochizuki, Rika; Moriishi, Kohji; Matsuura, Yoshiharu

    2003-01-01

    Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy. PMID:12941888

  4. 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.

  5. 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 ...

  6. Bioaccumulation and depuration of anthracene in Penaeus monodon (Fibricius) through food ingestion.

    PubMed

    Ong, Pei Thing; Yong, Jaw Chuen; Chin, Kam Yew; Hii, Yii Siang

    2011-07-01

    Understanding on the bioaccumulation and depuration of PAHs (polycyclic aromatic hydrocarbons) in Penaeus monodon is important in seafood safety because it is one of the most popular seafood consumed worldwide. In this study, we used anthracene as the precursor compound for PAHs accumulation and depuration in the shrimp. Commercial feed pellets spiked with anthracene were fed to P. monodon. At 20 mg kg(-1) anthracene, P. monodon accumulated 0.1% of the anthracene from the feed. P. monodon deputed the PAH two times faster than its accumulation. The shrimp reduced its feed consumption when anthracene content in the feed exceeded 20 mg kg(-1). At 100 mg kg(-1) anthracene, P. monodon started to have necrosis tissues on the posterior end of their thorax. The bioaccumulation factor (BAF), uptake rate constant (k(1)) and depuration rate constant (k(2)) of anthracene in P. monodon were 1.15×10(-3), 6.80×10(-4) d(-1) and 6.28×10(-1) d(-1), respectively. The depuration rate constant is about thousand times higher than the uptake rate constant and this indicated that this crustacean is efficient in depurating hydrocarbons from their tissue.

  7. Baculovirus expression: old dog, new tricks

    PubMed Central

    Berger, Imre; Poterszman, Arnaud

    2015-01-01

    Since its inception more than 30 years ago, the baculovirus expression vector system (BEVS) has been used prolifically to produce heterologous proteins for research and development. In the cell, a cornerstone of biological activity are multiprotein complexes, catalyzing essential functions. BEVS has been uniquely successful to unlock such complex assemblies for high-resolution structural and functional analysis. Synthetic biology approaches have been implemented to optimize multigene assembly methods, accelerating upstream processes. Specialized baculoviral genomes are being created with functions tailored to enhance production of particular target protein classes. Here we comment on current and emerging developments in the field and their potential to accelerate protein complex research. PMID:26488462

  8. Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.

    PubMed

    Lin, Chi-Hung; Jarvis, Donald L

    2013-05-10

    Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher

  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. Genome scale transcriptomics of baculovirus-insect interactions.

    PubMed

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

    2013-11-12

    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.

  11. Effective transduction of osteogenic sarcoma cells by a baculovirus vector.

    PubMed

    Song, Sun U; Shin, Seok-Hwan; Kim, Soon-Ki; Choi, Gwang-Seong; Kim, Woo-Chul; Lee, Moon-Hee; Kim, Sei-Joong; Kim, In-Ho; Choi, Mi-Sook; Hong, Young-Jin; Lee, Kwan-Hee

    2003-03-01

    Efficient gene delivery of a baculovirus-derived vector (BV-p53-lacZ) to a human osteogenic sarcoma cell line, Saos-2, was serendipitously found while evaluating the vector for gene delivery to human p53-null tumour cells in a previous study. Therefore, we investigated other human, rat and mouse osteogenic sarcoma and other types of tumour cell lines for transduction efficiency via baculovirus vectors containing a lacZ reporter gene under the control of either a cytomegalovirus or Rous sarcoma virus promoter. The expression of beta-galactosidase protein, assessed by X-Gal staining and beta-galactosidase ELISA, demonstrated an extremely high level of transduction efficiency in some osteogenic sarcoma cell lines, such as U-2OS, Saos-2 and Saos-LM2. These human osteogenic sarcoma cell lines showed levels of beta-galactosidase expression 5-40 times greater than HepG2 cells, which were previously thought to be the mammalian cells most susceptible to baculovirus-mediated gene delivery. The level of acetylated histone proteins in these tumour lines did not correlate well with the high level of reporter gene expression. These results strongly suggest that some osteogenic sarcoma cells are highly susceptible to baculovirus-mediated gene delivery and that a baculovirus-derived vector is an efficient gene delivery vehicle into human osteogenic sarcoma cells.

  12. 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.

  13. 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

  14. Expansion of the Litopenaeus vannamei and Penaeus monodon peptidomes using transcriptome shotgun assembly sequence data.

    PubMed

    Christie, Andrew E

    2014-09-15

    The shrimp Litopenaeus vannamei and Penaeus monodon are arguably the most important commercially farmed crustaceans. While expansion of their aquaculture has classically relied on improvements to rearing facilities, these options have largely been exhausted, and today a shift in focus is occurring, with increased investment in manipulating the shrimp themselves. Hormonal control is one strategy for increasing aquaculture output. However, to use it, one must first understand an animal's native hormonal systems. Here, transcriptome shotgun assembly (TSA) data were used to expand the peptidomes for L. vannamei and P. monodon. Via an established bioinformatics workflow, 41 L. vannamei and 25 P. monodon pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 158 and 106 distinct peptide structures for these species, respectively. The identified peptides included isoforms of allatostatin A, B and C, as well as members the bursicon, CAPA, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone, diuretic hormone 31, eclosion hormone, FLRFamide, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, red pigment concentrating hormone, RYamide, SIFamide, short neuropeptide F and tachykinin-related peptide families. While some of the predicted peptides are known L. vannamei and/or P. monodon isoforms (which vet the structures of many peptides identified previously via mass spectrometry and other means), most are described here for the first time. These data more than double the extant catalogs of L. vannamei and P. monodon peptides and provide platforms from which to launch future physiological studies of peptidergic signaling in these two commercially important species.

  15. 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

  16. Genome sequence of a baculovirus pathogenic for Culex nigripalpus.

    PubMed

    Afonso, C L; Tulman, E R; Lu, Z; Balinsky, C A; Moser, B A; Becnel, J J; Rock, D L; Kutish, G F

    2001-11-01

    In this report we describe the complete genome sequence of a nucleopolyhedrovirus that infects larval stages of the mosquito Culex nigripalpus (CuniNPV). The CuniNPV genome is a circular double-stranded DNA molecule of 108,252 bp and is predicted to contain 109 genes. Although 36 of these genes show homology to genes from other baculoviruses, their orientation and order exhibit little conservation relative to the genomes of lepidopteran baculoviruses. CuniNPV genes homologous to those from other baculoviruses include genes involved in early and late gene expression (lef-4, lef-5, lef-8, lef-9, vlf-1, and p47), DNA replication (lef-1, lef-2, helicase-1, and dna-pol), and structural functions (vp39, vp91, odv-ec27, odv-e56, p6.9, gp41, p74, and vp1054). Auxiliary genes include homologues of genes encoding the p35 antiapoptosis protein and a novel insulin binding-related protein. In contrast to these conserved genes, CuniNPV lacks apparent homologues of baculovirus genes essential (ie-1 and lef-3) or stimulatory (ie-2, lef-7, pe38) for DNA replication. Also, baculovirus genes essential or stimulatory for early-late (ie-1, ie-2), early (ie-0 and pe-38), and late (lef-6, lef-11, and pp31) gene transcription are not identifiable. In addition, CuniNPV lacks homologues of genes involved in the formation of virogenic stroma (pp31), nucleocapsid (orf1629, p87, and p24), envelope of occluded virions (odv-e25, odv-e66, odv-e18), and polyhedra (polyhedrin/granulin, p10, pp34, and fp25k). A homologue of gp64, a budded virus envelope fusion protein, was also absent, although a gene related to the other category of baculovirus budded virus envelope proteins, Ld130, was present. The absence of homologues of occlusion-derived virion (ODV) envelope proteins and occlusion body (OB) protein (polyhedrin) suggests that both CuniNPV ODV and OB may be structurally and compositionally different from those found in terrestrial lepidopteran hosts. The striking difference in genome

  17. 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.

  18. 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...

  19. 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...

  20. Stability of serum-free and purified baculovirus stocks under various storage conditions.

    PubMed

    Jorio, Hasnaa; Tran, Rosa; Kamen, Amine

    2006-01-01

    In a context of large-scale production of baculoviruses in serum-free media for use as gene delivery vectors, the stability of these viruses has become an important factor. The development of robust processes heavily relies on baculovirus stock stability. In the present work, we studied over a period of 300 days the stability of baculovirus vectors produced in serum-free media stored at 4, -20, or -80 degrees C or in liquid nitrogen. The viral stocks investigated were either crude baculovirus supernatant, baculovirus supernatant concentrated 10 times and diafiltered against fresh serum-free media by tangential flow filtration, or baculovirus purified by size exclusion chromatography. The results showed that baculovirus supernatant and diafiltered concentrate stored at 4 degrees C underwent a progressive loss of infectivity after a period of 100 and 50 days of storage, respectively. Aggregation has been recognized as the probable mechanism for the loss of infectivity. Baculovirus stocks were unstable at -20 degrees C, whereas in liquid nitrogen they retained infectivity after successive freeze thaw cycles. Concentration and diafiltration of baculovirus supernatant prior to storing at -80 degrees C contributed to improving viral stock stability over time. Glycerol as well as DMSO and sucrose have proven to be equally effective as additives to maintain the purified baculovirus stability after storage at -80 degrees C or in liquid nitrogen.

  1. 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.

  2. Dietary biotin requirement for maximum growth of juvenile grass shrimp, Penaeus monodon.

    PubMed

    Shiau, S Y; Chin, Y H

    1998-12-01

    A feeding trial was conducted to estimate the minimal dietary biotin requirement for juvenile grass shrimp, Penaeus monodon. Purified diets with eight levels (0, 0.2, 0.5, 1.0, 3.0, 6.0, 10.0 and 20.0 mg/kg) of supplemental biotin were fed to P. monodon (mean weight 0. 26 +/- 0.01 g) for 8 wk. Each diet was fed to three replicate groups of shrimp. Shrimp fed diets supplemented with biotin (0.2-20.0 mg/kg) had significantly (P < 0.05) higher weight gain, feed efficiency and protein efficiency ratio than those fed the unsupplemented control diet. Weight gain was high in shrimp fed 3. 0-10.0 mg biotin/kg diet and lowest in shrimp fed monodon is 2.0-2.4 mg/kg.

  3. Quantifying the vitamin K requirement of juvenile marine shrimp (Penaeus monodon) with menadione.

    PubMed

    Shiau, S Y; Liu, J S

    1994-02-01

    A feeding trial was conducted to estimate the optimum dietary vitamin K requirement for juvenile marine shrimp, Penaeus monodon. Purified diets with eight levels (0, 5, 10, 20, 50, 80, 160 and 320 mg/kg) of supplemental menadione were fed to P. monodon (mean weight 1.33 +/- 0.05 g) for 12 wk. Each diet was fed to three replicate groups of shrimp. Shrimp fed diets supplemented with vitamin K (5-320 mg/kg) had significantly (P < 0.05) higher weight gain and feed efficiency than those fed the unsupplemented control diet. Shrimp fed diets supplemented with > or = 50 mg/kg vitamin K had higher protein efficiency ratios than shrimp fed the control diet. Calcium deposition in shrimp generally increased as dietary vitamin K supplementation increased. Vitamin K-dependent carboxylase activity was highest in shrimp fed the control diet, followed by shrimp fed 5-160 mg menadione/kg diet, and lowest in those fed 320 mg menadione/kg diet. Vitamin K-dependent protein precursor concentrations were high in shrimp fed 0-10 mg menadione/kg diet, lower in those fed 20 mg/kg, and lowest in shrimp fed > or = 50 mg/kg diet. Protein efficiency ratio and vitamin K-dependent protein precursor concentration analyzed by broken-line regression indicated that the adequate dietary vitamin K concentration in growing P. monodon is 30-40 mg/kg diet.

  4. Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon.

    PubMed

    Nunan, Linda M; Noble, Brenda; Le Groumellec, Marc; Lightner, Donald V

    2003-03-17

    A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.

  5. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-01-24

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

  6. 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.

  7. Baculovirus studies in new, indigenous lepidopteran cell lines.

    PubMed

    Pant, U; Sudeep, A B; Athawale, S S; Vipat, V C

    2002-01-01

    Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

  8. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    PubMed

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  9. 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.

  10. Genetic engineering: Baculoviruses as expression vectors. (Latest citations from the Life Sciences Collection data base). Published Search

    SciTech Connect

    Not Available

    1992-05-01

    The bibliography contains citations concerning the use of baculoviruses in genetic engineering. Baculoviruses produce large quantities of a specific gene. Topics include genetic replication, expression of selected genes in host cells, and protein expression using baculoviruses. Baculovirus introduction into mammals causing antibody expression is considered, and implications on vaccine programs are briefly discussed. (Contains a minimum of 112 citations and includes a subject term index and title list.)

  11. Influence of probiotic bacterium Bacillus cereus isolated from the gut of wild shrimp Penaeus monodon in turn as a potent growth promoter and immune enhancer in P. monodon.

    PubMed

    NavinChandran, Manohar; Iyapparaj, Palanisamy; Moovendhan, Subramanian; Ramasubburayan, Ramasamy; Prakash, Santhiyagu; Immanuel, Grasian; Palavesam, Arunachalam

    2014-01-01

    A probiotic bacterium isolated from the gut of wild shrimp Penaeus monodon rendered maximum antagonistic activity against shrimp pathogens and was capable of producing extracellular enzymes. The probiotic bacterium was identified as Bacillus cereus through 16S rRNA sequencing. The lyophilized B. cereus was supplemented with shrimp basal diet at four different concentrations (0.1–0.4%/100 g feed) in D1–D4 diets. The viability of probiotic bacterium in the test diets was evaluated during the study period at various time intervals. The viability ranged from 50.24 ± 1.42 to 180.34 ± 1.30 CFU/g in D1 to D3 diets on the 30th day, whereas it was slightly declined from 45.23 ± 1.30 to 169.13 ± 1.18 CFU/g during the 90th day of storage. A control diet (C), devoid of probiotic supplementation was also simultaneously prepared. During experimentation, P. monodon postlarvae (PL-15) were cultured in individual one tonne capacity FRP tanks in triplicates provided with equal amount of substratum (clay soil) and fed with these respective diets at ad libitum for 90 days. Survival was high (82.0 ± 1.60%) in D4 diet fed shrimp as against a low survival of 65.0 ± 1.33% displayed by control diet fed shrimp. Overall growth responses inferred that a maximum production of 10.45 ± 0.275 g, SGR of 4.40 ± 0.179% and a better FCR of 1.27 ± 0.081 were obtained in D4 diet fed shrimp. However, the water quality parameters showed nonsignificant (P > 0.05) variations among the control and the probiotic treated groups. The tested immunological parameters such as Total haemocyte count, phenoloxidase activity, respiratory burst activity, lysozyme activity, plasma protein concentration and bactericidal activity were higher in D4 diet fed P. monodon, when compared to that of other diets fed shrimp. It is therefore suggested that lyophilized probiotic B. cereus at a concentration of 0.4%/100 g feed was efficient in stimulating the growth and immunity in shrimp.

  12. 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...

  13. 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....

  14. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 po...

  15. 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

  16. Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp (Penaeus monodon).

    PubMed

    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.

  17. 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.

  18. 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

  19. Downregulation of a chitin deacetylase-like protein in response to baculovirus infection and its application for improving baculovirus infectivity.

    PubMed

    Jakubowska, Agata K; Caccia, Silvia; Gordon, Karl H; Ferré, Juan; Herrero, Salvador

    2010-03-01

    Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.

  20. Quantification of vitamin C requirements for juvenile shrimp (Penaeus monodon) using polyphosphorylated L-ascorbic acid.

    PubMed

    Chen, H Y; Chang, C F

    1994-10-01

    The vitamin C requirements of marine shrimp (Penaeus monodon) for optimal growth were evaluated in a 15-wk feeding trial using polyphosphorylated L-ascorbic acid (C2PP), a stable derivative of L-ascorbic acid (C1). Juvenile shrimp (0.55 +/- 0.01 g) were fed purified diets containing graded levels (0, 50, 100, 200 and 400 mg/kg diet) of supplemental C2PP or a high dosage of C1 (2500 mg/kg diet). Their weight gain, survival, feed efficiency and C1 storage in hepatopancreas and muscle were used to quantify the requirements. The growth of the shrimp fed the unsupplemented diet was significantly lower than those of the supplemented groups except the 100 mg C2PP/kg diet group. The C1 concentrations in muscle and hepatopancreas were greatest in shrimp fed 200 or 400 C2PP mg/kg diet. The dietary level required for juvenile P. monodon was found to be 209 mg/kg diet, based on the broken-line model analysis of weight gain, and was 220 mg/kg diet and 210 mg/kg diet, based on the analyses of C1 concentrations of hepatopancreas and muscle, respectively. Shrimp fed unsupplemented diet showed a significantly higher mortality than the supplemented groups. Most of the shrimp in the unsupplemented group that died suffered incomplete molting. No other overt deficiency sign was observed in any group.

  1. Behavioural and biochemical alterations in Penaeus monodon post-larvae diet-exposed to inorganic mercury.

    PubMed

    Harayashiki, Cyntia Ayumi Yokota; Reichelt-Brushett, Amanda J; Liu, Lei; Butcher, Paul

    2016-12-01

    Mercury is a metal naturally present in the environment with concentrations in aquatic systems increasing annually due to human activities. This represents a great concern mainly due to its high toxicity to organisms and consequences for human health. Most studies regarding the toxic effect of mercury have focussed on freshwater species using water as the exposure and uptake pathway. In contrast, the present study investigated the effects of dietary exposure of mercury to the marine crustacean Penaeus monodon post-larvae during 96 h to evaluate changes in behaviour (swimming activity and risk taken) and in biochemical biomarkers [acetylcholinesterase (AChE) and glutathione S-transferase (GST)]. Results showed a decrease in swimming activity with an increase in mercury exposure, but no changes were observed regarding the behavioural response 'risk taken'. Prawns from medium (0.56 μg g(-1)) and high (1.18 μg g(-1)) treatments had their GST activity reduced in relation to the beginning of experiment (time 0), while AChE activity was increased in the low (0.15 μg g(-1)) treatment in relation to time 0. In the present study, behaviour analysis were clearer than biochemical biomarkers and results might indicate P. monodon populations from a mercury contaminated environment might be at risk, since the behavioural alterations observed increases the risk of predation.

  2. Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.

    PubMed

    Zhao, C; Fu, M J; Qiu, L H

    2016-09-16

    Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.

  3. Protection of shrimp Penaeus monodon from WSSV infection using antisense constructs.

    PubMed

    Ahanger, Sajad; Sandaka, Supriyanka; Ananad, Deepika; Mani, Madhu K; Kondadhasula, Ravinder; Reddy, Chandra Sekhar; Marappan, Makesh; Valappil, Rajendran K; Majumdar, Kshitish C; Mishra, Rakesh K

    2014-02-01

    White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

  4. Estimation of the dietary riboflavin required to maximize tissue riboflavin concentration in juvenile shrimp (Penaeus monodon).

    PubMed

    Chen, H Y; Hwang, G

    1992-12-01

    The riboflavin requirements of marine shrimp (Penaeus monodon) were evaluated in a 15-wk feeding trial. Juvenile shrimp (initial mean weight, 0.13 +/- 0.05 g) were fed purified diets containing seven levels (0, 8, 12, 16, 20, 40 and 80 mg/kg diet) of supplemental riboflavin. There were no significant differences in weight gains, feed efficiency ratios and survival of shrimp over the dietary riboflavin range. The riboflavin concentrations in shrimp bodies increased with the increasing vitamin supplementation. Hemolymph (blood) glutathione reductase activity coefficient was not a sensitive and specific indicator of riboflavin status of the shrimp. The dietary riboflavin level required for P. monodon was found to be 22.3 mg/kg diet, based on the broken-line model analysis of body riboflavin concentrations. Shrimp fed unsupplemented diet (riboflavin concentration of 0.48 mg/kg diet) for 15 wk showed signs of deficiency: light coloration, irritability, protuberant cuticle at intersomites and short-head dwarfism.

  5. Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon.

    PubMed

    Leelatanawit, Rungnapa; Sittikankeaw, Kanchana; Yocawibun, Patchari; Klinbunga, Sirawut; Roytrakul, Sittiruk; Aoki, Takashi; Hirono, Ikuo; Menasveta, Piamsak

    2009-01-01

    Isolation and characterization of genes involving gonadal development are an initial step towards understanding reproductive maturation and sex determination of the giant tiger shrimp (Penaeus monodon). In the present study, 896 clones from the testis cDNA library were sequenced. A total of 606 ESTs (67.6%) significantly matched sequences in the GenBank (E-value <1e-04) whereas 290 ESTs (32.4%) were newly unidentified transcripts. The full length cDNA of genes functionally involved in testicular development including cyclophilin A (PMCYA), small ubiquitin-like modifier 1 (PMSUMO-1), ubiquitin conjugating enzyme E2, dynactin subunit 5, cell division cycle 2 (cdc2) and mitotic checkpoint BUB3 were discovered. In addition, Tra-2, a gene involving sex determination cascades, was successfully characterized by RACE-PCR and first reported in crustaceans. Expression analysis indicated that a homologue of low molecular weight neurofilament protein XNF-L (termed P. monodon testis-specific transcript 1, PMTST1; N=8 for each sex) was only expressed in testes but not ovaries. PMCYA, thyroid hormone receptor-associated protein complex 240 kDa component (Trap240), multiple inositol polyphosphate phosphatase 2 (MIPP2) and heat shock-related 70 kDa protein 2 (HSP70-2), but not PMSUMO-1, PMTra-2 and prohibitin2 were differentially expressed between ovaries and testes of P. monodon. Expression of PMTST1 was up-regulated but that of the remaining genes in testes of P. monodon broodstock was down-regulated after shrimp were molted (P<0.05). Significant reduction of PMSUMO-1 and increment of prohibitin2 transcripts in domesticated broodstock (P<0.05) suggested that these reproductively related genes may be used as biomarkers to evaluate reduced degrees of the reproductive maturation in domesticated P. monodon.

  6. Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

    PubMed

    Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

    2014-05-01

    Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions.

  7. Increase in Gut Microbiota after Immune Suppression in Baculovirus-infected Larvae

    PubMed Central

    Jakubowska, Agata K.; Vogel, Heiko; Herrero, Salvador

    2013-01-01

    Spodoptera exigua microarray was used to determine genes differentially expressed in S. exigua cells challenged with the species-specific baculovirus SeMNPV as well as with a generalist baculovirus, AcMNPV. Microarray results revealed that, in contrast to the host transcriptional shut-off that is expected during baculovirus infection, S. exigua cells showed a balanced number of up- and down-regulated genes during the first 36 hours following the infection. Many immune-related genes, including pattern recognition proteins, genes involved in signalling and immune pathways as well as immune effectors and genes coding for proteins involved in the melanization cascade were found to be down-regulated after baculovirus infection. The down-regulation of immune-related genes was confirmed in the larval gut. The expression of immune-related genes in the gut is known to affect the status of gut microorganisms, many of which are responsible for growth and development functions. We therefore asked whether the down-regulation that occurs after baculovirus infection affects the amount of gut microbiota. An increase in the gut bacterial load was observed and we hypothesize this to be as a consequence of viral infection. Subsequent experiments on virus performance in the presence and absence of gut microbiota revealed that gut bacteria enhanced baculovirus virulence, pathogenicity and dispersion. We discuss the host immune response processes and pathways affected by baculoviruses, as well as the role of gut microbiota in viral infection. PMID:23717206

  8. Baculovirus-based genome editing in primary cells.

    PubMed

    Mansouri, Maysam; Ehsaei, Zahra; Taylor, Verdon; Berger, Philipp

    2017-01-22

    Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).

  9. Baculovirus-mediated interferon alleviates dimethylnitrosamine-induced liver cirrhosis symptoms in a murine model.

    PubMed

    Nishibe, Y; Kaneko, H; Suzuki, H; Abe, T; Matsuura, Y; Takaku, H

    2008-07-01

    The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis induced by the mutagen dimethylnitrosamine. Intraperitoneal injection of AcMNPV induced an immune response. The baculovirus was taken up by the liver and spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat liver cirrhosis.

  10. The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

    PubMed

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-12-11

    Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

  11. The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

    PubMed

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

    2013-10-25

    Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

  12. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    DTIC Science & Technology

    1990-02-01

    T’ansfocl~on Baculovitus Genom . DMA Homoogos vSpodopt...a A~cob~n.ion ~ecob~naonlFrugipoida Coll VERtO Coll IPlatt" Assay 4 3 -GA). PA Gene Postive...localization was not determined. Baculovirus recombinant- genome DNA into SF-9 cells followed by homologous re- infected SF-9 cells were also...baculovirus genome and pAcYMI. The PA gene epitopes defined by a battery of monoclonal antibodies were was inserted into the baculovirus polyhcdrin gene under

  13. Baculovirus as a gene delivery vector for cartilage and bone tissue engineering.

    PubMed

    Lin, Chin-Yu; Lu, Chia-Hsin; Luo, Wen-Yi; Chang, Yu-Han; Sung, Li-Yu; Chiu, Hsin-Yi; Hu, Yu-Chen

    2010-06-01

    Baculovirus is an effective vector for gene delivery into various mammalian cells, including chondrocytes and mesenchymal stem cells, and has been employed for diverse applications. By gene delivery and expression of the growth factor, recombinant baculovirus has been shown to modulate the differentiation state of the cells and stimulates the production of extracellular matrix and tissue formation, hence repairing the damaged cartilage and bone in vivo. This article reviews the studies pertaining to the applications of baculovirus-mediated gene delivery in cartilage and bone tissue engineering and discusses recent progress, future applications and potential hurdles.

  14. 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

  15. 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

  16. Baculovirus-mediated expression of GPCRs in insect cells.

    PubMed

    Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

    2015-01-01

    G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations.

  17. Estimation of baculovirus titer based on viable cell size.

    PubMed

    Janakiraman, Vasantharajan; Forrest, William F; Seshagiri, Somasekar

    2006-01-01

    In this paper, a simple and rapid protocol for determination of baculovirus titers based on increasing viable insect cell size/diameter following virus infection is presented. There are different methods available for determining virus titers such as plaque assays end-point dilution, quantitative real-time polymerase chain reaction and flow cytometry. However, most of these methods are time consuming and labor intensive. The titer estimation method presented here can be completed in approximately 28 h from start to finish. In this method, the Vi-CELL (Beckman Coulter) was used to measure cell diameter change over a range of virus dilutions, following infection. The cell diameter change data were used to compute the virus titer using a statistical method called the method of moments that we have described previously.

  18. 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.

  19. 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

  20. Expression of bioactive recombinant bovine interferon-gamma using baculovirus.

    PubMed

    Gentilomi, Giovanna; Lelli, Rossella; D'Angelo, Mirella; Langella, Vincenzo; Monaco, Federica; Portanti, Ottavio; Luciani, Mirella; Mirasoli, Mara; Roda, Aldo; Zerbini, Marialuisa; Musiani, Monica

    2006-01-01

    The precise role of bovine interferon-gamma (BoIFN-gamma) in disease and therapy is still poorly defined. Clearly it is involved in defence against parasites, bacteria, viruses and possibly tumor cells. This paper reports the expression of BoIFN-gamma in a baculovirus system to generate a fully functional recombinant protein. Bovine interferon-gamma cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) encoding for a putative 166 amino acid protein (22KDa) was cloned and expressed into baculovirus transfer vector pBlueBac 4.5/V5 His. This vector was co-transfected with Autografa californica multiple nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugiperda cells (Sf9) and the recombinant virus, named AcBoIFN-gamma, was then recovered. Recombinant BoIFN-gamma (rBoIFN-gamma His) was accumulated in the serum-free medium of AcBoIFN-gamma-infected cells. The nickel affinity spin column purified rBoIFN-gamma His was shown to be a glycosylated 20-22 KDa protein as confirmed by SDS-PAGE glycan determination and showed antiviral activity in vitro against the bovine viral diarrhoea-mucosal disease virus (BVD/MD). The production of this bioactive rBoIFN-gamma His will allow us to explore this cytokine as a potential vaccine adjuvant or therapeutic agent for bovine diseases.

  1. 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

  2. Pathology Associated with White Spot Virus (WSV) Infection in Wild Broodstock of Tiger Prawns (Penaeus monodon)

    PubMed Central

    Kua, Beng Chu; Rashid, Noraziah Mat

    2012-01-01

    A total of six wild broodstocks of tiger prawns, Penaeus monodon, were found positive for White Spot Virus (WSV) with an IQ2000 detection kit. Using histopathology, the intranuclear inclusion of haemocyte due to WSV infection was observed in the epithelium cells of the antennal gland, stomach and gills. This result confirmed that the wild broodstocks were positive with WSV without showing any white spot. Additionally, histopathological examination also revealed an accumulation of haemocytes around the hepatopancreatic tubules resulting from bacterial infection. Encapsulation and nodule formation, as well as related necrosis, were also observed around the hepatopancreatic tubules infected with a metazoan parasite. Encysted tylocephalum larval cestodes were observed in the hepatopancreas, with haemocytic aggregation being observed around the infected tubules. These findings showed some bacterial and parasitic infections which, in addition to the viral infection itself, could contribute to the 80% mortality rate in wild broodstocks infected with WSV. PMID:24575228

  3. Pathology Associated with White Spot Virus (WSV) Infection in Wild Broodstock of Tiger Prawns (Penaeus monodon).

    PubMed

    Kua, Beng Chu; Rashid, Noraziah Mat

    2012-05-01

    A total of six wild broodstocks of tiger prawns, Penaeus monodon, were found positive for White Spot Virus (WSV) with an IQ2000 detection kit. Using histopathology, the intranuclear inclusion of haemocyte due to WSV infection was observed in the epithelium cells of the antennal gland, stomach and gills. This result confirmed that the wild broodstocks were positive with WSV without showing any white spot. Additionally, histopathological examination also revealed an accumulation of haemocytes around the hepatopancreatic tubules resulting from bacterial infection. Encapsulation and nodule formation, as well as related necrosis, were also observed around the hepatopancreatic tubules infected with a metazoan parasite. Encysted tylocephalum larval cestodes were observed in the hepatopancreas, with haemocytic aggregation being observed around the infected tubules. These findings showed some bacterial and parasitic infections which, in addition to the viral infection itself, could contribute to the 80% mortality rate in wild broodstocks infected with WSV.

  4. Dietary vitamin C and its derivatives affect immune responses in grass shrimp, Penaeus monodon.

    PubMed

    Lee, Min Hsien; Shiau, Shi Yen

    2002-02-01

    Effects of L-ascorbic acid (AA) and its four derivatives, namely L-ascorbyl-2-sulfate (C2S), L-ascorbyl-2-polyphosphate (C2PP), L-ascorbyl-2-monophosphate-Na (C2MP-Na) and L-ascorbyl-2-monophosphate-Mg (C2MP-Mg) on the immune responses of juvenile grass shrimp, Penaeus monodon, were studied. The vitamin C deprived diet together with diets supplemented with either adequate or high (five times adequate) levels of AA, C2S, C2PP, C2MP-Na and C2MP-Mg were each fed to triplicate groups of shrimp (mean initial weight: 0.37 +/- 0.01 g) for 8 weeks. Significantly (P<0.01) higher weight gain (WG), feed efficiency (FE), survival, total haemocyte count (THC), superoxide anion (O2) production ratio and phenoloxidase (PO) activity were observed in shrimp fed diets supplemented with adequate and high levels of ascorbate than shrimp fed the vitamin C deprived diet, regardless of the ascorbate source. Among the ascorbate sources, shrimp fed C2MP-Mg and C2PP containing diets had higher THC than shrimp fed AA, C2S and C2MP-Na containing diets, regardless of the supplementation level. Shrimp fed adequate levels of C2MP-Mg and C2PP and high levels of C2MP-Mg containing diets had higher O2 production ratios than shrimp fed AA and C2S containing diets. Shrimp fed adequate levels of C2MP-Mg and C2PP and high levels of C2PP containing diets had higher PO activity than shrimp fed AA, C2S and C2MP-Na containing diets. These data suggest that dietary ascorbate enhances immune responses in P. monodon and different ascorbate sources may affect the immune responses differently.

  5. 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

  6. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia.

    PubMed

    Tang, Kathy F J; Lightner, Donald V

    2006-06-01

    We found an infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequence within the shrimp genome in populations of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in samples of P. monodon from Madagascar and Tanzania. These sequences vary considerably (14 and 8%, respectively) from that of IHHNV found in association with viral epidemics. Laboratory bioassays were carried out with P. monodon and Litopenaeus vannamei to determine if either of these IHHNV-related sequences is infectious. We used juvenile and adult P. monodon containing the virus-related sequences from four geographic regions to generate inocula and tissues for feeding. Specific pathogen free P. monodon and L. vannamei were used as indicator shrimp. During the 2-4 week bioassays, none of the indicator shrimp showed signs of infection or disease. Results of both PCR assays and histological examination of the indicator shrimp were negative for IHHNV infection, indicating that the Africa type IHHNV-related sequences are not infectious. With the shrimp containing the Madagascar type IHHNV-related sequence (designated as type A), we performed genome walking at the 3' end of the virus-related sequence and found that this virus-related sequence is part of the P. monodon genome. A fragment of 1.9 kb flanking sequence was cloned and sequenced. Sequence analysis showed that this flanking sequence contains shrimp microsatellite DNA. Also, its translated amino acid sequence was highly similar to a retrotransposon. This result provides molecular evidence that the type A IHHNV-related sequence is shrimp DNA. This sequence was found in the P. monodon collected from Africa and Australia.

  7. First record of a parasitic septate gregarines (Apicomplexa: Sporozoea) in the shrimp Peneaus monodon in Sundarbans of West Bengal.

    PubMed

    Chakraborti, J; Bandyopadhyay, P K

    2010-04-01

    Investigations on the incidence of septate gregarines in shrimp have immense importance because of severe pathogenicity of the parasite. The septate gregarines infect the midgut of shrimp Peneaus monodon and severe infection disturbs the intestinal tissues. Mostly gregarines of the genus Nematopsis have been identified from cultured peneaid shrimp. It has worldwide in distribution. In India, gregarine parasites have so far been reported from penaeid shrimps of Bombay and Kerala. The species which was isolated from the midgut of shrimp Peneaus monodon collected from Kharibari area of Sunderbans. 9 out of 20 i.e. 45% of the randomly sampled hosts were found to be infected with a species of the genus Nematopsis. Different developmental stages including trophozoites, sporadins, and gametocysts of the Nematopsis sp. infecting the shrimp have been isolated. No correlations have been established between incidence of infection and environmental parameters.

  8. Dietary copper requirement of juvenile grass shrimp, Penaeus monodon, and effects on non-specific immune responses.

    PubMed

    Lee, Min-Hsien; Shiau, Shi-Yen

    2002-10-01

    An 8-week feeding trial was conducted to determine the dietary copper (Cu) requirement and its effect on the non-specific immune responses of juvenile grass shrimp, Penaeus monodon. Purified diets with seven levels (0, 10, 20, 30, 40, 80, 160 mg Cu kg diet(-1) of supplemental Cu were fed to P. monodon (mean initial weight 0.29 +/- 0.004 g). Each diet was fed to three replicate groups of shrimp. The rearing water contained 1.53 microg Cu 1(-1). Shrimp fed diets supplemented with 10 and 20 mg Cu kg diet(-1) had significantly (P < 0.01) greater weight gain, feed efficiency (FE) and protein efficiency ratio (PER) than those fed the unsupplemented control diet and diets supplemented with > or = 40 mg Cu kg diet(-1). Whole body Cu concentration in shrimp generally increased as dietary Cu supplementation increased. Total haemocyte count (THC) was higher in shrimp fed diets supplemented with 10-30 mg Cu kg diet(-1) than shrimp fed the unsupplemented control diet and diets supplemented with > or = 40 mg Cu kg diet(-1). Intracellular superoxide anion (O2-) production ratios were significantly higher in shrimp fed diets supplemented with 10-30 mg Cu kg diet(-1) than shrimp fed the diet supplemented with 160 mg Cu kg diet(-1). Analysis by polynomial regression of weight gain percent, FE and by linear regression of the whole-body Cu retention of shrimp indicated that the adequate dietary Cu concentration in growing P. monodon is about 15-21 mg Cu kg diet (-1). The immune indicators suggest that an adequate dietary Cu concentration for non-specific immune responses in P. monodon is about 10-30 mg Cu kg diet(-1).

  9. 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.

  10. Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon.

    PubMed

    Thammasorn, Thitiporn; Somchai, Parinyachat; Laosutthipong, Chaowanee; Jitrakorn, Sarocha; Wongtripop, Somjai; Thitamadee, Siripong; Withyachumnarnkul, Boonsirm; Saksmerprome, Vanvimon

    2013-10-01

    We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11-17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.

  11. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    PubMed Central

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  12. 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.

  13. 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

  14. Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Sánchez-Ramos, Ismael; Escribano, José M

    2013-06-10

    The promoter sequences of the encoding genes for the three most abundant hexamerins of the Lepidoptera Trichoplusia ni were isolated and cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-derived baculovirus expression vector. From the sequences analyzed, the DNA region driving the expression of the Basic juvenile hormone-suppressible protein 2 (BJHSP-2), denominated pB2, presented the highest promoter strength in the context of the baculovirus vector in Sf21 insect cells. This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter (polh), but surprisingly stopped at 48h post-infection. A mapping of pB2 essential promoter elements determined that a region of about 400bp, denominated pB29, retained and even increased the transcriptional activity with respect to the parental full-length sequence. Finally, several chimeric combinations of the insect-derived pB2 with the virus-derived conventional polh or p10 promoters were constructed and incorporated into an AcMNPV baculovirus vector. The pB2-p10 combination showed increased recombinant protein expression at early times post-infection and similar expression levels at very late times post-infection in Sf21 cells with respect to conventional late promoters. To the best of our knowledge, pB2 is the first promoter isolated from the Lepidoptera T. ni, the natural host of AcMNPV, to be assayed in a baculovirus expression vector.

  15. Detection of single and mixed covert baculovirus infections in eastern spruce budworm, Choristoneura fumiferana populations.

    PubMed

    Kemp, Elizabeth M; Woodward, David T; Cory, Jenny S

    2011-07-01

    We surveyed for covert baculovirus infections in the eastern spruce budworm, Choristoneura fumiferana (Clemens) and compared the prevalence of virus detected in a laboratory and a field population. DNA was extracted from budworm adults and then PCR with degenerate primers was used to identify individuals carrying baculovirus DNA. Multiplex PCR was then applied to the positive samples to distinguish between the multiple baculovirus types that could potentially be found in C. fumiferana populations. Covert infections were found in both the laboratory and the field population of C. fumiferana, although the frequency of infection and the composition of viruses found were very different. Overall 28% of insects from the laboratory population were positive for baculovirus DNA. Individual adults supported both single and mixed covert infections with CfMNPV plus CfDEFNPV, CfDEFNPV plus a GV and mixtures of all three viruses together. However, the majority of insects supported single virus infections, and surprisingly this virus was CfDEFNPV, a virus that is reported not to have per os activity in C. fumiferana larvae. Insects from field populations showed a very different pattern; 70.5% of individuals were baculovirus positive and all of these were positive for CfDEFNPV only.

  16. 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.

  17. 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

  18. Molecular biology of the baculovirus occlusion-derived virus envelope.

    PubMed

    Braunagel, Sharon C; Summers, Max D

    2007-10-01

    Study of the biology of the occlusion-derived virus (ODV) of the baculovirus Autographa californica nucleopolyhedrovirus provides opportunities to reveal new discoveries into the mechanism of several cellular pathways. The synchronous pulse of multiple ODV envelope proteins that integrate into the endoplasmic reticulum (ER) and traffic to the nuclear membranes on their way to the ODV envelope provide a unique tool to study the mechanisms of integral membrane protein trafficking from the ER to the outer and inner nuclear membrane. Studies of the formation of virus-induced, intranuclear membrane microvesicles provide insight on mechanisms that alter fluidity and regulate budding of the inner nuclear membrane. Since ODV is specially adapted for primary infection of the insect gut, studies of the structure and function of ODV envelope proteins reveals insights on the mechanism of viral invasion of the gut and this knowledge is fundamental for the development of new strategies for insect control. This review focuses on recent advances in understanding the source of the ODV envelope and the molecular events that sort and traffic integral membrane proteins from the ER to the ODV envelope. The composition of ODV is reviewed, however it is worth noting that the function of many ODV proteins are currently unknown.

  19. 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.

  20. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

    PubMed

    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  1. [Rapid and efficient expression of foreign genes in mammalian cells by baculovirus vectors].

    PubMed

    Cheng, Tong; Xu, Chen-Yu; Wang, Ying-Bin; Chen, Min; Wu, Ting; Xie, Xiao-Yan; Zhang, Jun; Xia, Ning-Shao

    2003-09-01

    The baculovirus insect cell expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, reports have described that recombinant baculoviruses can transduce a broad spectrum of primary and established mammalian cells, which shows the baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. In this report, we further research the modification of baculovirus vector and the way to deliver exogenous gene into mammalian cells. On the base of Bac-to-Bac baculovirus insect cell expression system, two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) were constructed containing different direction of CMV promoters which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells with relatively low efficiency. The culture supernatant of Sf9 cells which have been infected by the recombinant baculoviruses for four days were collected and the titers of the viruses in culture supernatant were determined by plaque assay on Sf9 cells. The HepG2 cells, an human hepatocellular carcinoma cell line, were directly incubated with the collected culture supernatant which contains the recombinant baculoviruses for 8 hours in 37 degrees C CO2 incubator (moi = 100). Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by flow cytometry (FCM) which detect the green fluorescence of individual cells. Results show that these two recombinant baculoviruses have similar gene-transfer and expression efficiency in HepG2 cells, which means the direction of CMV promoters has no effects on reporter gene expression. The optimal transduction conditions of incubating the mammalian cells with the culture supernatant of Sf9 cells infected by recombinant baculoviruses for four days were determined by FCM assay in HepG2 cells. The HepG2 cells inoculated in 24-well plate (5 x 10(4)/well) were incubated with the

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

    PubMed

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

    2014-07-20

    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.

  3. Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System.

    PubMed

    Fornwald, James A; Lu, Quinn; Boyce, Frederick M; Ames, Robert S

    2016-01-01

    BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. BacMams have become an integral part of the recombinant mammalian gene expression toolbox in research labs worldwide. Construction of transfer vectors is straightforward using basic molecular biology protocols. Virus generation is based on common methods used with the baculovirus insect cell expression system. BacMam transduction of mammalian cells requires minimal modifications to familiar cell culture methods. This chapter highlights the BacMam transfer vector pHTBV.

  4. Tissue-specific expression and regulation of the haemolymph clottable protein of tiger shrimp (Penaeus monodon).

    PubMed

    Yeh, Maw-Sheng; Huang, Chang-Jen; Cheng, Jin-Hwa; Tsai, Inn-Ho

    2007-08-01

    The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml(-1) to above 12 mg ml(-1) after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.

  5. Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants.

    PubMed

    Yogeeswaran, Aiyakani; Velmurugan, Subramanian; Punitha, Stanislas Mary Josephine; Babu, Mariavincent Michael; Selvaraj, Thangaswamy; Kumaran, Thangamani; Citarasu, Thavasimuthu

    2012-06-01

    To improve the immune response in tiger shrimp Penaeus monodon against WSSV infection, juveniles (350 ± 10 mg) were vaccinated with formalin-inactivated WSSV and fed with herbal immunostimulants. The methanolic extracts of herbal immunostimulants such as Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa, Withania somnifera and Zingiber officinalis were incorporated in formulated diets at different concentrations; 250 (ED(1)), 500 (ED(2)), 1000 (ED(3)) and 2000 (ED(4)) mg kg(-1) of feed and fed for 60 days after vaccination. After 30 and 60 days intervals of feeding, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps fed with control diets (C(1)) succumbed to death within 5 days after WSSV challenge, when no vaccination and immunostimulations were given. The other control groups (C(2) and C(3)) had slight improvements in all parameters including survival. The percentage survival was significantly (P < 0.05) increased to 30, 50 and 60% in the ED(2), ED(3) and ED(4) diets respectively after 60 days challenging. The better haematological, biochemical and immunological parameters were also found in the herbal extracts supplemented diets fed vaccinated shrimps. The present study revealed that the combined effect of immunostimulation and vaccination helped to boost the immune system against WSSV infection and hence this application can be adopted for shrimp culture.

  6. Highly Directional Sonar Beam of Narwhals (Monodon monoceros) Measured with a Vertical 16 Hydrophone Array.

    PubMed

    Koblitz, Jens C; Stilz, Peter; Rasmussen, Marianne H; Laidre, Kristin L

    2016-01-01

    Recordings of narwhal (Monodon monoceros) echolocation signals were made using a linear 16 hydrophone array in the pack ice of Baffin Bay, West Greenland in 2013 at eleven sites. An average -3 dB beam width of 5.0° makes the narwhal click the most directional biosonar signal reported for any species to date. The beam shows a dorsal-ventral asymmetry with a narrower beam above the beam axis. This may be an evolutionary advantage for toothed whales to reduce echoes from the water surface or sea ice surface. Source level measurements show narwhal click intensities of up to 222 dB pp re 1 μPa, with a mean apparent source level of 215 dB pp re 1 μPa. During ascents and descents the narwhals perform scanning in the vertical plane with their sonar beam. This study provides valuable information for reference sonar parameters of narwhals and for the use of acoustic monitoring in the Arctic.

  7. Ultrastructural effect of gamma radiation on grass shrimps ( Penaeus monodon fabricius)

    NASA Astrophysics Data System (ADS)

    Perng, Fang-Shiow; Yang, Jui-Sen

    The effect of ionizing radiation (2, 5, and 10 KGy of gamma rays) on muscle ultrastructure of grass shrimps ( Penaeus monodon Fabricius) tails at ambient or frozen (-18°C) temperature was investigated by transmission electron microscopy (TEM). No significant change was found in muscle ultrastructure of shrimp meat irradiated at doses of 2 or 5 KGy and then stored at 4°C for 0 and 8 days. However, in shrimps which were irradiated at ambient temperature at a dose of 10 KGy, the margin of A-band of the myofibrils in longitudinal sections exhibited an irregular zig-zag pattern. In cross section some myosin filaments in the A-band regions were missing. A possible interpretation of these results would be that 10 KGy gamma irradiation dose at ambient temperature depolymerized myosin in the A-band from the tail of thick filament. Actin damage was also observed in some cross sections. Irradiation damage of sarcoplasmic membranes appeared in the specimens irradiated at a dose of 10KGy at ambient temperature. Shrimps frozen at -18°C and then irradiated with 10 KGy did not exhibit filament damage. Freezing apparently protected shrimp meat from irradiation injury.

  8. New insights into the spermatogenesis of the black tiger prawn, Penaeus monodon.

    PubMed

    Feng, Tianyi; Paterson, Brian; Johnston, Stephen

    2017-02-05

    This study reports a comprehensive description of penaeid spermatogenesis (Penaeus monodon) by light and transmission electron microscopy. A conspicuous characteristic of spermatocytogenesis was a ring-like structure with high electron-density adjacent to the nucleus of a primary spermatocyte. During the spermiogenesis from stage I (StI) to stage VI spermatid (StVI), the formation of the acrosome and decondensation of the nucleus were the most notable morphological transformations. StIs were small and compact and they were contained in the syncytia. In the cytoplasm of StII, mitochondrion-like bodies (MLB) participated the extension of perinuclear multi-layered lamellae. The association of MLBs and endoplasmic reticula appeared to contribute to the formation of small cytoplasmic pre-acrosomal vesicles (PV) which coalesced into an acrosomal chamber (AC) at the periphery of StIII. A dense anterior acrosomal body (AB) was formed in the enlarged AC in StIV. The nuclear envelope became disintegrated in StV. At last, an AB-derived spiky acrosome was emerged from AC in StVI. Sperm nuclei became increasingly decondensed during the entire process of spermiogenesis and the nuclear components in the testicular spermatozoa appeared to only contain chains of DNA and nucleosome-contained chromatin.

  9. Highly Directional Sonar Beam of Narwhals (Monodon monoceros) Measured with a Vertical 16 Hydrophone Array

    PubMed Central

    Koblitz, Jens C.; Stilz, Peter; Rasmussen, Marianne H.; Laidre, Kristin L.

    2016-01-01

    Recordings of narwhal (Monodon monoceros) echolocation signals were made using a linear 16 hydrophone array in the pack ice of Baffin Bay, West Greenland in 2013 at eleven sites. An average -3 dB beam width of 5.0° makes the narwhal click the most directional biosonar signal reported for any species to date. The beam shows a dorsal-ventral asymmetry with a narrower beam above the beam axis. This may be an evolutionary advantage for toothed whales to reduce echoes from the water surface or sea ice surface. Source level measurements show narwhal click intensities of up to 222 dB pp re 1 μPa, with a mean apparent source level of 215 dB pp re 1 μPa. During ascents and descents the narwhals perform scanning in the vertical plane with their sonar beam. This study provides valuable information for reference sonar parameters of narwhals and for the use of acoustic monitoring in the Arctic. PMID:27828956

  10. Enhanced wound healing activity of desert locust (Schistocerca gregaria) vs. shrimp (Penaeus monodon) chitosan based scaffolds.

    PubMed

    Marei, Narguess H; El-Mazny, W; El-Shaer, Aida; Zaki, Kareem Dorri; Hussein, Zahra S; Abd-El-Samie, Emtithal M

    2017-04-01

    Chitosan (CS) has received great attention in tissue engineering, especially in wound healing acceleration. In this study, chitin was isolated from desert locust (Schistocerca gregaria) and shrimp (Penaeus monodon) then deacetylated to chitosan. Then, chitosan was characterized by degree of deacetylation (DD), molecular weight (M.Wt), swelling index (SI), Fourier transform infrared (FTIR) and X ray diffraction (XRD). The chitosan was then casted into 2D scaffolds and was pictured using scanning electron microscope (SEM). In a comparative study, primary cell cultures of neonatal (1-2day old) mice skin tissue, supplemented with 10% fetal calf serum, were seeded onto locust chitosan based scaffolds (LCSBS) and shrimp chitosan based scaffold (SCSBS). Their attachment percentage was determined after 1h. The cell proliferation rate was tested for 5days on LCSBS and SCSBS. Wound healing activity progress of LCSBS and SCSBS was tested in vivo using histopathology, and results revealed that seeded and unseeded LCSBS accelerated healing in contrast to SCSBS. The data demonstrated that LCSBS shows a high degree of biocompatibility in vivo. These results suggest that LCSBS is a potential substitute for the development of low cost implantable materials to accelerate wound healing.

  11. 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

  12. 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

  13. Vitamin E requirements of juvenile grass shrimp, Penaeus monodon, and effects on non-specific immune responses.

    PubMed

    Lee, Min-Hsien; Shiau, Shi-Yen

    2004-04-01

    A feeding trial was conducted to determine the dietary vitamin E (DL-alpha-tocopheryl acetate, dl-alpha-TOA) requirement and its effect on the non-specific immune responses of juvenile grass shrimp, Penaeus monodon. Purified diets with eight levels (0, 25, 50, 75, 100, 150, 200, 400 mg vitamin E kg diet-1) of supplemental dl-alpha-TOA were fed to P. monodon (mean initial weight 0.29 +/- 0.01 g) for eight weeks. Each diet was fed to three replicate groups of shrimp. Weight gains and total haemocyte count (THC) were higher (P < 0.05) in shrimp fed diets supplemented with 75 and 100 mg vitamin E kg diet-1 than in shrimp fed diets supplemented with monodon and that 179 mg vitamin E kg diet-1 is required to maximise tissue vitamin E concentration.

  14. Solvent extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger shrimp Penaeus monodon.

    PubMed

    Kanjana, Kulwadee; Radtanatip, Tawut; Asuvapongpatana, Somluk; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2011-01-01

    Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.

  15. 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.

  16. 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.

  17. 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.

  18. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    PubMed

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  19. 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.

  20. 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

  1. Complex dynamics of defective interfering baculoviruses during serial passage in insect cells.

    PubMed

    Zwart, Mark P; Pijlman, Gorben P; Sardanyés, Josep; Duarte, Jorge; Januário, Cristina; Elena, Santiago F

    2013-03-01

    Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.

  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. Enhanced Baculovirus-Mediated Transduction of Human Cancer Cells by Tumor-Homing Peptides

    PubMed Central

    Mäkelä, Anna R.; Matilainen, Heli; White, Daniel J.; Ruoslahti, Erkki; Oker-Blom, Christian

    2006-01-01

    Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy. PMID:16775347

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

    PubMed

    Toth, Ann M; Geisler, Christoph; Aumiller, Jared J; Jarvis, Donald L

    2011-12-01

    In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

  11. Suppression subtractive hybridization (SSH) for isolation and characterization of genes related to testicular development in the giant tiger shrimp Penaeus monodon.

    PubMed

    Leelatanawit, Rungnapa; Klinbunga, Sirawut; Aoki, Takashi; Hirono, Ikuo; Valyasevi, Rudd; Menasveta, Piamsak

    2008-11-30

    Suppression subtractive hybridization (SSH) cDNA libraries of the giant tiger shrimp, Penaeus monodon, were constructed. In total, 178 and 187 clones from the forward and reverse SSH libraries, respectively, of P. monodon were unidirectionally sequenced. From these, 37.1% and 53.5% Expressed Sequence Tags (ESTs) significantly matched known genes (E-value < 1e-04). Three isoforms of P. monodon progestin membrane receptor component 1: PM-PGMRC1-s (1980 bp), PM-PGMRC1-m (2848 bp), and PM-PGMRC1-l (2971 bp), with an identical ORF of 573 bp corresponding to a deduced polypeptide of 190 amino acids, were successfully identified by RACE-PCR. Interestingly, PMPGMRC1 showed a greater expression level in testes of juvenile than broodstock P. monodon (P < 0.05). Dopamine administration (10(-6) mol/shrimp) resulted in up-regulation of PMPGMRC1 in testes of juveniles at 3 hrs post treatment (P < 0.05), but had no effect on PM-Dmc1 (P > 0.05).

  12. 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

  13. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

    PubMed Central

    2013-01-01

    Background Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are

  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. Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening.

    PubMed

    Xu, Xiaodong; Chen, Yuanrong; Zhao, Yu; Liu, Xiaofen; Dong, Beitao; Jones, Ian M; Chen, Hongying

    2013-01-01

    In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

  16. Homologous genetic recombination in the yellow head complex of nidoviruses infecting Penaeus monodon shrimp.

    PubMed

    Wijegoonawardane, Priyanjalie K M; Sittidilokratna, Nusra; Petchampai, Natthida; Cowley, Jeff A; Gudkovs, Nicholas; Walker, Peter J

    2009-07-20

    Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp. It is one of six known genotypes in the yellow head complex of nidoviruses which also includes mildly pathogenic gill-associated virus (GAV, genotype 2) and four other genotypes (genotypes 3-6) that have been detected only in healthy shrimp. In this study, comparative phylogenetic analyses conducted on replicase- (ORF1b) and glycoprotein- (ORF3) gene amplicons identified 10 putative natural recombinants amongst 28 viruses representing all six genotypes from across the Indo-Pacific region. The approximately 4.6 kb genomic region spanning the two amplicons was sequenced for three putative recombinant viruses from Vietnam (genotype 3/5), the Philippines (genotype 5/2) and Indonesia (genotype 3/2). SimPlot analysis using these and representative parental virus sequences confirmed that each was a recombinant genotype and identified a recombination hotspot in a region just upstream of the ORF1b C-terminus. Maximum-likelihood breakpoint analysis predicted identical crossover positions in the Vietnamese and Indonesian recombinants, and a crossover position 12 nt upstream in the Philippine recombinant. Homologous genetic recombination in the same genome region was also demonstrated in recombinants generated experimentally in shrimp co-infected with YHV and GAV. The high frequency with which natural recombinants were identified indicates that genetic exchange amongst genotypes is occurring commonly in Asia and playing a significant role in expanding the genetic diversity in the yellow head complex. This is the first evidence of genetic recombination in viruses infecting crustaceans and has significant implications for the pathogenesis of infection and diagnosis of these newly emerging invertebrate pathogens.

  17. 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.

  18. Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

    PubMed Central

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

    2013-01-01

    Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities. PMID:23577162

  19. Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

    PubMed

    Yeh, M S; Huang, C J; Leu, J H; Lee, Y C; Tsai, I H

    1999-12-01

    To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

  20. Immunomodulation by dietary beta-1, 3-glucan in the brooders of the black tiger shrimp Penaeus monodon.

    PubMed

    Chang, C F; Chen, H Y; Su, M S; Liao, I C

    2000-08-01

    The present study evaluated the effectiveness of beta-1,3-glucan derived from Schizophyllum commune in enhancing shrimp survival as well as haemocyte phagocytosis and superoxide anion production in brooder Penaeus monodon. Pond-reared P. monodon adults (135 +/- 25 g) stocked in outdoor or indoor tanks were fed either a test diet containing beta-1,3-glucan (2.0 g kg(-1) or a glucan-free control diet for 40 days. Their survival was compared. The brooders reared in indoor tanks were analysed at days 0, 1, 3, 6, 12, 24, 30 and 40 for their haemocyte phagocytic activity and superoxide anion production. The results showed that regardless of indoor or outdoor rearing the survival rate of shrimp fed the glucan diet was significantly higher (P<0.001) than that of the control group. The brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production when glucan was administered in their diets. The immunostimulatory enhancement peaked at day 24 after starting the dietary exposure and subsequently decreased to the pre-feeding level at the end of the 40 days feeding trial.

  1. Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

    PubMed

    Nunan, Linda M; Poulos, Bonnie; Redman, Rita; Le Groumellec, Marc; Lightner, Donald V

    2003-01-22

    Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

  2. Seasonal incidence of protozoan parasites of the black tiger shrimp (Penaeus monodon) of Sundarbans, West Bengal, India.

    PubMed

    Chakraborti, Jayati; Bandyapadhyay, Probir K

    2011-06-01

    There is a delicate balance between the host, pathogen and environment. Aquatic organisms, including shellfish, respond directly to climatic changes in their biological environment as their metabolic processes are influenced by temperature, salinity, and oxygen levels. Certain environmental conditions are more conducive to diseases than others among which water temperature is significantly associated with disease outbreak. The present study showed that Peneaus monodon of Sundarbans serve as a host for many protozoan parasites and epibionts including ciliates, gregarines and microsporidia. The protozoan parasites also require a particular environmental condition for their maximum growth and survival. The intensity of infection significantly increases with rise in temperature (P < 0.05) following a definite trend but no significant relationship between infection rate of ciliates and pH of water. In case of gregarine parasites significance (P < 0.05) exists among infection rate and temperature as well as pH of the farm water. Microsporidian parasites do not follow any significant seasonal trend in infecting the host P. monodon.

  3. Experimental exposure of Artemia to hepatopancreatic parvo-like virus and subsequent transmission to post-larvae of Penaeus monodon.

    PubMed

    Sivakumar, V K; Sarathi, M; Venkatesan, C; Sivaraj, A; Hameed, A S Sahul

    2009-11-01

    The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvo-like virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV. All the five developmental stages of Artemia were challenged with HPV both by immersion and oral infection routes. The viral infectivity to Artemia was studied by PCR but not much difference in mortality between control and challenge groups were observed. To confirm the vector status of Artemia for HPV, the HPV exposed Artemia were fed to postlarval forms of Penaeus monodon. Post-larvae of P. monodon were fed with HPV exposed Artemia and could get infected upon feeding on them. Mortality was observed in the post-larvae, which were fed with HPV exposed Artemia, and whereas no mortality was observed in post-larvae fed with Artemia not exposed to HPV and these post-larvae were PCR negative for HPV, as well. Results of this experiment suggest that Artemia might be a possible horizontal transmission pathway for HPV. Further research however is required with histology, immunohistochemistry and transmission electron microcopy to determine whether the Artemia are actually infected with this virus or whether they are simply mechanical carriers. This will enable us to understand better whether Artemia is a carrier of this virus and if so the mechanism involved.

  4. Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV-vaccines and live white spot syndrome virus.

    PubMed

    Kulkarni, Amod D; Kiron, Viswanath; Rombout, Jan H W M; Brinchmann, Monica F; Fernandes, Jorge M O; Sudheer, Naduvilamuriparampu S; Singh, Bright I S

    2014-07-01

    White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate 'vaccines', WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.

  5. 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

  6. 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...

  7. Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system.

    PubMed

    Mon, Hiroaki; Lee, Jaeman; Fukushima, Mai; Nagata, Yudai; Fujii, Mie; Xu, Jian; Nishi, Oumi; Iiyama, Kazuhiro; Kusakabe, Takahiro

    2013-08-01

    Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca(2+) and Sr(2+) were able to replace Mg(2+) and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.

  8. Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

    PubMed

    Graham, L A; Bendena, W G; Walker, V K

    1996-02-01

    The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

  9. Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm.

    PubMed

    Usami, Akihiro; Ishiyama, Seiji; Enomoto, Chiaki; Okazaki, Hironobu; Higuchi, Keiko; Ikeda, Mashahiro; Yamamoto, Takeshi; Sugai, Mutsumi; Ishikawa, Yukiko; Hosaka, Yumiko; Koyama, Teruyuki; Tobita, Yoneko; Ebihara, Syoko; Mochizuki, Toshiko; Asano, Yoshimi; Nagaya, Hidekazu

    2011-02-01

    Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.

  10. Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.

    PubMed

    Argyle, D J; Harris, M; Lawrence, C; McBride, K; Barron, R; McGillivray, C; Onions, D E

    1998-07-08

    We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.

  11. Baculovirus infection triggers a positive phototactic response in caterpillars to induce 'tree-top' disease.

    PubMed

    van Houte, Stineke; van Oers, Monique M; Han, Yue; Vlak, Just M; Ros, Vera I D

    2014-12-01

    Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions ('tree-top' disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.

  12. Display of a Maize cDNA library on baculovirus infected insect cells

    PubMed Central

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-01-01

    Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence. PMID:18700036

  13. Dengue Type-2 Virus Envelope Protein Made Using Recombinant Baculovirus Protects Mice Against Virus Challenge

    DTIC Science & Technology

    1994-01-01

    the envelope (E) glycoprotein of dengue 2 virus was cloned into baculovirus (IAutographa californical nuclear polyhedrosis virus, AcNPV). The...polyclonal, anti- dengue type 2 antibody and a dengue type 2-specific, neutralizing monoclonal antibody. Balb/c mice immunized with the recombinant...antigen produced only non-neutralizing antibody against dengue 2 virus but were partially protected against morbidity and mortality after

  14. The effect of cell line, phylogenetics and medium on baculovirus budded virus yield and quality.

    PubMed

    Matindoost, Leila; Hu, Hao; Chan, Leslie C L; Nielsen, Lars K; Reid, Steven

    2014-01-01

    The performance of bioprocesses involving baculoviruses largely depends on an efficient infection of cells by concentrated budded virus (BV) inoculums. Baculovirus expression vector systems have been established using Autographa californica nucleopolyhedrovirus (AcMNPV), a group I NPV that displays rapid virus kinetics, whereas bioprocesses using group II baculovirus-based biopesticides such as Helicoverpa armigera nucleopolyhedrovirus (HearNPV) have the limitation of low levels of BV, as these viruses often display poor BV production kinetics. In this study, the effect of key parameters involved in the quality of progeny virions, including cell line, virus phylogenetics and medium, on viral DNA replication, virus trafficking to the extracellular environment, and the yield of recombinant protein or polyhedra were investigated in synchronous infections of HearNPV and AcMNPV. HearNPV showed higher vDNA replication in its optimum medium, SF900III, when compared to AcMNPV, but both viruses had similar specific extracellular virion content. However, the ratio of AcMNPV extracellular virions to the total number of progeny virions produced was higher, and their quality was tenfold higher than that of HearNPV extracellular virions. The results of infection of two different cell lines, High Five and Sf9, with AcMNPV, along with HearNPV infection of HzAM1 cells in three different media, suggest that the host cells and the nutritional state of the medium as well as the phylogenetics of the virus affect the BV yields produced by different baculovirus/cell line/medium combinations.

  15. 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.

  16. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    PubMed Central

    Chateigner, Aurélien; Bézier, Annie; Labrousse, Carole; Jiolle, Davy; Barbe, Valérie; Herniou, Elisabeth A.

    2015-01-01

    Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential. PMID:26198241

  17. Evaluation of Cre Recombinase Delivery in Mammalian Cells using Baculovirus Infection

    PubMed Central

    Erbs, Eric; Pradhan, Amynah A.; Matifas, Audrey; Kieffer, Brigitte L.; Massotte, Dominique

    2013-01-01

    In vivo conditional knock-out of a protein is a method of choice to decipher its biological function. It can be achieved by encoding the cre-recombinase on a recombinant virus to exert spatio-temporal control of its expression and enzymatic activity and, subsequently, of the target gene deletion. Recombinant baculoviruses have been successfully used to express a wide range of proteins in insect cells. More recently, their potential to infect mammalian cells has been addressed but, so far, their ability to yield a conditional knock-out as a result of efficient in vivo cre-recombinase gene delivery has not been examined. Cre-recombinase fused to the green fluorescent protein was cloned under the control of the CAG promoter in a recombinant Autographa californica baculovirus expressing the vesicular stomatitis virus envelope G protein for increased mammalian cell infection. Gene delivery was evaluated in vitro in mammalian cells, neuroblastoma and mouse primary neuronal cultures as well as in vivo in the mouse brain. Infection with adeno-associated viruses encoding the cre-recombinase fused to the green fluorescent protein was performed as a positive control. Our results indicate that baculovirus infection leads to functional cre-recombinase expression in non-neuronal and neuroblastoma cell lines but not in mouse primary neuronal cultures or brain. PMID:23732834

  18. Domain 2 of a Kazal serine proteinase inhibitor SPIPm2 from Penaeus monodon possesses antiviral activity against WSSV.

    PubMed

    Visetnan, Suwattana; Donpudsa, Suchao; Supungul, Premruethai; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien

    2014-12-01

    A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2.

  19. Copper accumulation and transport in a marine food chain composed of Platymonas subcordiformis, brachionus plicatilis and Penaeus monodon

    NASA Astrophysics Data System (ADS)

    Cai, A.-Gen; Chen, Wei-Qi; Li, Wen-Quan

    1997-09-01

    Accumulation, transport and toxicity of Cu in the food chain consisting of Platymonas subcordiformis, Brachionus plicatilis and Penaeus monodon were studied. Effects of Cu on the growth of organisms in the food chain were investigated and the inhibiting effect concentration (EC50) of Cu was then determined according to the dynamics of the relative number of cells or total individuals of organisms, expressed in percentages with reference to the controlled system, under different culture conditions. On the basis of the variations in accumulation and percentages of accumulation of Cu in the biological phase, the relationship between the accumulation of Cu in organisms and its toxicity was analyzed and the main approach for determining the transport of Cu in the food chain was then discussed.

  20. Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon).

    PubMed Central

    Fuerst, J A; Sambhi, S K; Paynter, J L; Hawkins, J A; Atherton, J G

    1991-01-01

    During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included. Images PMID:1781677

  1. 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

  2. Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon).

    PubMed

    Sengprasert, Panjana; Amparyup, Piti; Tassanakajorn, Anchalee; Wongpanya, Ratree

    2015-06-01

    Calmodulin (CaM), a ubiquitous intracellular calcium (Ca(2+)) sensor in all eukaryotic cells, is one of the well-known signaling proteins. Previously, CaM gene has shown a high transcriptional level in hemocyte of the pathogen infected shrimp, suggesting that shrimp CaM does not only regulate Ca(2+) metabolism, but is also involved in immune response cascade. In the present study, the CaM gene of shrimp Penaeus monodon was identified and the recombinant P.monodon CaM (rPmCaM) was produced and biochemically characterized. The identification of CaM-binding proteins was also performed. The PmCaM cDNA consisted of an open reading frame of 447 bp encoding for 149 amino acid residues with a calculated mass of 16,810 Da and an isoelectric point of 4.09. Tissue distribution showed that the PmCaM transcript was expressed in all examined tissues. The results of gel mobility shift assay, circular dichroism spectroscopy and fluorescence spectroscopy all confirmed that the conformational changes of the rPmCaM were observed after the calcium binding. According to the gene silencing of PmCaM transcript levels, the shrimp's susceptibility to pathogenic Vibrio harveyi infection increased in comparison with that of the control groups. Protein pull-down assay and LC-MS/MS analysis were performed to identify rPmCaM-binding proteins involved in shrimp immune responses and transglutaminase, elongation factor 1-alpha, elongation factor 2 and actin were found. However, by computational analysis, only the first three proteins contained CaM-binding domain. These findings suggested that PmCaM may play an important role in regulation of shrimp immune system.

  3. Anti-white spot syndrome virus activity of Ceriops tagal aqueous extract in giant tiger shrimp Penaeus monodon.

    PubMed

    Sudheer, N S; Philip, Rosamma; Bright Singh, I S

    2012-09-01

    White spot syndrome virus (WSSV), the most contagious pathogen of cultured shrimp, causes mass mortality, leading to huge economic loss to the shrimp industry. The lack of effective therapeutic or prophylactic measures has aggravated the situation, necessitating the development of antiviral agents. With this objective, the antiviral activity in the aqueous extract of a mangrove plant Ceriops tagal in Penaeus monodon was evaluated. The Ceriops tagal aqueous extract (CTAE) was non-toxic to shrimps at 50 mg/ml when injected intramuscularly at a dosage of 10 μL/animal (0.5 mg/animal) and showed a protective effect against WSSV at 30 mg/ml when mixed with WSSV suspension at a 1:1 ratio. When the extract was administered along with the diet and the animals were challenged orally, there was a dose-dependent increase in survival, culminating in 100 % survival at a concentration of 500 mg/kg body weight/day. Neither hypertrophied nuclei nor the viral envelope protein VP28 could be demonstrated in surviving shrimps using histology and indirect immunofluorescence histochemistry (IIFH), respectively. To elucidate the mode of action, the temporal expression of WSSV genes and shrimp immune genes, including antimicrobial peptides, was attempted. None of the viral genes were found to be expressed in shrimps that were fed with the extract and challenged or in those that were administered CTAE-exposed WSSV. The overall results suggest that the aqueous extract from C. tagal can protect P. monodon from white spot syndrome virus infection.

  4. Poly-β-hydroxybutyrate (PHB) accumulating Bacillus spp. improve the survival, growth and robustness of Penaeus monodon (Fabricius, 1798) postlarvae.

    PubMed

    Laranja, Joseph Leopoldo Q; Ludevese-Pascual, Gladys L; Amar, Edgar C; Sorgeloos, Patrick; Bossier, Peter; De Schryver, Peter

    2014-10-10

    Low larval survival resulting from suboptimal culture conditions and luminous vibriosis poses a major problem for the larviculture of penaeid shrimp. In this study, a poly-β-hydroxybutyrate (PHB) accumulating mixed bacterial culture (mBC; 48.5% PHB on cell dry weight) and two PHB accumulating bacterial isolates, Bacillus sp. JL47 (54.7% PHB on cell dry weight) and Bacillus sp. JL1 (45.5% PHB on cell dry weight), were obtained from a Philippine shrimp culture pond and investigated for their capacity to improve growth, survival and robustness of Penaeus monodon postlarvae (PL). Shrimp PL1 and shrimp PL30 were provided with the PHB containing bacterial cultures in the feed for 30 days followed by, respectively, a challenge with pathogenic Vibrio campbellii and exposure to a lethal dose of ammonia. Prior to the pathogenic challenge or ammonia stress, growth and survival were higher for shrimp receiving the PHB accumulating bacteria as compared to shrimp receiving diets without bacterial additions. After exposure to the pathogenic challenge the shrimp fed PHB accumulating bacteria showed a higher survival as compared to non-treated shrimp, suggesting an increase in robustness for the shrimp. Similar effects were observed when shrimp PL30 were provided with the PHB accumulating bacterial cultures during a challenge with pathogenic V. campbellii through the water. The survival of shrimp exposed to lethal ammonia stress showed no significant difference between PHB accumulating bacteria-fed shrimp and non-PHB treated shrimp. The data illustrate that bacilli capable of accumulating PHB can provide beneficial effects to P. monodon post-larvae during culture in terms of growth performance, survival and resistance against pathogenic infection and ammonia stress. Further investigations are required to verify the PHB effect of the bacterial cultures on the shrimp.

  5. Baculovirus Superinfection: A Probable Restriction Factor on the Surface Display of Proteins for Library Screening

    PubMed Central

    Xu, Xiaodong; Chen, Yuanrong; Zhao, Yu; Liu, Xiaofen; Dong, Beitao; Jones, Ian M.; Chen, Hongying

    2013-01-01

    In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides. PMID:23365677

  6. Two new anti-apoptotic proteins of white spot syndrome virus that bind to an effector caspase (PmCasp) of the giant tiger shrimp Penaeus (Penaeus) monodon.

    PubMed

    Lertwimol, Tareerat; Sangsuriya, Pakkakul; Phiwsaiya, Kornsunee; Senapin, Saengchan; Phongdara, Amornrat; Boonchird, Chuenchit; Flegel, Timothy W

    2014-05-01

    White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55-214) of Penaeus monodon caspase (PmCasp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length PmCasp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with PmCasp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon (Pm caspase) did not block apoptosis induced by PmCasp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon.

  7. Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH).

    PubMed

    Preechaphol, Rachanimuk; Klinbunga, Sirawut; Khamnamtong, Bavornlak; Menasveta, Piamsak

    2010-10-01

    Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0%) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein Mprecursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thrcheckpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

  8. A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response.

    PubMed

    Karuppannan, Anbu K; Qiang, Jia; Chang, C C; Kwang, Jimmy

    2013-11-04

    Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.

  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.

  10. Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

    PubMed

    Garretson, Tyler A; McCoy, Jason C; Cheng, Xiao-Wen

    2016-11-01

    Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking

  11. Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus.

    PubMed

    Peng, Ke; van Oers, Monique M; Hu, Zhihong; van Lent, Jan W M; Vlak, Just M

    2010-09-01

    Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% beta-mercaptoethanol with heating at 50 degrees C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.

  12. 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.

  13. MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

    PubMed

    Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

    2013-06-01

    MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

  14. In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter

    PubMed Central

    Luz-Madrigal, Agustín; Clapp, Carmen; Aranda, Jorge; Vaca, Luis

    2007-01-01

    Background Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed in vitro in different mammalian cell lines and in vivo in adult rat retinal vasculature. Results BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, n = 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. In vivo transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, n = 10) in blood vessels, confirming the endothelial phenotype of the transduced cells. Conclusion Taken together, our results indicate that the restricted expression in endothelial cells

  15. 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%.

  16. Production and purification of recombinant enteropeptidase expressed in an insect-baculovirus cell system.

    PubMed

    Azhar, Mahammad; Somashekhar, R

    2015-01-01

    Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.

  17. Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Ojala, Kirsi; Michel, Patrik O; Vuento, Matti; Oker-Blom, Christian

    2002-12-01

    Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

  18. 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

  19. Baculovirus envelope protein ODV-E66 is a novel chondroitinase with distinct substrate specificity.

    PubMed

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

    2011-08-19

    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.

  20. Baculovirus vector as an avian influenza vaccine: hemagglutinin expression and presentation augment the vaccine immunogenicity.

    PubMed

    Chen, Chi-Yuan; Lin, Shih-Yeh; Cheng, Ming-Chu; Tsai, Ching-Ping; Hung, Chang-Lin; Lo, Kai-Wei; Hwang, Yung; Hu, Yu-Chen

    2013-03-10

    Baculovirus simultaneously displaying and expressing the avian influenza virus (AIV) hemagglutinin (HA) protein can induce potent anti-HA humoral and cellular immune responses. Based on the hypothesis that improving the antigen expression and presentation can further boost the AIV vaccine efficacies, we first constructed a baculoviral vector (Bac-HAW) with HA gene fused with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) near its 3' end and expressed under the control of the hybrid CAG promoter. The WPRE fusion improved the HA expression and augmented the humoral and Th1 cellular immune responses after intramuscular administration into BALB/c mice. With Bac-HAW as the backbone, we next constructed Bac-HAMW which harbored the HA gene flanked with the signal sequence (MHCIss) and trafficking domain (MITD) of MHC class I molecule. In comparison with Bac-HAW, Bac-HAMW ameliorated the HA peptide presentation, significantly elevated the HA-specific humoral response (total IgG, IgG2a and hemagglutination inhibition titers) and favorably boosted the Th1 and IFN-γ(+)/CD8(+) T cell responses without extraneous adjuvants. These data collectively confirmed that enhancement of antigen expression and presentation by combining the WPRE and MHCIss/MITD fusion can potentiate the immunogenicity of the baculovirus-based vaccine, and implicates the potential of Bac-HAMW as an appealing AIV vaccine.

  1. Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin.

    PubMed

    Ali, M P; Kato, Tatsuya; Park, Enoch Y

    2015-12-01

    Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a "head-to-tail" shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.

  2. Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

    PubMed

    Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

    2004-10-01

    Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

  3. Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system.

    PubMed

    Qi, Qi; Yao, Lunguang; Liang, Zhisheng; Yan, Donghua; Li, Zhuo; Huang, Yadong; Sun, Jingchen

    2016-12-01

    Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257 bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure. About 1 mg/larva human type II collagen is purified from silkworm skin, which shows a putative large scale of collagen production way. An activity assay of recombinant human type II collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. Scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the baculovirus-silkworm multigene expression system can be used as an efficient platform for express active human type II collagen and other complicated eukaryotic proteins.

  4. A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity

    PubMed Central

    2014-01-01

    Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml. PMID:24735532

  5. Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus

    PubMed Central

    O’Flynn, Neil M. J.; Patel, Avnish; Kadlec, Jan; Jones, Ian M.

    2012-01-01

    The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry. PMID:23035899

  6. A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities.

    PubMed

    Somprasong, Nawarat; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee

    2006-01-01

    A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.

  7. Antibacterial function of herbal extracts on growth, survival and immunoprotection in the black tiger shrimp Penaeus monodon.

    PubMed

    AftabUddin, Sheikh; Siddique, Mohammad Abdul Momin; Romkey, Shaharin Salma; Shelton, William L

    2017-03-29

    This study examined the effects of an herbal extract composed of nine herbs i.e Aloe vera, Andrographis pariculata, Annona squamosa, Azadirachta indica, Citrus aurantifolia, Coriandrum sativum, Ocimum sanctum, Ollium cepa and Psidium guajava on growth, survival rate and immunoprotection against pathogenic Vibrio harveyi in the tiger shrimp Penaeus monodon. The petroleum ether, methanol and N-hexen extracts of different herbal plants were selected, processed and thoroughly mixed in equal proportions and added to the shrimp diets at a concentration of 1.0, 2.5 and 5.0 mL kg(-1). After 60 days of feeding, shrimps were challenged with V. harveyi bacteria (1 × 10(7) cells mL(-1)), which were isolated and propagated from the infected shrimps. The shrimps fed on diets with methanolic extraction of 2.5 mL kg(-1) had significantly (P < 0.001) higher survival rate (76%), specific growth rate (4.26 ± 0.11%) and better food conversion ratio (1.5) than the other groups. This study indicates that addition of methanolic herbal extracts of 2.5 mL kg(-1) can positively influence the immune response of tiger shrimp against V. harveyi infection.

  8. 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.

  9. A novel gonad-specific Argonaute 4 serves as a defense against transposons in the black tiger shrimp Penaeus monodon.

    PubMed

    Leebonoi, Wantana; Sukthaworn, Suchitraporn; Panyim, Sakol; Udomkit, Apinunt

    2015-02-01

    Argonaute is a key protein of the small-RNA guided gene regulation process. The Argonaute family is generally divided into two subfamilies; AGO and PIWI. In this study, a cDNA encoding a novel type of Argonaute (PmAgo4) in the black tiger shrimp Penaeus monodon was identified and characterized. PmAgo4 cDNA contained an open reading frame of 2433 nucleotides that can be translated into a deduced amino acid with the conserved PAZ and PIWI domains. PmAgo4 was phylogenetically clustered with the AGO subfamily while exhibited a gonad-specific expression pattern similar to that of proteins in the PIWI subfamily. The expression of PmAgo4 did not change significantly in response to either double-stranded RNA or yellow head virus injection suggesting that PmAgo4 may not be the main AGO proteins that play a role in dsRNA-mediated gene silencing or antiviral defense. Interestingly, PmAgo4 appeared to participate in the control of transposons since the activation of both DNA transposon and retrotransposon was detected in the testis of PmAgo4-knockdown shrimp. Our study thus provided the first evidence for an unusual type of the AGO proteins that was predominantly expressed in shrimp gonad and implication of its role in protecting the shrimp genome against an invasion of transposons.

  10. 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...

  11. 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.

  12. High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

    PubMed

    Wu, Tzong-Yuan; Chen, Hsin-An; Li, Feng-Yin; Lin, Ching-Ting; Wu, Chi-Ming; Hsieh, Feng-Chia; Tzen, Jason Tze-Cheng; Hsieh, Sheng-Kuo; Ko, Jiunn-Liang; Jinn, Tzyy-Rong

    2013-02-01

    Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

  13. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Baculoviruses are microbial agents that affects mosquito and lepidoptera larvae. They are characterized by rod-shaped virions containing circular double-stranded DNA and are the most studied insect viruses, due to their role as biological pesticides. The aim of this study was to assess the occurrenc...

  15. 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...

  16. Expression Profile of Penaeus monodon Ubiquitin Conjugating Enzyme (PmUbc) at Protein Level in White spot syndrome virus Challenged Shrimp.

    PubMed

    Keezhedath, Jeena; Kurcheti, Pani Prasad; Pathan, Mujahid Khan; Babu, Gireesh P; Tripathi, Gayatri; Sudhagar, Arun; Rao, Srinivas P

    2013-06-01

    White spot syndrome virus (WSSV) is one of the major pathogens in shrimp aquaculture. Four proteins of WSSV are predicted to encode a RING H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimps 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, expression profile of Penaeus monodon Ubiquitin conjugating enzyme (PmUbc) was studied at protein level in WSSV challenged shrimp. A time point analysis of the expression of PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge in P. monodon. Recombinant PmUbc (rPmUbc) was produced in prokaryotic expression vector, BL21 (DE3) pLys S. The PmUbc expression pattern was studied by ELISA with rPmUbc antibodies raised in rabbit. A significant increase in PmUbc expression at 24 h post infection (hpi) was observed followed by a decline till 72 hpi. Since the up-regulation and a tremendous decline of PmUbc protein expression was observed at 24 and in 72 hpi respectively in ELISA, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. Many findings have shown that viral infection can up-regulate expression of ubiquitin and that the ubiquitin system plays a key role in the course of viral infection. The present study reveals the expression patterns of PmUbc at protein level in WSSV infected P. monodon. However, further studies are to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this major culprit in shrimp culture industry.

  17. Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

    PubMed

    Zhou, Kaimin; Zhou, Falin; Huang, Jianhua; Yang, Qibin; Jiang, Song; Qiu, Lihua; Yang, Lishi; Zhu, Caiyan; Jiang, Shigui

    2017-03-01

    Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%-88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

  18. 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

    2015-09-25

    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.

  19. 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.

  20. 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

  1. Expression of Aleutian mink disease parvovirus proteins in a baculovirus vector system.

    PubMed Central

    Christensen, J; Storgaard, T; Bloch, B; Alexandersen, S; Aasted, B

    1993-01-01

    We have previously published a detailed transcription map of Aleutian mink disease parvovirus (ADV) and proposed a model for the translation of the two virion structural proteins (VP1 and VP2) and three nonstructural proteins (NS-1, NS-2, and NS-3) (S. Alexandersen, M. E. Bloom, and S. Perryman, J. Virol. 62:3684-3994, 1988). To verify and further characterize this model, we cloned the predicted open reading frames for NS-1, NS-2, NS-3, VP1-VP2, and VP2 alone into a recombinant baculovirus and expressed them in Sf9 insect cells. Expression of VP1-VP2 or VP2 alone in cDNA and in the genomic form was achieved. The expressed proteins had molecular weights similar to those of the corresponding proteins of wild-type ADV-G, although the ratio of VP1 to VP2 was altered. The recombinant baculovirus-expressed ADV VP1 and VP2 showed nuclear localization in Sf9 cells and were able to form particles indistinguishable, by electron microscopy, from wild-type virus. The large nonstructural protein, NS-1, showed predominantly nuclear localization in Sf9 cells when analyzed by immunofluorescence and had a molecular weight similar to that of wild-type ADV NS-1. Moreover, expression of NS-1 in Sf9 cells caused a change in morphology of the cells and resulted in 10-times-lower titers of recombinant baculovirus during infection, suggesting a cytostatic or cytotoxic action of this protein. The smaller NS-2 gene product seems to be located in the cytoplasm. When analyzed by Western immunoblotting, NS-2 comigrated with an approximately 16-kDa band seen in lysates of ADV-infected feline kidney cells. The putative NS-3 gene product exhibited a diffuse distribution in Sf9 cells and had a molecular weight of approximately 10,000. All of the expressed ADV-encoded proteins were recognized by sera from ADV-infected mink. Thus, expression of ADV cDNAs allowed assignment of the different mRNAs to the viral proteins observed during ADV infection in cell culture and supported our previously proposed

  2. Development of an in situ toxicity assay system using recombinant baculoviruses.

    PubMed

    Grant, D F; Greene, J F; Pinot, F; Borhan, B; Moghaddam, M F; Hammock, B D; McCutchen, B; Ohkawa, H; Luo, G; Guenthner, T M

    1996-02-23

    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 more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.

  3. Production of CCHF virus-like particle by a baculovirus-insect cell expression system.

    PubMed

    Zhou, Zhao-Rui; Wang, Man-Li; Deng, Fei; Li, Tian-Xian; Hu, Zhi-Hong; Wang, Hua-Lin

    2011-10-01

    Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).

  4. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

    PubMed Central

    Kitts, P A; Ayres, M D; Possee, R D

    1990-01-01

    Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location. Images PMID:2216760

  5. 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.

  6. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

    PubMed

    Hummel, K B; Martin, M L; Auperin, D D

    1992-09-01

    A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

  7. Preparation of connexin43-integrated giant Liposomes by a baculovirus expression-liposome fusion method.

    PubMed

    Kamiya, Koki; Tsumoto, Kanta; Arakawa, Satoko; Shimizu, Shigeomi; Morita, Ikuo; Yoshimura, Tetsuro; Akiyoshi, Kazunari

    2010-12-01

    Connexin-43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression-liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43-mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43-expressing U2OS cells (human osteosarcoma cell). The functional connexin-containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins.

  8. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.

    PubMed

    Mitchell, Jonathan K; Friesen, Paul D

    2012-12-01

    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.

  9. 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

  10. 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.

  11. Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

    PubMed

    Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

    2015-06-01

    White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

  12. Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon).

    PubMed

    Shekhar, M S; Dillikumar, M; Vinaya Kumar, K; Gopikrishna, G; Rajesh, S; Kiruthika, J; Ponniah, A G

    2012-12-01

    Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 μg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 μg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.

  13. The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp.

    PubMed

    van de Braak, C B T; Botterblom, M H A; Taverne, N; van Muiswinkel, W B; Rombout, J H W M; van der Knaap, W P W

    2002-10-01

    In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. A rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. It is proposed that the spheroids have a degradation function for both bacterial and viral material

  14. Putative spawner-isolated mortality virus associated with mid-crop mortality syndrome in farmed Penaeus monodon from northern Australia.

    PubMed

    Owens, L; Haqshenas, G; McElnea, C; Coelen, R

    1998-11-30

    Beginning in 1994, farms in northern Australia experienced a higher than normal mortality rate in 12 to 15 g prawns from growout ponds. The farmers named this problem mid-crop mortality syndrome (MCMS). Intramuscular injection of filtered (450 nm), cell-free extracts of moribund prawns from these ponds killed healthy prawns between 5 to 30 d post-injection. A 20 nm virus was visualized by electron microscopy from a 1.4 g ml-1 band recovered from caesium chloride gradients of extracts from the moribund prawns. DNA was extracted from this band, restriction enzyme digested and ligated into pGEM7zf(+) vector. A digoxigenin-labelled polymerase chain reaction (PCR)-generated, gene probe was subsequently prepared by amplifying an inserted sequence (approximately 2 kb) of one selected clone specific for the virus. Specimens of the moribund prawns stained positively by in situ DNA hybridization in endodermal tissues, including the apical ends of hepatopancreatic tubules, the midgut and hindgut caecae, the midgut, and the hindgut folds. In prawns that showed haemocytic enteritis, some haemocytes in the affected midgut showed limited staining. The positively-staining cells showed no cytolysis. In prawns injected with cell-free viral extracts, additional tissues were positive by probe analysis, including strong staining in the male reproductive tract, specifically in the terminal ampoule and the medial vas deferens. Limited staining also occurred in the ovary and in both the stromal matrix and spheroid cells of the lymphoid organ. It was evident that the infection was enteric by natural pathways and systemic by injection. Historical specimens of Penaeus monodon experimentally infected with spawner-isolated mortality virus (SMV) were probe-positive in exactly the same pattern as the naturally and experimental MCMS prawns. Altogether, the evidence suggested that the MCMS agent was a parvo-like virus very similar or identical to SMV.

  15. 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%).

  16. A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon.

    PubMed

    Monwan, Warunthorn; Amparyup, Piti; Tassanakajon, Anchalee

    2017-02-01

    Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

  17. 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.

  18. PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

    PubMed

    Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2014-01-01

    Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410-fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

  19. Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine.

    PubMed

    Sudheer, N S; Poulose, Gigi; Thomas, Ancy; Viswanath, Kiron; Kulkarni, Amod; Narayanan, R B; Philip, Rosamma; Singh, I S Bright

    2015-05-01

    White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×10(9) DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×10(6) DNA copies of inactivated virus preparation (IVP) day(-1) for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.

  20. White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy.

    PubMed

    Mishra, S S; Shekhar, M S

    2005-07-01

    Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

  1. Molecular cloning, expression and functional analysis of three subunits of protein phosphatase 2A (PP2A) from black tiger shrimps (Penaeus monodon).

    PubMed

    Zhao, Chao; Wang, Yan; Fu, Mingjun; Yang, Keng; Qiu, Lihua

    2017-02-01

    Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.

  2. Osmo and ionic regulation of black tiger prawn (Penaeus monodon Fabricius 1798) juveniles exposed to K(+) deficient inland saline water at different salinities.

    PubMed

    Tantulo, Uras; Fotedar, Ravi

    2007-02-01

    An 11-day trial was conducted to investigate the osmoregulatory capacity (OC) and regulation of K(+), Na(+), Ca(2+) and Mg(2+) of Penaeus monodon juveniles when exposed to K(+) deficient inland saline water (ISW) of four different salinities (5, 15, 25 and 35 ppt). The survival of juveniles showed a positive linear relationship (R(2) ranging from 0.72 to 0.98) with salinity. At the end of the trial, juveniles were able to survive only in 5 ppt of ISW and showed no changes in OC when transferred from ocean water (OW) to ISW. Further, the OC of juveniles in 5 ppt of ISW was significantly different (P<0.05) from the OC of juveniles exposed to 15, 25 and 35 ppt and exhibited strong serum K(+), Na(+), Ca(2+) and Mg(2+) regulation monitored over 16 h. In contrast, at 35 ppt, significant decrease (P<0.05) in serum K(+) and Mg(2+) concentrations and accumulation of serum Na(+) concentration occurred after 16 h of exposure to ISW. At higher salinity, an increase in serum Na(+) concentration leads to an increase in the serum osmolality of the juveniles, which in turn causes decrease in the OC of the juveniles. The results of this study suggest that K(+) deficiency in ISW has a negative effect on survival, OC and the ability of P. monodon juveniles to regulate serum Na(+), K(+), Ca(2+) and Mg(2+) concentrations. These effects are compounded as salinity increases.

  3. Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

    PubMed Central

    Wu, Yueh-Lung; Wu, Carol-P; Huang, Yu-Hui; Huang, Sheng-Ping; Lo, Huei-Ru; Chang, Hao-Shuo; Lin, Pi-Hsiu; Wu, Ming-Cheng; Chang, Chia-Jung

    2014-01-01

    ABSTRACT The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143

  4. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    PubMed

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

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

    PubMed

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

    2012-02-01

    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 P6(2) or P6(4), 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 Å(3) Da(-1).

  6. Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.

    PubMed

    Chen, Wen-Shuo; Chang, Yen-Chung; Chen, Ying-Ju; Chen, Yu-Jie; Teng, Chao-Yi; Wang, Chung-Hsiung; Wu, Tzong-Yuan

    2009-08-01

    Recombinant baculoviruses are suitable for the high-level production of large multi-protein complexes. A tri-cistronic expression vector was constructed by the inclusion of two internal ribosome entry sites (IRESs). In this novel polycistronic vector, one single polyhedrin promoter controlled the transcription of a tri-cistronic transcript. Also, the first cistron was translated through a cap-dependent mechanism, while the second and third cistrons were translated by the IRESs derived from the 5' UTR of Rhopalosiphum padi virus (RhPV) and Perina nuda virus (PnV), respectively. The ratio of tri-cistronic gene expression levels produced by the three translational initiation modules is about 2:1:1 (cap:PnV IRES:RhPV IRES). This study indicates that polycistronic genes can be co-expressed at the translational level as in prokaryotic expression system by baculovirus biotechnology.

  7. Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system.

    PubMed

    Reiländer, H; Haase, W; Maul, G

    1996-02-06

    A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

  8. 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

  9. 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.

  10. 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.

  11. Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

    PubMed Central

    Dong, Zhan-Qi; Hu, Nan; Dong, Fei-Fan; Chen, Ting-Ting; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2017-01-01

    Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research. PMID:28393927

  12. 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.

  13. 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.

  14. Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks.

    PubMed

    Sokolenko, Stanislav; George, Steve; Wagner, Andreas; Tuladhar, Anup; Andrich, Jonas M S; Aucoin, Marc G

    2012-01-01

    The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.

  15. Dietary effect of Sargassum wightii fucoidan to enhance growth, prophenoloxidase gene expression of Penaeus monodon and immune resistance to Vibrio parahaemolyticus.

    PubMed

    Sivagnanavelmurugan, Madasamy; Thaddaeus, Bergmans Jude; Palavesam, Arunachalam; Immanuel, Grasian

    2014-08-01

    The polysaccharide fucoidan from brown seaweed Sargassum wightii was extracted and it was incorporated with pellet diets at three concentrations (0.1, 0.2 & 0.3%). The fucoidan incorporated diets were fed to shrimp Penaeus monodon for 60 days and the growth performance was assessed. The weight gain and SGR of control group was 6.83 g and 9.72%, respectively, but the weight gain and SGR of various concentrations (0.1-0.3%) of fucoidan incorporated diets fed groups of shrimp was increased from 7.30 to 8.20 g and 9.83 to 10.03%, respectively. After 60 days of feeding experiment, the relative quantification of prophenoloxidase gene of experimental groups over control group was analysed by RT-PCR and it was ranged between 2.13 and 7.95 fold increase within 33.52-34.61 threshold cycles, respectively at 0.1-0.3% concentrations of fucoidan. After 60 days of feeding experiment, the P. monodon were challenged with shrimp pathogen Vibrio parahaemolyticus and the mortality percentage was recorded daily up to 21 days. The reduction in mortality percentage of experimental groups over control group was recorded from 44.56 to 72.79%, respectively in 0.1-0.3% of fucoidan incorporated diets fed groups. During challenge experiment, all the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity, phagocytic activity, bactericidal activity and bacterial clearance ability of experimental groups were significantly (P < 0.05) increased than control group. The V. parahaemolyticus load was enumerated from the infected shrimp at every 10 days intervals during challenge experiment. In control group, the Vibrio load was increased in hepatopancreas and muscle tissues from 10th to 21st days of challenge test. But in the experimental groups, the Vibrio load in both the tissues decreased positively from 10th to 21st days of challenge duration. It is concluded that the S. wightii fucoidan had enhanced the innate immunity and

  16. 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.

  17. Comparative proteomics reveal fundamental structural and functional differences between the two progeny phenotypes of a baculovirus.

    PubMed

    Hou, Dianhai; Zhang, Leike; Deng, Fei; Fang, Wei; Wang, Ranran; Liu, Xijia; Guo, Lin; Rayner, Simon; Chen, Xinwen; Wang, Hualin; Hu, Zhihong

    2013-01-01

    The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently.

  18. Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

    PubMed

    Cromlish, W A; Payette, P; Kennedy, B P

    1999-11-15

    We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments. Assays are carried out in a 96-well format, with cells expressing the PTP of interest. Cells are preincubated with or without inhibitor and challenged with substrate, and the phosphatase activity is determined spectrophotometrically by monitoring the conversion of p-nitrophenyl phosphate to p-nitrophenol at OD405. Documented PTP inhibitors have been used to validate this assay system. This study demonstrates that a direct readout of PTP activity in intact cells can be achieved, thus providing a useful cell-based screen for determining selective inhibitors of PTPs.

  19. 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.

  20. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    PubMed

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  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.

  2. 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.

  3. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

    PubMed Central

    Condreay, J. Patrick; Witherspoon, Sam M.; Clay, William C.; Kost, Thomas A.

    1999-01-01

    Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression. PMID:9874783

  4. Actin-based motility drives baculovirus transit to the nucleus and cell surface

    PubMed Central

    Ohkawa, Taro; Volkman, Loy E.

    2010-01-01

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses. PMID:20660627

  5. A rapid method for estimation of baculovirus titer based on viable cell size.

    PubMed

    Janakiraman, Vasantharajan; Forrest, William F; Chow, Bernard; Seshagiri, Somasekar

    2006-03-01

    Baculovirus protein expression system is a powerful tool for producing recombinant proteins. To optimize conditions for efficient recombinant protein expression, it is important to determine titer of virus stock for arriving at an optimal multiplicity of infection (MOI) that maximizes recombinant protein expression. Traditionally plaque assays have been used for titer determination. Other methods such as endpoint dilution, quantitative real-time polymerase chain reaction and flow cytometry have been developed to aid the determination of virus titers. However, most of these methods are time-consuming and labor intensive. In this regard, a simple and rapid method for determination of virus titers based on the cytopathic effects that lead to viable cell size increase following virus infection is presented in this paper. In this study, the Vi-CELL (Beckman Coulter) was used to measure cell-diameter over a range of virus dilutions, following infection. Applying statistical modeling techniques, the viable cell-diameter data was used to estimate the virus titer. The results indicated that the viable cell-diameter based titer estimation to be reliable and comparable to titers determined by the traditional plaque assay.

  6. 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.

  7. Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

    PubMed

    Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

    2013-08-01

    The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs.

  8. 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.

  9. 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

  10. Increase of dietary vitamin C improves haemocyte respiratory burst response and growth of juvenile grass shrimp, Penaeus monodon, fed with high dietary copper.

    PubMed

    Lee, Min-Hsien; Shiau, Shi-Yen

    2003-04-01

    Effects of dietary vitamin C (l-ascorbyl-2-monophosphate-Mg, C2MP-Mg) on growth, tissue copper (Cu) accumulation, and haemocyte superoxide anion production of juvenile grass shrimp, Penaeus monodon, fed with either adequate or high (8 x adequate) dietary Cu were studied. Three experimental diets were used: basal diet supplemented with adequate levels of both C2MP-Mg (40 mg kg diet(-1)) and Cu (20mg kg diet(-1)) (NC-NCu); basal diet supplemented with adequate C2MP-Mg and high Cu (8 x adequate) (NC-HCu); and basal diet supplemented with high C2MP-Mg (5 x adequate) and high Cu (HC-HCu). These were each fed to triplicate groups of shrimp (mean initial weight: 0.29+/-0.01 g) for 8 weeks. Highest (P< 0.01) weight gain, feed efficiency (FE) and protein efficiency ratio (PER) were observed in shrimp fed NC-NCu diet, intermediate in shrimp fed HC-HCu diet, and lowest in shrimp fed NC-HCu diet. Cu concentrations in hepatopancreas, muscle and haemolymph were highest in shrimp fed NC-HCu diet, followed by shrimp fed HC-HCu diet, and lowest for shrimp fed NC-NCu diet. Survival, total haemocyte count (THC) and intracellular superoxide anion (O-2) production were higher in shrimp fed NC-NCu diet than shrimp fed NC-HCu diet, whereas hepatosomatic index (HSI) was higher in shrimp fed NC-HCu diet than shrimp fed NC-NCu diet. However, all these parameters were similar in shrimp fed NC-NCu diet and shrimp fed HC-HCu diet. These data suggest that increase of dietary vitamin C improved haemocyte respiratory burst response and growth and prevented tissue Cu accumulation in P. monodon fed with high dietary Cu.

  11. The effect of choline and cystine on the utilisation of methionine for protein accretion, remethylation and trans-sulfuration in juvenile shrimp Penaeus monodon.

    PubMed

    Richard, Lenaïg; Vachot, Christiane; Surget, Anne; Rigolet, Vincent; Kaushik, Sadasivam J; Geurden, Inge

    2011-09-01

    This 35-d feeding experiment examined in juvenile shrimp Penaeus monodon (3·3 g initial body weight) the effects of methionine (Met), choline and cystine on protein accretion and the activity of two key enzymes of remethylation (betaine-homocysteine methyltransferase; BHMT) and trans-sulfuration (cystathionine β-synthase; CBS). The interaction between Met and choline was tested using semi-purified diets either adequate or limiting (30 or 50 %) in total sulphur amino acid (SAA) content with a constant cystine:Met ratio. The diets contained either basal or excess choline (3 v. 7 g/kg feed). Cystine was added to two other 30 and 50 % Met-limiting diets to adjust the SAA supply to that of the control diet in order to evaluate the interaction between Met and cystine. As expected, N accretion was significantly lower with the SAA-limiting diets but increased back to control levels by the extra choline or cystine, demonstrating their sparing effect on Met utilisation for protein accretion. We show, for the first time, the activities of BHMT and CBS in shrimp hepatopancreas. Only BHMT responded to the SAA deficiencies, whereas the extra choline and cystine did not stimulate remethylation or down-regulate trans-sulfuration. Our data also suggest the capacity of P. monodon to synthesise taurine, being significantly affected by the cystine level in the 30 % SAA-limiting diets. Further research is warranted to better understand the metabolic regulation of taurine synthesis in shrimp and of the observed Met-sparing effects.

  12. Toxic effects of blooms of marine species of Oscillatoriales on farmed prawns (Penaeus monodon, Penaeus japonicus) and brine shrimp (Artemia salina).

    PubMed

    Smith, P T

    1996-08-01

    Benthic and planktonic blooms of species of Oscillatoriales coincided with mortalities of Penaeus monodon during four episodes at Australian prawn farms. Oscillatoria corakiana was the dominant planktonic species at 65-90,000 cells/ml, but Spirulina sp., Lyngbya sp., Oscillatoria sp. and Nodularia sp. were also identified from the water column, benthic layers or surface mats. The levels and variety of Vibrionaceae in prawn tissue, suggest that mortalities were caused by secondary infections of bacteria. However, experimental results indicate that toxicity of the blooms of Oscillatoriales was the primary cause of disease. Pond water and extracts from a tank culture of benthic Oscillatoriales caused mortalities when injected into P. monodon and P. japonicus. Immersion of artemia in extracts from the tank culture also caused mortalities, with L.D50 values for the supernatant extract of 70 mg/litre for artemia cysts and 50 mg/litre for adult artemia, and LD50 values for the pellet extract of 110 mg/litre for artemia cysts and 200 mg/litre for adult artemia. Experiments with artemia suggested the blooms of Oscillatoriales produced water-soluble, heat-labile toxin/s. Mortalities may have been caused by a neurotoxin because: (a) there was a lack of histopathological evidence of damage to the digestive tracts of prawns during each episode; and (b) artemia cysts immersed in extracts of Oscillatoriales died before they developed digestive tracts. PSP toxin, anatoxin-a, homoanatoxin-a and microcystins were not detected when pond water from a diseased pond was tested. It is proposed that sub-lethal levels of toxin weakened the prawns, causing reduced feeding behaviour and an impaired immune system. As a result, prawns were prone to secondary infection by pathogenic bacteria. Because Oscillatoriales are ubiquitous in prawn farms, the findings have significant implications for the assessment of disease in the prawn farming industry.

  13. 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.

  14. 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.

  15. Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

    PubMed

    Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

  16. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  17. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    PubMed

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  18. Baculovirus surface display of sigmaC and sigmaB proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model.

    PubMed

    Lin, Yueh H; Lee, Long H; Shih, Wen L; Hu, Yu C; Liu, Hung J

    2008-11-25

    Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The sigmaC and sigmaB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express sigmaC and sigmaB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB have been successfully constructed. After infection, both His6-tagged recombinant sigmaC (rsigmaC) and sigmaB (rsigmaB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rsigmaC and rsigmaB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rsigmaC and rsigmaB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-sigmaC and BacSC-sigmaB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the

  19. Human olfactory receptors: recombinant expression in the baculovirus/Sf9 insect cell system, functional characterization, and odorant identification.

    PubMed

    Matarazzo, Valéry; Ronin, Catherine

    2013-01-01

    Cell surface expression of recombinant olfactory receptors (ORs) is a major limitation in characterizing their functional nature. We have shown that the recombinant expression of a human OR, OR 17-210, in the baculovirus/Sf9 insect cell system allows this protein to be expressed at the cell surface. We used Ca(2+) imaging to demonstrate that recombinant OR 17-210 produces cellular activities upon odorant stimulation with ketones. Furthermore, this expression and functional system has been used to show that the preincubation of Human Odorant Binding Protein 2A decrease the calcium response of OR 17-210 following stimulation by acetophenone and beta ionone.

  20. 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.

  1. 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.

  2. Active Depletion of Host Cell Inhibitor-of-Apoptosis Proteins Triggers Apoptosis upon Baculovirus DNA Replication▿

    PubMed Central

    Vandergaast, Rianna; Schultz, Kimberly L. W.; Cerio, Rebecca J.; Friesen, Paul D.

    2011-01-01

    Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects. PMID:21653668

  3. 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.

  4. [The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus].

    PubMed

    Cao, Lei; Yi, Yao; Song, Jing-Dong; Tian, Miao-Miao; Tian, Rui-Guang; Meng, Qing-Ling; Qiu, Feng; Jia, Zhi-Yuan; Bi, Sheng-Li

    2012-05-01

    To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

  5. Characterization of novel components of the baculovirus per os infectivity factor complex.

    PubMed

    Peng, Ke; van Lent, Jan W M; Boeren, Sjef; Fang, Minggang; Theilmann, David A; Erlandson, Martin A; Vlak, Just M; van Oers, Monique M

    2012-05-01

    Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

  6. Protein-Protein Interactions of the Baculovirus Per Os Infectivity Factors in the PIF Complex.

    PubMed

    Zheng, Qin; Shen, Yunwang; Kon, Xiangshuo; Zhang, Jianjia; Feng, Min; Wu, Xiaofeng

    2017-01-28

    After ingestion of occlusion bodies, the occlusion-derived viruses (ODVs) of baculoviruses establish the first round of infection within the larval host midgut cells. Several ODV envelope proteins, called per os infectivity factors (PIFs), have been shown to be essential for oral infection. Eight PIFs have been identified to date, including P74, PIFs1-6, and Ac110. At least six PIFs: P74, PIFs1-4, PIF6, together with three other ODV-specific proteins: Ac5, P95 (Ac83), and Ac108, have been reported to form a complex on the ODV surface. In this study, in order to understand the interactions of these PIFs, the direct protein-protein interactions of the nine components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex were investigated using yeast two-hybrid (Y2H) combined with bimolecular fluorescence complementation (BiFC) assay. Six direct interactions comprising PIF1-PIF2, PIF1-PIF3, PIF1-PIF4, PIF1-P95, PIF2-PIF3, and PIF3-PIF4, were identified in Y2H analysis, and these results were further verified by BiFC. For P74, PIF6, Ac5 and Ac108, no direct interaction was identified. P95 (Ac83) was identified to interact with PIF1 and further Y2H analysis of the truncations and deletion mutants showed that the predicted P95 chitin-binding domain and PIF1 100-200aa were responsible for P95 interaction with PIF1. Furthermore, a summary of the protein-protein interactions of PIFs reported so far, comprising 10 reciprocal interactions and 2 self-interactions, is presented, which will facilitate our understanding of the characteristic of PIF complex.

  7. Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori.

    PubMed

    Sugiura, Nobuo; Ikeda, Motoko; Shioiri, Tatsumasa; Yoshimura, Mayumi; Kobayashi, Michihiro; Watanabe, Hideto

    2013-12-01

    Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

  8. Recombinant protein production by the baculovirus-insect cell system in Basal media without serum supplementation.

    PubMed

    Nishikawa, Norikatsu; Yamaji, Hideki; Fukuda, Hideki

    2003-11-01

    The production of beta-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the beta-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted beta-galactosidase production, their removal by medium replacement on post-infection day 1 gave a beta-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing beta-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.

  9. Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

    PubMed

    Parrington, M; Cockle, S; Wyde, P; Du, R P; Snell, E; Yan, W Y; Wang, Q; Gisonni, L; Sanhueza, S; Ewasyshyn, M; Klein, M

    1997-01-01

    Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

  10. Baculovirus-Mediated miRNA Regulation to Suppress Hepatocellular Carcinoma Tumorigenicity and Metastasis

    PubMed Central

    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

  11. Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica.

    PubMed

    Haas, A L; Katzung, D J; Reback, P M; Guarino, L A

    1996-04-30

    The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.

  12. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    PubMed

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  13. 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

  14. Characterization of a protein tyrosine phosphatase as a host factor promoting baculovirus replication in silkworm, Bombyx mori.

    PubMed

    Wang, Fei; Xue, Renju; Li, Xianyang; Hu, Cuimei; Xia, Qingyou

    2016-04-01

    The relevance of protein tyrosine phosphatase (PTP) to host-pathogen interaction is highlighted in mammalian studies, whereas less is known in insects. Here we presented the categorization of the PTP complement of silkworm and characterized their homologous relationship with human and fruit fly PTPs. Among the 36 PTP genes, ptp-h, which was proposed to be the origin of baculovirus ptp belongs to atypical VH1-like dual-specific PTP subset and encodes a catalytic active protein. The maximum expression level of Bmptp-h was at 5th instar and in fat body. Bombyx mori nucleopolyhedrovirus (BmNPV) infection potently induced its expression in silkworm larvae and in BmE cells. Knock-down of Bmptp-h by RNA interference significantly inhibited viral replication, and over-expression enhanced viral replication as determined by viral DNA abundance and BmNPV-GFP positive cells. These results suggest that BmPTP-h might be one of the host factors that is beneficial to baculovirus infection by promoting viral replication.

  15. 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.

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

    PubMed

    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(P48-KO-PH-GFP) was unable to propagate in cell culture, while a 'repair' Bacmid vAc(P48-REP-PH-GFP) was able to replicate in a manner similar to a wild-type Bacmid vAc(PH-GFP). Titration assays and Western blotting confirmed that vAc(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.

  17. Production of DUSP1 protein using the baculovirus insect cell expression system and its in vitro effects on cancer cells.

    PubMed

    Cheng, Peng; Zhu, Shuying; Jun, Li; Huang, Lihua; Hong, Yahui

    2015-06-01

    The aim of the present study was to produce the human dual specificity phosphatase 1 (DUSP1) protein with biological activity and to investigate its in vitro effects on cancer cells. DUSP1 protein was expressed in the baculovirus expression system and purified by Ni-affinity chromatography followed by dialysis in PBS. The purified protein was verified by SDS-PAGE and western blot analysis. Six cancer cell lines were then cultured in the presence of DUSP1 for various periods of time, and the phosphorylated extracellular signal-regulated kinase (p-ERK) content in each cell line was subsequently determined by western blot analysis. Compared to the β-actin level, the amount of p-ERK markedly decreased after 1 h, indicating that DUSP1 suppressed the expression of p-ERK in 6 cancer cell lines examined. Human cervical cancer cells were also collected and counted following co-culture with DUSP1 to examine its effect on the growth rate of cancer cells. A baculovirus expression system for the production of DUSP1 protein was successfully constructed. The p-ERK content was found to be significantly decreased when the cancer cell lines were exposed to DUSP1. The capability of binary fission was reduced when the cells were examined under a microscope. The proliferation of human cervical cancer cells was also inhibited by DUSP1.

  18. 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

  19. 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.

  20. 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

  1. The effect of spent medium recycle on cell proliferation, metabolism and baculovirus production by the lepidopteran Se301 cell line infected at very low MOI.

    PubMed

    Beas-Catena, Alba; Sánchez-Mirón, Asterio; Garía-Camacho, Francisco; Contreras-Gómez, Antonio; Molina-Grima, Emilio

    2013-12-01

    The aim of this paper was to study the effect of spent medium recycle on Spodoptera exigua Se301 cell line proliferation, metabolism, and baculovirus production when grown in batch suspension cultures in Ex-Cell 420 serum-free medium. The results showed that the recycle of 20% of spent medium from a culture in mid-exponential growth phase improved growth relative to a control culture grown in fresh medium. Although both glucose and glutamine were still present at the end of the growth phase, glutamate was always completely exhausted. The pattern of the specific glucose and lactate consumption and production rates, as well as the specific glutamine and glutamate consumption rates, suggests a metabolic shift at spent medium recycle values of over 60%, with a decrease in the efficiency of glucose utilization and an increase in glutamate consumption to fuel energy metabolism. Baculovirus infection provoked a change in the metabolic pattern of Se301 cells, although a beneficial effect of spent medium recycle was also observed. Both growth rate and maximum viable cell density decreased relative to uninfected cultures. The efficiency of glucose utilization was dramatically reduced in those cultures containing the lowest percentages of spent medium, whereas glutamine and glutamate consumption was modulated, thereby suggesting that infected cells were devoted to virus replication, retaining their ability to incorporate the nutrients required to support viral replication. Recycle of 20% of spent medium increased baculovirus production by around 90%, thus showing the link between cell growth and baculovirus production.

  2. 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

  3. 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...

  4. 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.

  5. 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...

  6. 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.

  7. Reduction of the infectivity of baculovirus stocks frozen at ultra-low temperature in serum-free media: The role of lipid emulsions.

    PubMed

    Eberhardt, Ignacio; Gioria, Verónica Viviana; Micheloud, Gabriela Analía; Claus, Juan Daniel

    2016-11-01

    The infectivity of stocks of baculoviruses produced in serum-free media is sensitive to freezing at ultra-low temperatures. The objective of this work was to elucidate the causes of such sensitivity, using as a model the freezing of stocks of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a baculovirus widely employed as biological insecticide. Titers of supernatants of cell cultures infected with AgMNPV in four different serum-free media supplemented with lipid emulsions were reduced by 50 to 90% after six months freezing. By using a full factorial experiment, freezing and lipid emulsion, as well as the interaction between them, were identified as the main factors reducing the viral titer. The virucidal effect of the lipid emulsion was reproduced by one of their components, the surfactant Polysorbate 80. Damaged viral envelopes were observed by transmission electron microscopy in most particles frozen in a medium supplemented with lipid emulsion or Polysorbate 80. Additionally, Polysorbate 80 also affected the infectivity of AgMNPV stocks that were incubated at 27°C. The identification of the roles played by the lipid emulsion and Polysorbate 80 is not only a contribution to the understanding of the mechanisms underlying the inactivation of baculovirus stocks produced in serum-free media during storage at ultra-low temperature, but is also an input for the rational development of new procedures aimed at improving both the preservation of baculovirus stocks and the composition of culture media for the production of baculovirus-based bioproducts in insect cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1559-1569, 2016.

  8. Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

    PubMed

    Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

    2017-03-10

    Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10(6)cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10(6)cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10(6)cells/mL showed comparable HA titers per cell to those of CCI of 2×10(6)cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable

  9. Identification of RAPD-SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp, Penaeus monodon Fabricius.

    PubMed

    Dutta, S; Biswas, S; Mukherjee, K; Chakrabarty, U; Mallik, A; Mandal, N

    2014-05-01

    White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost-effective strategy for long-term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10(3) fold higher in disease susceptible than that in disease resistant shrimps using real-time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease-free shrimp aquaculture industry.

  10. PmTBC1D20, a Rab GTPase-activating protein from the black tiger shrimp, Penaeus monodon, is involved in white spot syndrome virus infection.

    PubMed

    Yingvilasprasert, Wanchart; Supungul, Premruethai; Tassanakajon, Anchalee

    2014-02-01

    TBC (TRE2/BUB2/CDC16) domain proteins contain an ≈ 200-amino-acid motif and function as Rab GTPase-activating proteins that are required for regulating the activity of Rab proteins, and so, in turn, endocytic membrane trafficking in cells. TBC domain family member 20 (TBC1D20) has recently been reported to mediate Hepatitis C virus replication. Herein, PmTBC1D20 identified from the black tiger shrimp, Penaeus monodon, was characterized and evaluated for its role in white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of PmTBC1D20 contains 2003 bp with a predicted 1443 bp open reading frame encoding a deduced 480 amino acid protein. Its transcript levels were significantly up-regulated at 24 and 48 h by ≈ 2.3- and 2.1-fold, respectively, after systemic infection with WSSV. In addition, depletion of PmTBC1D20 transcript in shrimps by double stranded RNA interference led to a decrease in the level of transcripts of three WSSV genes (VP28, ie1 and wsv477). This suggests the importance of PmTBC1D20 in WSSV infection. This is the first report of TBC1D20 in a crustacean and reveals the possible mechanism used by WSSV to modulate the activity of the host protein, PmTBC1D20, for its benefit in viral trafficking and replication.

  11. The expression and purification of WSSV134 from white spot syndrome virus and its inhibitory effect on caspase activity from Penaeus monodon.

    PubMed

    Bowornsakulwong, Thanunthon; Charoensapsri, Walaiporn; Rattanarojpong, Triwit; Khunrae, Pongsak

    2017-02-01

    WSSV134 or VP36A protein of white spot syndrome virus was previously reported to be able to reduce apoptosis in Sf-9 cells transfected with caspase of Penaeus monodon (PmCasp). The protein was therefore believed to have a role in supporting the survival of WSSV inside the host cells during infection. However, the anti-apoptosis activity of WSSV134 involved in the inhibition of PmCasp is still unclear. In this study, we produced a recombinant WSSV134 (rWSSV134) and tested for its ability to inhibit PmCasp in vitro. The results from a caspase inhibition assay revealed that rWSSV134 could inhibit PmCasp in a dose-dependent manner. Since WSSV134 was predicted to contain three potential caspase binding sites, corresponding to the D54, D104 and D259, we then employed site-directed mutagenesis to investigate the involvement of these sites in PmCasp inhibition. D54A and D259A mutants could still inhibit PmCasp while D104A mutant lacks this activity. Our results confirmed that the WSSV134 is an inhibitor for PmCasp and that residue D104 is important for PmCasp inhibition.

  12. Accumulation, metabolism, and food-chain transfer of chlorinated and brominated contaminants in subadult white whales (Delphinapterus leucas) and narwhals (Monodon monoceros) from Svalbard, Norway.

    PubMed

    Wolkers, H; Lydersen, C; Kovacs, K M; Burkow, I; van Bavel, B

    2006-01-01

    The concentrations and patterns of polychlorinated biphenyls (PCBs), chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs) were studied in white whales (Delphinapterus leucas) and narwhals (Monodon monoceros) from Svalbard, Norway. In addition, their main food items were included in the study. In the whales, a broad range of pollutants was found in relatively high concentrations. PCBs and pesticides were approximately 3000 and 8000 ng/g lipid, respectively, for white whales and three times higher for narwhals. PBDEs 47 were approximately 70 ng/g lipid for white whales and 170 ng/g lipid for narwhals. Compared with other marine mammals from the same area, contaminant levels are among the highest levels ever measured. These high levels are likely in part because of a decreased capacity to metabolize contaminants. Metabolic indices indicated that most compounds accumulate to the same degree in white whales and narwhals, but for some toxaphenes and chlordanes, narwhals might have a decreased metabolism and consequently a higher accumulation. The three-times-higher contaminant levels in blubber of narwhals was further explained by substantially higher contaminant levels in their more benthic diet. The high levels and broad pattern of accumulating pollutants make white whales and narwhals excellent indicators for a wide range of contaminants in the Arctic.

  13. Ecological niches and areas of overlap of the squat lobster ‘munida’ ( Pleuroncodes monodon) and anchoveta ( Engraulis ringens) off Peru

    NASA Astrophysics Data System (ADS)

    Gutiérrez, Mariano; Ramirez, Argiro; Bertrand, Sophie; Móron, Octavio; Bertrand, Arnaud

    2008-10-01

    The world’s largest mono-specific fishery, the Peruvian anchovy or anchoveta ( Engraulis ringens) fishery, has been the subject of many studies since the 1960s. Details of its relationship with other species have mainly focused on alternations with sardine, Sardinops sagax, and little effort has so far been paid to interactions with other species sharing the same ecosystem. This is the case for Pleuroncodes monodon, the crustacean squat lobster or ’munida’, which has become highly abundant along the Peruvian coast since the mid-1990s. Munida is now an important prey for seabirds, mammals and coastal predatory fish. Knowledge of patterns of distribution and ecological niche of munida is scarce however off Peru. Here we describe and compare spatial patterns of distribution of anchoveta and munida and their ecological niches based on data from 26 acoustic surveys performed along the Peruvian coast between 1998 and 2006. The results indicate that munida and anchoveta share ecological niches but that munida is restricted to the coldest part of the productive cold coastal waters whereas anchoveta do not present any temperature preference over a large range (14-23 °C). The recent increase in munida abundance off Peru is concomitant with colder conditions; with their onset munida extended its range from central Chile northwards. Off Peru the very shallow oxycline keeps munida from its usual bottom habitat and has forced it to adopt pelagic behaviour.

  14. 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

  15. Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses.

    PubMed

    Lynn, Dwight E

    2003-07-01

    Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.

  16. 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.

  17. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

    PubMed

    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  18. Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

    PubMed

    Sullender, W M; Britt, W J

    1996-04-01

    The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

  19. Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

    PubMed Central

    2012-01-01

    Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule. PMID:22953695

  20. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

    PubMed Central

    Medin, J A; Hunt, L; Gathy, K; Evans, R K; Coleman, M S

    1990-01-01

    Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins. Images PMID:2181448

  1. 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

  2. Induction of a subnuclear structure by the simultaneous expression of baculovirus proteins, IE1, LEF3, and P143 in the presence of hr

    SciTech Connect

    Nagamine, Toshihiro . E-mail: tnaga@riken.jp; Kawasaki, Yu; Matsumoto, Shogo

    2006-09-01

    Baculoviruses elicit the formation of a nuclear domain, called the virogenic stroma, in which viral DNA replication and nucleocapsid assembly occur. We had previously reported that nuclear focus formation of a transcriptional activator, IE1, is triggered by its binding to a viral DNA element, hr, and predicted that this hr-induced IE1 focus is an initial scaffold for the virogenic stroma. However, LEF3, a component of the virogenic stroma, did not localize to the IE1 foci. In exploring a mediator for its localization, we found that a baculovirus DNA helicase (P143), in combination with IE1 and hr, induced a subnuclear structure to which LEF3 localized and also that another component of the virogenic stroma, DBP, is able to localize to this structure. These results reveal that only four viral molecules are necessary to establish a nuclear domain which possesses a recruiting ability for a component of the virogenic stroma.

  3. High-level protein expression following single and dual gene cloning of infectious bronchitis virus N and S genes using baculovirus systems.

    PubMed

    Abdel-Moneim, Ahmed S; Giesow, Katrin; Keil, Günther M

    2014-03-01

    Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

  4. Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system.

    PubMed

    Song, Mira; Park, Da-Young; Kim, Youngkwan; Lee, Kyung-Jin; Lu, Zhe; Ko, Kinarm; Choo, Young Kug; Han, Yeon Soo; Ahn, Mi-Hyun; Oh, Doo-Byoung; Ko, Kisung

    2010-08-01

    Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P(10) and Polyhedrin promoters in the pFastBac dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb(I)) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb(I) from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb(I) had insect specific glycan structures that differed from their mammalian counterparts, mAb(I) similarly interacted with CD64 (FcgammaRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.

  5. A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

    PubMed

    Wu, Chunxiao; Wang, Shu

    2012-01-01

    Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

  6. Assessment of WSSV prevalence and distribution of disease-resistant shrimp among the wild population of Penaeus monodon along the west coast of India.

    PubMed

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

    2014-06-01

    Shrimp aquaculture is threatened by many diseases, among which white spot disease (WSD) caused by white spot syndrome virus (WSSV) is the leading one. Information related to the geographical distribution and seasonal prevalence of WSD is necessary to obtain a clear understanding of the disease biology in shrimp. Identification of WSD-resistant individual shrimp with DNA markers is also an important technique to develop better WSD-free shrimp health management. The present study aim is to estimate the occurrence of WSSV in Penaeus monodon qualitatively and quantitatively during three different seasons during the years 2011 to 2013 along the west coast of India. Additionally, the disease resistance prevalence using previously developed 71 bp microsatellite and 457 bp RAPD-SCAR DNA markers is also investigated. Samples were collected throughout the year from four locations along the west coast of India: Kochi, Kerala; Mangalore, Karnataka; Vasco-da-Gama, Goa; and Veraval, Gujarat. The results depicted that the average WSSV prevalence, as determined by the nested PCR method and taken cumulatively over the four locations, was the lowest (0%) during the post-monsoon season and the highest (31.6%) during the monsoon season. The WSD prevalence was observed to increase when the latitude was decreased along the west coast of India (from Veraval to Kochi). Out of the three different seasons, the average WSSV copy number was the highest (approximately 10(3) copies μg(-1) shrimp genomic DNA) during the monsoon season. The disease-resistant prevalence, as determined using the developed DNA markers, was found to be the highest in Vasco-da-Gama (59.5%) and the lowest in Kochi (40.9%). The present study suggests better options for the efficient collection of disease-free and disease-resistant brood stocks, which would be a more cost-effective and safer approach toward disease prevention over conventional trends of seed generation from unselected wild brood stock.

  7. Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

    PubMed

    Pham, K-C; Tran, H T T; Van Doan, C; Le, P H; Van Nguyen, A T; Nguyen, H A; Hong, H A; Cutting, S M; Phan, T-N

    2017-03-01

    In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 10(9 ) CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 10(9 ) CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 10(7)  CFU per g and ≥1 × 10(6)  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 10(9)  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 10(6)  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps.

  8. The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response.

    PubMed

    Tavarone, Eugenia; Molina, Guido Nicolás; Amalfi, Sabrina; Peralta, Andrea; Molinari, Paula; Taboga, Oscar

    2017-02-17

    In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.

  9. 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.

  10. 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

  11. Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells.

    PubMed

    Fu, Yuejun; Lin, Taotao; Liang, Aihua; Hu, Fengyun

    2016-05-01

    Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.

  12. High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.

    PubMed

    Sisk, W P; Bradley, J D; Leipold, R J; Stoltzfus, A M; Ponce de Leon, M; Hilf, M; Peng, C; Cohen, G H; Eisenberg, R J

    1994-02-01

    Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.

  13. Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins.

    PubMed Central

    Thomsen, D R; Roof, L L; Homa, F L

    1994-01-01

    The capsid of herpes simplex virus type 1 (HSV-1) is composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, which are the products of six HSV-1 genes. Recombinant baculoviruses were used to express the six capsid genes (UL18, UL19, UL26, UL26.5, UL35, and UL38) in insect cells. All constructs expressed the appropriate-size HSV proteins, and insect cells infected with a mixture of the six recombinant baculoviruses contained large numbers of HSV-like capsids. Capsids were purified by sucrose gradient centrifugation, and electron microscopy showed that the capsids made in Sf9 cells had the same size and appearance as authentic HSV B capsids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the protein composition of these capsids was nearly identical to that of B capsids isolated from HSV-infected Vero cells. Electron microscopy of thin sections clearly demonstrated that the capsids made in insect cells contained the inner electron-translucent core associated with HSV B capsids. In infections in which single capsid genes were left out, it was found that the UL18 (VP23), UL19 (VP5), UL38 (VP19C), and either the UL26 (VP21 and VP24) or the UL26.5 (VP22a) genes were required for assembly of 100-nm capsids. VP22a was shown to form the inner core of the B capsid, since in infections in which the UL26.5 gene was omitted the 100-nm capsids that formed lacked the inner core. The UL35 (VP26) gene was not required for assembly of 100-nm capsids, although assembly of B capsids was more efficient when it was present. These and other observations indicate that (i) the products of the UL18, UL19, UL35, and UL38 genes self-assemble into structures that form the outer surface (icosahedral shell) of the capsid, (ii) the products of the UL26 and/or UL26.5 genes are required (as scaffolds) for assembly of 100-nm capsids, and (iii) the interaction of the outer surface of the capsid with the scaffolding proteins requires the product

  14. 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

  15. Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

    PubMed

    Aparna, Gunda; Bhuyan, Abani K; Sahdev, Sudhir; Hasnain, Seyed E; Kaufman, Randal J; Ramaiah, Kolluru V A

    2003-12-30

    Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

  16. Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production

    PubMed Central

    Liu, Yang; de Jong, Jondavid; Nagy, Éva; Theilmann, David A.

    2016-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is in the family Baculoviridae, genus Alphabaculovirus. AcMNPV me53 is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Although me53 is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions, and peptides fused with hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCE Baculovirus me53 is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as

  17. [Characterization of hepatitis C virus structural proteins and HCV-like particles produced in recombinant baculovirus infected insect cells].

    PubMed

    Belzhelarskaia, S N; Koroleva, N N; Popenko, V V; Drutsa, V L; Orlova, O V; Rubtsov, P M; Kochetkov, S N

    2010-01-01

    Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.

  18. 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

  19. 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.

  20. Component co-expression and purification of recombinant human pyruvate dehydrogenase complex from baculovirus infected SF9 cells.

    PubMed

    Jiang, Yong; Wang, Juan; Zhang, Guofeng; Oza, Khyati; Myers, Linda; Holbert, Marc A; Sweitzer, Sharon

    2014-05-01

    The mammalian pyruvate dehydrogenase complex (PDC) is a multi-component mitochondrial enzyme that plays a key role in the conversion of pyruvate to acetyl-CoA connecting glycolysis to the citric acid cycle. Recent studies indicate that targeting the regulation of PDC enzymatic activity might offer therapeutic opportunities by inhibiting cancer cell metabolism. To facilitate drug discovery in this area, a well defined PDC sample is needed. Here, we report a new method of producing functional, recombinant, high quality human PDC complex. All five components were co-expressed in the cytoplasm of baculovirus-infected SF9 cells by deletion of the mitochondrial localization signal sequences of all the components and E1a was FLAG-tagged to facilitate purification. The protein FLAG tagged E1a complex was purified using FLAG-M2 affinity resin, followed by Superdex 200 sizing chromatography. The E2 and E3BP components were then Lipoylated using an enzyme based in vitro process. The resulting PDC is over 90% pure and homogenous. This non-phosphorylated, lipoylated human PDC was demonstrated to produce a robust detection window when used to develop an enzyme coupled assay of PDHK.

  1. A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements.

    PubMed

    Kawashima, Tomoko; Osanai, Mizuko; Futahashi, Ryo; Kojima, Tetsuya; Fujiwara, Haruhiko

    2007-07-01

    Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions.

  2. Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

    PubMed

    Muraki, Michiro; Honda, Shinya

    2011-11-01

    To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.

  3. Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs.

    PubMed

    Westenberg, Marcel; Soedling, Helen M; Mann, Derek A; Nicholson, Linda J; Dolphin, Colin T

    2010-09-01

    Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

  4. Construction of a Baculovirus-Silkworm Multigene Expression System and Its Application on Producing Virus-Like Particles

    PubMed Central

    Su, Shuo; Yao, Ning; He, Jian; Peng, Li; Sun, Jingchen

    2012-01-01

    A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes. PMID:22403668

  5. Significant reduction of fungal disease symptoms in transgenic lupin (Lupinus angustifolius) expressing the anti-apoptotic baculovirus gene p35.

    PubMed

    Wijayanto, Teguh; Barker, Susan J; Wylie, Stephen J; Gilchrist, David G; Cowling, Wallace A

    2009-10-01

    Narrow-leafed lupin (NLL; Lupinus angustifolius) is a recently domesticated but anciently propagated crop with significant value in rotation with cereals in Mediterranean climates. However, several fungal pathogens, traditionally termed necrotrophs, severely affect broad-acre production and there is limited genetic resistance in the NLL germplasm pool. Symptoms of many of these diseases appear as localized areas of dead cells exhibiting markers of programmed cell death. Based on our previous research, we hypothesized that engineered expression of the baculovirus anti-apoptotic p35 gene might reduce symptoms of these diseases. Using Agrobacterium tumefaciens-mediated transformation of a cultivar highly susceptible to several pathogens, 14 independent NLL lines containing both the p35 and bar genes were obtained (p35-NLL). Integration and expression of the transgenes were confirmed by polymerase chain reaction (PCR), progeny testing, Southern blot, Northern blot and reverse transcriptase-PCR analyses. Fecundity and nodulation were not altered in these lines. Third or fourth generation p35-NLL lines were challenged with necrotrophic fungal pathogens (anthracnose in stem and leaf, and Pleiochaeta root rot and leaf brown spot) in controlled environment conditions. Several p35-NLL lines had significantly reduced disease symptoms. Interestingly, as with natural resistance, no single line was improved for all three diseases which possibly reflecting spatial variation of p35 expression in planta. These data support an alternative molecular definition for 'necrotrophic disease' in plants and suggest new routes for achieving resistance against a range of pathogens.

  6. Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV)

    PubMed Central

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M.; Sugumar, Vimal; Taju, Gani; Sahul Hameed, A. S.

    2017-01-01

    Background White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. Methods/Principal Findings Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional “gold standard test”, viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen’s kappa coefficient of 0.983 suggested "very good agreement” between the developed LFIA and the conventional one-step PCR. Conclusion The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry. PMID:28046005

  7. 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.

  8. A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon.

    PubMed

    Lv, Jing; Pan, Yu; Ju, Huijun; Zhou, Jinxin; Cheng, Dengfeng; Shi, Hongcheng; Zhang, Yifan

    2017-03-06

    Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18(th) and 196(th) day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo.

  9. Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system.

    PubMed

    Li, Xing-hua; Wang, Dan; Zhou, Fang; Yang, Hua-jun; Bhaskar, Roy; Hu, Jia-biao; Sun, Chun-guang; Miao, Yun-gen

    2010-12-01

    Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and β-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.

  10. 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.

  11. 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.

  12. A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

    PubMed Central

    Lv, Jing; Pan, Yu; Ju, Huijun; Zhou, Jinxin; Cheng, Dengfeng; Shi, Hongcheng; Zhang, Yifan

    2017-01-01

    Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo. PMID:28262785

  13. Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp.

    PubMed

    Panichareon, Benjaporn; Khawsak, Paisarn; Deesukon, Warin; Sukhumsirichart, Wasana

    2011-12-01

    A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40fg, 50fg, and 500fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers.

  14. Study on the Distribution of Disease-Resistant Shrimp Identified by DNA Markers in Respect to WSSV Infection in Different Seasons Along the Entire East Coast of India Aiming to Prevent White Spot Disease in Penaeus monodon.

    PubMed

    Mallik, A; Chakrabarty, U; Dutta, S; Mondal, D; Mandal, N

    2016-02-01

    White spot disease caused by white spot syndrome virus (WSSV) is responsible for harming shrimp aquaculture industry and results in a pandemic throughout the world. Undeniably, the knowledge on geographic distribution, transmission, virulence, and seasonal prevalence of this disease alongside information on the distribution of disease-resistant shrimps may be helpful to understand important aspects of disease biology. This study was intended to estimate WSSV prevalence by qualitative and quantitative PCR method among the Penaeus monodon samples collected from four different places namely Digha, West Bengal; Chilika, Orissa; Visakhapatnam, Andhra Pradesh; and Chennai, Tamil Nadu at three different seasons in the period of 2011-2013 from east coast of India. Along with this, the disease-resistant prevalence was also investigated using earlier developed 71 bp microsatellite and 457 bp RAPD-SCAR DNA marker among the collected shrimps. Qualitative PCR depicted that the cumulative WSSV prevalence at four places was the lowest (0%) at pre-monsoon, whereas, it was the highest (21.2%) during post-monsoon season. Quantitative real-time PCR showed the average copy number of WSSV to be the highest (~10(3) copy μg(-1) shrimp genomic DNA) at post-monsoon season. Additionally, estimated disease-resistant prevalence was the highest in Visakhapatnam (79%) and lowest in Digha (21%). It is well known to all that a trait cannot be identified using a single genetic pattern. This study will significantly contribute insight to develop specific pathogen-resistant (SPR) seeds of P. monodon simultaneously using two DNA markers that would be a cost-effective and safer approach towards disease prevention instead of conventional trends of seed generation from unselected wild broodstock.

  15. 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.

  16. Evaluating a naturally occurring baculovirus for extended biological control of the black cutworm (Lepidoptera: Noctuidae) in golf course habitats.

    PubMed

    Bixby-Brosi, Andrea J; Potter, Daniel A

    2010-10-01

    Golf courses are a potential market for microbial insecticides, but how intensive management of such sites interacts with efficacy of entomopathogens is poorly known. We evaluated Agrotis ipsilon nucleopolyhedrovirus (AgipMNPV) for suppressing black cutworms, Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae), in turf representative of golf course habitats and on whole tees under actual play. In independent trials on sand- or soil-based putting greens and surrounds, or fairway-height creeping bentgrass (Agrostis stolonifera L.), < or = 1-wk-old AgipMNPV residues (10 x 10(8) occlusion bodies [OBs] per m2) typically gave 50-60% lethal infection of introduced third instars. In most cases, however, there was no residual control beyond 2-4 wk. Spraying fairway-height bentgrass with AgipMNPV alone (10 x 10(9) OBs per m2) gave 90, 85, and 7% infection of second instars introduced 4 d, 3 wk, or 5 wk later, but adjuvants (optical brightener, lignin, or both) intended to synergize and protect the virus from UV degradation did not extend infectivity. Fresh (< 1-wk-old) AgipMNPV residues killed 76-86% of neonates hatching from eggs on tees under play, but levels of control plummeted within a few weeks. Three species of braconids, an encyrtid Copidosoma bakeri (Howard), and a tachinid, Bonnetia comta (Fallen) collectively killed 24-31% of larvae recovered from those tees. AgipMNPV seems better suited for targeted control of early instars than for season-long control. Golf turf is a severe environment for baculoviruses so several applications per growing season would probably be needed to maintain high enough titers on grass foliage to effectively control caterpillar pests.

  17. Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

    PubMed Central

    Vihko, P; Kurkela, R; Porvari, K; Herrala, A; Lindfors, A; Lindqvist, Y; Schneider, G

    1993-01-01

    Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A. Images PMID:8430088

  18. Angiopoietin-1-expressing adipose stem cells genetically modified with baculovirus nanocomplex: investigation in rat heart with acute infarction.

    PubMed

    Paul, Arghya; Nayan, Madhur; Khan, Afshan Afsar; Shum-Tim, Dominique; Prakash, Satya

    2012-01-01

    The objective of this study was to develop angiopoietin-1 (Ang1)-expressing genetically modified human adipose tissue derived stem cells (hASCs) for myocardial therapy. For this, an efficient gene delivery system using recombinant baculovirus complexed with cell penetrating transactivating transcriptional activator TAT peptide/deoxyribonucleic acid nanoparticles (Bac-NP), through ionic interactions, was used. It was hypothesized that the hybrid Bac- NP(Ang1) system can efficiently transduce hASCs and induces favorable therapeutic effects when transplanted in vivo. To evaluate this hypothesis, a rat model with acute myocardial infarction and intramyocardially transplanted Ang1-expressing hASCs (hASC-Ang1), genetically modified by Bac-NP(Ang1), was used. Ang1 is a crucial pro-angiogenic factor for vascular maturation and neovasculogenesis. The released hAng1 from hASC-Ang1 demonstrated profound mitotic and anti-apoptotic activities on endothelial cells and cardiomyocytes. The transplanted hASC-Ang1 group showed higher cell retention compared to hASC and control groups. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with hASC-Ang1 treatment compared to infarcted hearts treated with hASC or the untreated group. Furthermore, the hASC-Ang1 group showed significantly higher cardiac performance in echocardiography (ejection fraction 46.28% ± 6.3%, P < 0.001 versus control, n = 8) than the hASC group (36.35% ± 5.7%, P < 0.01, n = 8), 28 days post-infarction. The study identified Bac-NP complex as an advanced gene delivery vehicle for stem cells and demonstrated its potential to treat ischemic heart disease with high therapeutic index for combined stem cell-gene therapy strategy.

  19. Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system.

    PubMed

    Liu, Yu-Kuo; Yang, Ching-Jen; Liu, Chao-Lin; Shen, Chia-Rui; Shiau, Lie-Ding

    2010-08-01

    Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.

  20. 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.

  1. Baculovirus VP80 protein and the F-actin cytoskeleton interact and connect the viral replication factory with the nuclear periphery.

    PubMed

    Marek, Martin; Merten, Otto-Wilhelm; Galibert, Lionel; Vlak, Just M; van Oers, Monique M

    2011-06-01

    Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.

  2. Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

    PubMed

    Kolpe, Annasaheb B; Kiener, Tanja K; Grotenbreg, Gijsbert M; Kwang, Jimmy

    2012-09-01

    Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

  3. 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.

  4. 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

  5. A peptide with similarity to baculovirus ODV-E66 binds the gut epithelium of Heliothis virescens and impedes infection with Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Sparks, Wendy O; Rohlfing, Amy; Bonning, Bryony C

    2011-05-01

    Baculoviruses infect their lepidopteran hosts via the midgut epithelium through binding of occlusion-derived virus (ODV) and fusion between the virus envelope and microvillar membranes. To identify genes and sequences that are involved in this process, a random phage display library was screened for peptides that bound to brush border membrane vesicles (BBMV) derived from the midgut epithelium of Heliothis virescens. Seventeen peptides that bound to BBMV were recovered. Two of these, HV1 and HV2, had sequence similarity to the ODV envelope protein ODV-E66 that is found in five species of alphabaculoviruses. Chemically synthesized versions of HV1 and HV2, and two peptides (AcE66A and AcE66B) derived from similar sequences of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV-E66, bound to unfixed cryosections of whole midgut tissues. AcE66A, but not HV1, bound to H. virescens gut BBMV proteins on a far-Western blot. Competition assays with HV1 and purified AcMNPV ODV resulted in decreased mortality of H. virescens larvae at a dose of 1 LD(50), and a significant increase in survival time at higher virus concentrations. These results suggest a role for ODV-E66 in baculovirus infection of lepidopteran larval midgut epithelium.

  6. Occlusion-derived baculovirus: interaction with human cells and evaluation of the envelope protein P74 as a surface display platform.

    PubMed

    Mäkelä, Anna R; Tuusa, Jenni E; Volkman, Loy E; Oker-Blom, Christian

    2008-06-01

    To develop complementary baculovirus-based tools for gene delivery and display technologies, the interaction of occlusion-derived baculovirus (ODV) with human cells, and the functionality of the P74 ODV envelope protein for display of the IgG-binding Z domains (ZZP74) were evaluated. The cellular binding of ODV was concentration-dependent and saturable. Only minority of the bound virions were internalized at both 37 and 4 degrees C, suggesting usage of direct membrane fusion as the entry mode. The intracellular transport of ODV was confined in vesicular structures peripheral to the plasma membrane, impeding subsequent nuclear entry and transgene expression. Transduction of ODV was not rescued by mimicking the preferred alkaline environment and lowered temperature of the ODV infective entry, or following treatment with the microtubule depolymerizing agent nocodazole or with the histone deacetylase inhibitor sodium butyrate. Similar to unmodified P74, the ZZP74 chimera localized in the intranuclear ring zone, and was enriched in virus-induced microvesicles. However, Western blotting of ODV and budded virions (BV), as well as viral envelope and nucleocapsid fractions combined with functional infection/transduction studies revealed incorporation of the ZZP74 fusion protein into viral nucleocapsids. The ZZP74 BV preserved normal infectivity, polypeptide profile, and morphology, but became incapable of entering and transducing human cells.

  7. Recombinant baculovirus as a highly potent vector for gene therapy of human colorectal carcinoma: molecular cloning, expression, and in vitro characterization.

    PubMed

    Paul, Arghya; Jardin, Barbara A; Kulamarva, Arun; Malhotra, Meenakshi; Elias, Cynthia B; Prakash, Satya

    2010-06-01

    Present therapeutic strategies for most cancers are restricted mainly to the primary tumors and are also not very effective in controlling metastatic states. Alternatively, gene therapy can be a potential option for treating such cancers. Currently mammalian viral-based cancer gene therapy is the most popular approach, but the efficacy has been shown to be quite low in clinical trials. In this study, for the first time, the insect cell-specific baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been evaluated as a vector for gene delivery to colorectal cancer cells. Experiments involving factorial design were employed to study the individual and combined effects of different parameters such as multiplicity of infection (MOI), viral incubation time and epigenetic factors on transduction efficiency. The results demonstrate that baculovirus gene delivery system holds immense potential for development of a new generation of highly effective virotherapy for colorectal, as well as other major carcinomas (breast, pancreas, and brain), and offers significant benefits to traditional animal virus-based vectors with respect to safety concerns.

  8. 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

    2015-07-31

    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.

  9. Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP.

    PubMed

    Hohdatsu, Tsutomu; Yamato, Hiroshi; Ohkawa, Tasuku; Kaneko, Miyuki; Motokawa, Kenji; Kusuhara, Hajime; Kaneshima, Takashi; Arai, Setsuo; Koyama, Hiroyuki

    2003-12-02

    The Type II feline infectious peritonitis virus (FIPV) infection of feline macrophages is enhanced by a monoclonal antibody (MAb) to the S protein of FIPV. This antibody-dependent enhancement (ADE) activity increased with the MAb that showed a neutralizing activity with feline kidney cells, suggesting that there was a distinct correlation between ADE activity and the neutralizing activity. The close association between enhancing and neutralizing epitopes is an obstacle to developing a vaccine containing only neutralizing epitopes without enhancing epitopes. In this study, we immunized cats with cell lysate with recombinant baculovirus-expressed N protein of the Type I FIPV strain KU-2 with an adjuvant and investigated its preventive effect on the progression of FIP. Cats immunized with this vaccine produced antibodies against FIPV virion-derived N protein but did not produce virus-neutralizing antibodies. A delayed type hypersensitivity skin response to N protein was observed in these vaccinated cats, showing that cell mediated immunity against the FIPV antigen was induced. When these vaccinated cats were challenged with a high dose of heterologous FIPV, the survival rate was 75% (6/8), while the survival rate in the control group immunized with SF-9 cell-derived antigen was 12.5% (1/8). This study showed that immunization with the cell lysate with baculovirus-expressed N protein was effective in preventing the progression of FIP without inducing ADE of FIPV infection in cats.

  10. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    PubMed

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  11. Baculovirus-mediated expression of human apolipoprotein E in Manduca sexta larvae generates particles that bind to the low density lipoprotein receptor.

    PubMed Central

    Gretch, D G; Sturley, S L; Friesen, P D; Beckage, N E; Attie, A D

    1991-01-01

    Human apolipoprotein E (apoE) is a ligand for the low density lipoprotein (LDL) receptor and mediates the catabolism of several classes of lipoprotein particles. Binding of apoE to the LDL receptor requires association of apoE with lipid in a vesicle or a lipoprotein particle. Because of this requirement, purified apoE or apoE derived directly from bacterial expression systems does not bind to the LDL receptor. To overcome this problem and to facilitate analysis of apoE structure, recombinant baculoviruses containing the human apoE cDNA fused to the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus were constructed. The recombinant viruses were used to infect larvae of the tobacco hornworm Manduca sexta in vivo. High levels of lipoprotein particles containing human apoE were present in the hemolymph of infected larvae. In contrast to apoE produced by recombinant baculovirus-infected insect cells in vitro, these particles were excellent ligands for the LDL receptor. Images PMID:1924311

  12. 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

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Vaca, Luis; 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.

  13. Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells

    PubMed Central

    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

  14. Secretory expression of porcine interferon-gamma in baculovirus using HBM signal peptide and its inhibition activity on the replication of porcine reproductive and respiratory syndrome virus.

    PubMed

    Wang, Yan-Bin; Wang, Zhen-Ya; Chen, Hong-Ying; Cui, Bao-An; Wang, Ya-Bin; Zhang, Hong-Ying; Wang, Rui

    2009-12-15

    The gene sequence encoding mature porcine interferon-gamma (PoIFN-gamma) fused with a C-terminal 6x histidine tag was cloned into the baculovirus pFastBac Dual vector of the Bac-to-Bac Baculovirus expression system under the control of PH promoter. The authentic signal sequence of porcine interferon-gamma was substituted with the honeybee melittin (HBM) signal sequence, and expressed in insect cells. The recombinant proteins were detected by SDS-PAGE and immunofluorescence assay. The nickel affinity column purified recombinant porcine interferon-gamma with HBM signal peptide (rPoIFN-gammaH) was shown to be a 19kDa protein as confirmed by Western blot analysis. The recombinant PoIFN-gammaH was shown to have cytokine activity, inhibiting the cytopathic effect of vesicular stomatitis virus (VSV) in PK-15 cells at about 1.07x10(6)U/mL. The 2(-7) dilution of the rPoIFN-gammaH in culture supernatant protected the MARC-145 cells from the cytopathic effect caused by 100TCID(50) of porcine reproductive and respiratory syndrome virus.

  15. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection.

    PubMed

    Plana Duran, J; Climent, I; Sarraseca, J; Urniza, A; Cortés, E; Vela, C; Casal, J I

    1997-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tübingen isolates and between 61-64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.

  16. Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells.

    PubMed

    Pan, Chuanying; Jia, Wenchao; Lu, Baisong; Bishop, Colin E

    2015-02-10

    Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)-fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36kD, 40kD, 24kD, and 36kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirus-infected Sf9 insect cells will be the method of choice for cellular reprogramming.

  17. 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

  18. Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: the case of melanocortin 4 receptors.

    PubMed

    Veiksina, Santa; Kopanchuk, Sergei; Rinken, Ago

    2014-01-01

    We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.

  19. [Construction of a recombinant baculovirus transfer vector with two promoters expressing the anti-human CD28 chimeric antibody by using TP-PCR method].

    PubMed

    Zhu, Yan; Chen, Yong-Jing; Qiu, Yu-Hua; Zheng, Feng-Feng; Zhu, Jiang

    2005-09-01

    CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.

  20. Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits.

    PubMed

    Zhang, Huawei; Li, Xiangmin; Peng, Guiqing; Tang, Chenkai; Zhu, Shixuan; Qian, Suhong; Xu, Jinfang; Qian, Ping

    2014-11-20

    Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.

  1. Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

    PubMed Central

    Mena, Jimmy A; Ramírez, Octavio T; Palomares, Laura A

    2007-01-01

    Background The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry. Results In single infections, the population infected with any of the recombinant baculoviruses followed the Poisson distribution, as the population expressing a recombinant protein exhibited a hyperbolic-type function with respect to the multiplicity of infection (MOI) up to 5 pfu/cell. In coinfections, the population fraction expressing each recombinant protein could not be anticipated from results of single infections, as in some cases interference and synergistic effects were found. Only cultures with a total MOI below 5 pfu/cell followed the Poisson distribution. For cultures with a MOI of bacGFPVP2 above that of bacVP6 (overall MOI above 5 pfu/cell), the total population expressing one or both recombinant proteins was as low as 50% below that predicted by Poisson. In contrast, the population fraction expressing VP6 increased in coinfections, compared to that in single infections. The largest population fraction simultaneously expressing both recombinant proteins was 58%, and corresponded to cultures infected at a MOI of 5 and 1 pfu/cell of bacGFPVP2 and bacVP6, respectively

  2. 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

  3. [Expression and subcellular localization of the ORF4 gene of barley yellow dwarf virus GAV strain in baculovirus-insect cell expression system].

    PubMed

    Xia, Zong-Liang; Wang, Mei-Ping; Liu, Quan-Jun; Wang, Dao-Wen

    2007-11-01

    According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four a-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.

  4. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  5. 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.

  6. Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1.

    PubMed

    Xu, Jian; Nagata, Yudai; Mon, Hiroaki; Li, Zhiqing; Zhu, Li; Iiyama, Kazuhiro; Kusakabe, Takahiro; Lee, Jae Man

    2013-07-01

    RNA interference (RNAi) is a biological phenomenon that silences the expression of genes of interest. Passive double-stranded RNA (dsRNA) uptake has been uniquely observed in Caenorhabditis elegans due to the expression of systemic RNAi defective-1 (SID-1). We report that ectopic expression of CeSID-1 endows the Sf9 cells with a capacity for soaking RNAi. Soaking the Sf9-SID1 with dsRNA corresponding to either exogenous or endogenous target genes induced a significant decrease in the amount of mRNA or protein. These results enabled us to modify the target proteins of baculovirus expression vector system in both quantities and posttranslational modifications. The current low-cost and high-efficiency RNAi system is useful for high-throughput gene function analysis and mass production of recombinant protein.

  7. Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.

    PubMed

    Ollo, R; Maniatis, T

    1987-08-01

    The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos. Characterization of the Krüppel protein extracted from infected cells showed that it is tightly bound to the nucleus, it binds to calf thymus DNA-cellulose, and it is phosphorylated. These results support the hypothesis that Krüppel is a regulatory protein that acts by binding DNA.

  8. 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.

  9. 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.

  10. A nanobiohybrid complex of recombinant baculovirus and Tat/DNA nanoparticles for delivery of Ang-1 transgene in myocardial infarction therapy.

    PubMed

    Paul, Arghya; Binsalamah, Zyad M; Khan, Afshan A; Abbasia, Sana; Elias, Cynthia B; Shum-Tim, Dominique; Prakash, Satya

    2011-11-01

    The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NP(Ang1) showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than Bac(Ang1), with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NP(Ang1) to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than Bac(Ang1)). Furthermore, the Bac-NP(Ang1) group showed significantly higher cardiac performance in echocardiography than Bac(Ang1) (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NP(Ang1) and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications.

  11. Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    PubMed Central

    Makvandi, Manoochehr; Teimoori, Ali; Neisi, Niloofar; Samarbafzadeh, Alireza

    2016-01-01

    Background The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5α and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results The lengths of subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. PMID:28138375

  12. Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA.

    PubMed

    Oem, Jae-Ku; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Yong-Joo; Kye, Soo-Jeong; Park, Jee-Yong; Song, Hee-Jong

    2007-05-16

    Non-infectious recombinant pentamer-like structures of the foot-and-mouth disease virus (FMDV) were expressed by baculovirus, and the antigenicity and immunogenicity of the proteins were analyzed in a blocking ELISA for the detection of FMDV antibodies. The recombinant pentamer-like structures were produced in insect (Sf9) cells that were inoculated with recombinant baculoviruses that expressed, simultaneously, the genes for the P1 and 3C proteins of FMDV from individual promoters. The FMDV pentamer-like structures were processed by viral 3C protease, as shown in Western blots, and were antigenic, as revealed by their reactivities in an indirect ELISA. Analysis by CsCl gradient centrifugation showed that the pentamer-like structures were similar to authentic pentameric subunits from FMDV in terms of sedimentation velocity. Furthermore, the pentamer-like structures induced high levels of FMDV-specific antibodies in mice following immunization. Observations made under the electron microscope revealed that the pentamer-like structures expressed by insect cells self-assembled to form pentameric subunits of 7-8 nm in diameter, which resemble the authentic FMDV (23+/-2 nm in diameter). The results indicate that these pentamer-like structures are as antigenic and immunogenic as authentic FMDV, although the former are smaller in size. Based on these results, a blocking ELISA was developed using the recombinant pentamer-like structure. The ELISA showed specificity of 99.5% and sensitivity of 98.5% when tested with FMDV antibody-negative and -positive sera, respectively. This blocking ELISA is highly specific and offers many advantages over the current ELISAs that use inactivated FMDV antigen. This is the first report of the production and diagnostic application of recombinant pentameric subunits of FMDV.

  13. Optimization of heme precursors for the expression of human cytochrome P450 2A13 and its co-expression with oxidoreductase in baculovirus/sf9 system.

    PubMed

    Lu, Hui-Yuan; Qiu, Liang-Lin; Yang, Xue-Jiao; Zhang, Xiao-Ming; Zhang, Zhan; Wang, Shou-Lin

    2013-06-01

    Human cytochrome P450 2A13 (CYP2A13), mainly expressed in respiratory tract, is active towards numerous toxicants. To establish the metabolism in vitro, we expressed CYP2A13 and NADPH-CYP450 oxidoreductase (POR) in a baculovirus/sf9 system. Due to the deficiency of sf9 cells in heme incorporation, we investigated the effects of different heme precursors on the expression of CYP2A13, POR and their co-expression. The present results showed that both CYP2A13 and POR were presented the highest expression levels or activity with 0.2 mM δ-aminolaevulinic acid (5-ALA), 0.02 mM Fe(3+) and 0.5-1.0 μg/ml hemin. The combination of 0.2 mM 5-ALA and 0.02 mM Fe(3+) significantly improved CYP2A13 expression and content compared with heme precursors alone, so was POR activity. A multiplicity of infection (MOI) value of 5 pfu/cell for CYP2A13 baculovirus particles induced very high CYP2A13 expression. When co-infected with different POR MOI values, a viral ratio of 5 : 2 was associated with the highest CYP2A13 activity, whereas POR activity dose dependently increased with POR MOI. Furthermore, the expressed CYP2A13 in the optimized conduction could eliminate its substrate aflatoxin B1 at a significantly higher than those in other condition (P < 0.01). Our results provide an efficient approach for expressing functionally characterized, highly active and homogeneous CYP2A13 proteins.

  14. Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems.

    PubMed

    Matindoost, Leila; Nielsen, Lars K; Reid, Steve

    2015-05-05

    To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as "good" and "bad" BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA was lower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields.

  15. Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression

    PubMed Central

    Hanson, Michael A.; Brooun, Alexei; Baker, Kent A.; Jaakola, Veli-Pekka; Roth, Chris; Chien, Ellen; Alexandrov, Alexander; Velasquez, Jeffrey; Davis, Leila; Griffith, Mark; Moy, Kin; Ganser-Pornillos, Barbie; Kuhn, Peter; Ellis, Sam; Yeager, Mark; Stevens, Raymond C.

    2007-01-01

    Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple construct expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and coupled with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human β2-adrenergic receptor (β2AR) and gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of fourteen β2AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild type β2AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecyl maltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell micro-expression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants. PMID:17723307

  16. Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression.

    PubMed

    Hanson, Michael A; Brooun, Alexei; Baker, Kent A; Jaakola, Veli-Pekka; Roth, Chris; Chien, Ellen Y T; Alexandrov, Alexander; Velasquez, Jeffrey; Davis, Leila; Griffith, Mark; Moy, Kin; Ganser-Pornillos, Barbie K; Hua, Yuanzi; Kuhn, Peter; Ellis, Sam; Yeager, Mark; Stevens, Raymond C

    2007-11-01

    Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.

  17. 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.

  18. The role of host cell physiology in the productivity of the baculovirus-insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Alves, Paula M

    2017-03-01

    The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.

  19. Characterization of the interaction between P143 and LEF-3 from two different baculovirus species: Choristoneura fumiferana nucleopolyhedrovirus LEF-3 can complement Autographa californica nucleopolyhedrovirus LEF-3 in supporting DNA replication.

    PubMed

    Chen, Tricia; Sahri, Daniela; Carstens, Eric B

    2004-01-01

    The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.

  20. 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

  1. Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

    PubMed

    Shippam-Brett, C E; Willis, L G; Theilmann, D A

    2001-05-01

    Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

  2. Development of monoclonal antibody-linked ELISA for sero-diagnosis of vesicular stomatitis virus (VSV-IN) using baculovirus expressed glycoprotein.

    PubMed

    Kweon, Chang Hee; Kwon, Byung Joon; Kim, In Joong; Lee, Se Young; Ko, Young Joon

    2005-12-01

    The gene encoding the envelope glycoprotein (GP) of vesicular stomatitis virus serotype, Indiana (VSV-IN), was expressed under the polyhedron promoter of baculovirus. The recombinant GP was applied as a diagnostic antigen for the detection of cattle and horse antibodies to VSV. In addition, the neutralizing monoclonal antibody (Mab) to GP of VSV-IN was used as trapping antibody in a Mab-linked indirect ELISA (MLI-ELISA) or detecting antibody in a Mab-linked competitive ELISA (MLC-ELISA). The diagnostic efficiencies of MLI-ELISA and MLC-ELISA were evaluated with currently available C-ELISA from OIE reference laboratory for vesicular stomatitis as a gold standard by using VSV-positive equine sera and negative bovine sera vaccinated against foot-and-mouth disease (FMD) in the field. When naturally infected equine sera and FMDV vaccinated bovine sera were tested, MLI-ELISA and MLC-ELISA showed relative sensitivities of 80% and 95% with relative specificity of 97% and 99%, respectively. However, both ELISAs cross-reacted with equine sera against New Jersey (VSV-NJ) serotype. The comparison of the two ELISAs revealed that MLC-ELISA was relatively more sensitive and specific than MLI-ELISA, indicating that MLC-ELISA can be applied to sero-diagnosis for VSV-IN infection.

  3. 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.

  4. 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.

  5. 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

  6. Evaluation of baculovirus-derived recombinant 53-kDa protein of Trichinella spiralis for detection of Trichinella-specific antibodies in domestic pigs by ELISA.

    PubMed

    Jung, Doreen; Teifke, Jens Peter; Karger, Axel; Michael, Kathrin; Venz, Simone; Wittmann, Wolfgang; Kindermann, Katharina; Nöckler, Karsten; Mundt, Egbert

    2007-02-01

    The complete gene encoding the 53-kDa protein derived from Trichinella spiralis was cloned and expressed using a baculovirus-based system. Characterization of a purified fusion protein consisting of the 53-kDa protein and the glutathione S-transferase protein showed unspecific reactivity with swine pre-immune serum in both enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Subsequently, a purified C-terminal 6xHis-tagged 53-kDa protein was used in an ELISA. The evaluation of the test using a negative serum panel showed a high specificity for the ELISA. Serum panels of pigs infected with T. spiralis of two independent experiments showed that pigs of one experiment were tested positive by the ELISA, whereas all sera of the second experiment were negative, indicating a low sensitivity of the ELISA. Furthermore, experimental evidence was found by using mass spectroscopy and Western blot analysis that the 53-kDa protein was not part of the excretory/secretory antigen of T. spiralis as shown in this study.

  7. Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.

    PubMed

    Yue, Wanfu; Miao, Yungen; Li, Xinghua; Wu, Xiaofeng; Zhao, Aichun; Nakagaki, Masao

    2006-11-01

    Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

  8. 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.

  9. Co-inoculation of baculovirus and FMDV vaccine in mice, elicits very early protection against foot and mouth disease virus without interfering with long lasting immunity.

    PubMed

    Quattrocchi, V; Molinari, P; Langellotti, C; Gnazzo, V; Taboga, O; Zamorano, P

    2013-05-31

    Baculoviruses (Bvs) potentiate the immune response against soluble antigens. We investigated whether Bv could be used as immunoactivator in foot-and-mouth disease (FMD) vaccines using the BALB/c mouse model. Mice were vaccinated with a single dose of inactivated FMDV (iFMDV), iFMDV+Bv, Bv, or culture medium. Humoral and cellular immune responses were higher in animals immunized with iFMDV+Bv than in mice vaccinated with iFMDV alone. Animals receiving iFMDV+Bv had significantly lower viremia at 2, 4 and 7dpv, than those immunized with iFMDV alone. In order to prolong the immune response, iFMDV oil vaccine was co-inoculated with Bv. Animals receiving iFMDV oil vaccine+Bv were protected two days earlier than those receiving the iFMDV oil vaccine alone. Both formulations protected until 14dpv, the last day of the experiment. This is the first report in which Bv is used as an adjuvant in a FMDV vaccine.

  10. Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge.

    PubMed

    Bhat, S A; Saravanan, P; Hosamani, M; Basagoudanavar, S H; Sreenivasa, B P; Tamilselvan, R P; Venkataramanan, R

    2013-12-01

    Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination.

  11. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    PubMed

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  12. Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

    PubMed

    Lerner, C G; Saiki, A Y

    1996-09-05

    DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

  13. Characterization of the baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain.

    PubMed

    Wilson, J A; Hill, J E; Kuzio, J; Faulkner, P

    1995-12-01

    The DNA sequence and transcription pattern of the p10 gene from the spruce budworm baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV) were analysed. The CfMNPV p10 gene codes for a protein 81 amino acids in length: this is the shortest p10 peptide identified thus far. A novel characteristic of the CfMNPV p10 gene is that it contains tandem late initiation sites (TAAG) having different temporal transcription patterns. Comparative analysis of CfMNPV p10 and the amino acid sequences of other p10 gene products showed that they each appear to have a similar N-terminal structure: an amphipathic alpha-helical terminus which condenses as coiled-coil multimers. Another feature of the p10 N terminus is that the central region of the coiled-coil domain resembles a bend or hairpin loop and could fold into a hairpin or form a bent rod structure. The condensation of p10 monomers to coiled-coil multimers is likely to be a step leading to the formation of p10 fibrous bodies in infected cells.

  14. 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

  15. Historic emergence, impact and current status of shrimp pathogens in the Americas.

    PubMed

    Lightner, D V; Redman, R M; Pantoja, C R; Tang, K F J; Noble, B L; Schofield, P; Mohney, L L; Nunan, L M; Navarro, S A

    2012-06-01

    Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the

  16. Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-3'-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA.

    PubMed

    Delaney, W E; Miller, T G; Isom, H C

    1999-08-01

    (-)-Beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 microM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same

  17. Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

    PubMed Central

    Godeau, F; Saucier, C; Kourilsky, P

    1992-01-01

    The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation. Images PMID:1335569

  18. Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a method to restore cell-specific beta-trace glycoprotein productivity at high cell density.

    PubMed

    Chico, E; Jäger, V

    2000-12-05

    The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

  19. Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P

    PubMed Central

    Bai, Fan; Caster, Hannah M.; Rubenstein, Peter A.; Dawson, John F.; Kawai, Masataka

    2014-01-01

    Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2+], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2+ sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers, and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0

  20. Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38

    PubMed Central

    Gebhardt, Manuela M.; Eberle, Karolin E.; Radtke, Pit; Jehle, Johannes A.

    2014-01-01

    The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth. PMID:25331863

  1. Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system.

    PubMed Central

    Morais, V A; Serpa, J; Palma, A S; Costa, T; Maranga, L; Costa, J

    2001-01-01

    The human alpha-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4 unit/l (1 mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were k(cat)=0.4 s(-1) and K(m)=0.87 mM for the SFT3 secreted by Tn cells, and k(cat)=0.09 s(-1) and K(m)=0.76 mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII. PMID:11171070

  2. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene.

    PubMed

    McCarthy, Christina B; Theilmann, David A

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC(ac142)(REP-ac143KO)). Fluorescence and light microscopy showed that infection by AcBAC(ac142)(REP-ac143KO) is limited to a single cell and titration assays confirmed that AcBAC(ac142)(REP-ac143KO) was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC(ac142)(REP-ac143KO) transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  3. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    SciTech Connect

    McCarthy, Christina B.; Theilmann, David A.

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC{sup ac142REP-ac143KO}). Fluorescence and light microscopy showed that infection by AcBAC{sup ac142REP-ac143KO} is limited to a single cell and titration assays confirmed that AcBAC{sup ac142REP-ac143KO} was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC{sup ac142REP-ac143KO} transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  4. Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form.

    PubMed

    Imamura, Morikazu; Kato, Nobuko; Iwamaru, Yoshifumi; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2017-01-02

    We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrP(Sc)) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrP(C) source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

  5. Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system.

    PubMed

    Grego, E; Uslenghi, F; Strasser, M; Luzzago, C; Frigerio, M; Peletto, S; Rosati, S

    2007-01-01

    The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.

  6. Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A.

    PubMed

    Jayakumar, Arumugam; Kang, Ya'an; Mitsudo, Kenji; Henderson, Ying; Frederick, Mitchell J; Wang, Mary; El-Naggar, Adel K; Marx, Ute C; Briggs, Katrina; Clayman, Gary L

    2004-05-01

    The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.

  7. Epidemiological study of Norwalk virus infections in Japan and Southeast Asia by enzyme-linked immunosorbent assays with Norwalk virus capsid protein produced by the baculovirus expression system.

    PubMed Central

    Numata, K; Nakata, S; Jiang, X; Estes, M K; Chiba, S

    1994-01-01

    In this study, we investigated Norwalk virus (NV) antigen and antibody to recombinant NV (rNV) in human populations in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs). Baculovirus-expressed recombinant NV (rNV) capsid protein was used for preparing antisera to rNV or used as an antigen for detecting antibody to rNV. The ELISAs were specific for NV and had sensitivities equivalent to or higher than those of the previously developed radioimmunoassays. In 159 stool samples obtained from children, mainly younger than 10 years old, with acute gastroenteritis due to small round structured viruses in Japan, only 1 was positive for NV antigen. The pattern of acquisition of antibody to rNV was quite different from those of antibodies to group A rotavirus and human calicivirus Sapporo (HuCV-Sa) strain. The prevalence of antibody to rNV remained at a low level throughout childhood and then showed a steep rise during school age and early adulthood in Japan. A high prevalence of antibody was observed in samples collected from healthy adults in Japan and Southeast Asia. These results suggested that NV infection is common in adults in Japan and Southeast Asia but may be rare in infants in Japan. The HuCV-Sa strain was negative by the ELISA, and no serological relationship between NV and the HuCV-Sa strain was found. NV may be quite different from the HuCV-Sa strain, although both viruses are classified in the family Caliciviridae. PMID:8126165

  8. Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems.

    PubMed

    Kim, Woe Yeon; Lee, Sun Yong; Jung, Young Jun; Chae, Ho Byoung; Nawkar, Ganesh M; Shin, Mi Rim; Kim, Sun Young; Park, Jin Ho; Kang, Chang Ho; Chi, Yong Hun; Ahn, Il Pyung; Yun, Dae Jin; Lee, Kyun Oh; Kim, Young-Myeong; Kim, Min Gab; Lee, Sang Yeol

    2011-12-09

    A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

  9. Expression of Baculovirus Anti-Apoptotic Genes p35 and op-iap in Cotton (Gossypium hirsutum L.) Enhances Tolerance to Verticillium Wilt

    PubMed Central

    Liang, Benguo; Li, Shanwei; Wu, Zhixia; Wang, Qianhua; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2010-01-01

    Background Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored. Methodology/Principal Findings In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T3 generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T1–T3 generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines's 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD). Conclusion/Significance Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants. PMID:21151969

  10. Cannibalism and virus production in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae fed with two leaf substrates inoculated with Baculovirus spodoptera.

    PubMed

    Valicente, F H; Tuelher, E S; Pena, R C; Andreazza, R; Guimarães, M R F

    2013-04-01

    Cannibalism in the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) (FAW), is a limiting factor in a baculovirus production system. To detect the impact of cannibalism, a two-step bioassay was conducted with different larval ages of FAW fed on two food sources (corn and castor bean leaves) contaminated with the S. frugiperda multiple-embedded nucleopolyhedrovirus. In a first bioassay, the food source affected the cannibalism, being higher for all larval ages tested (5-, 6- and 7-day-old larvae) in larvae fed on corn than on those fed on castor bean leaves. Larval mortality, weight equivalent and larval equivalents (LEs) per hectare decreased as the larval age increased. Larval weight, occlusion bodies (OBs)/larva and total OBs increased when the larval age increased. In a second bioassay, in which only 6- and 7-day-old larvae were used because of the performance in the first bioassay, the cannibalism rates were affected by the interaction between food sources and time of feeding (48 and 72 h), reaching the highest values for 6- and 7-day-old larvae fed on corn leaves for 72 h. Mortality of the FAW was affected by the interaction between food sources, larval age and time of feeding. The lowest mortalities were on 7-day-old larvae when they were fed on castor bean leaves for 48 and 72 h. Larval weight, OBs/larva, total OBs and LEs were affected by the interaction between food sources and larval age. A significant correlation was observed between larval weight and OBs/larva that fed on both food sources, suggesting that larval weight can be used to achieve a concentration to be sprayed in 1 ha.

  11. Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.

    PubMed

    Cadepond, F; Binart, N; Chambraud, B; Jibard, N; Schweizer-Groyer, G; Segard-Maurel, I; Baulieu, E E

    1993-11-15

    Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

  12. Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38.

    PubMed

    Gebhardt, Manuela M; Eberle, Karolin E; Radtke, Pit; Jehle, Johannes A

    2014-11-04

    The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.

  13. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

    PubMed Central

    Vialard, J; Lalumière, M; Vernet, T; Briedis, D; Alkhatib, G; Henning, D; Levin, D; Richardson, C

    1990-01-01

    An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified

  14. 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.

  15. Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication

    PubMed Central

    Zhu, Mengxiao; Deng, Riqiang

    2016-01-01

    ABSTRACT An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae. IMPORTANCE Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a

  16. Rapid screening of purification strategies for the capture of a human recombinant F(ab')2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS.

    PubMed

    Pezzini, J; Brenac Brochier, V; Barrouillet, M P; Cerruti, M; Clofent-Sanchez, G; Schapman, A; Topol, A; Robert, R; Cabanne, C; Cerruti, P; Santarelli, X

    2009-08-15

    The development of a capture step of a human recombinant F(ab')(2) produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab')(2) was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.

  17. 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

  18. Disruption of the baculovirus core gene ac78 results in decreased production of multiple nucleocapsid-enveloped occlusion-derived virions and the failure of primary infection in vivo.

    PubMed

    Li, Sai-Nan; Wang, Jin-Yu; Yuan, Mei-Jin; Yang, Kai

    2014-10-13

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac78 gene is one of the baculovirus core genes. Recent studies showed that ac78 is essential for budded virion (BV) production and the embedding of occlusion-derived virion (ODV) into occlusion body during the AcMNPV life cycle. Here, we report that an ac78-knockout AcMNPV (vAc78KO) constructed in this study had different phenotypes than those described in the previous studies. A few infectious BVs were detected using titer assays, immunoblot analyses and plaque assays, indicating that ac78 is not essential for BV formation. Electron microscopy confirmed that the ac78 deletion did not affect nucleocapsid assembly and ODV formation. However, the numbers of multiple nucleocapsid-enveloped ODVs and ODV-embedded occlusion bodies were significantly decreased. Subsequently, the highly conserved amino acid residues 2-25 and 64-88 of Ac78, which are homologous to an oxidoreductase and cytochrome c oxidase, respectively, were demonstrated to play a crucial role in the morphogenesis of multiple nucleocapsid-enveloped ODV. Immunoblot analysis found that Ac78 was an ODV envelope-associated protein. Consistently, amino acid residues 56-93 of Ac78 were identified as an inner nuclear membrane sorting motif, which may direct the localization of Ac78 to the ODV envelope. In vivo infectivity assays showed that the occlusion bodies of vAc78KO were unable to establish primary infection in the midgut of Trichoplusia ni larvae. Taken together, our results suggest that ac78 plays an important role in BV production and proper multiple nucleocapsid-enveloped ODV formation, as well as AcMNPV primary infection in vivo.

  19. Assessment of the protection afforded by triple baculovirus recombinant coexpressing H5, N3, M1 proteins against a homologous H5N3 low-pathogenicity avian influenza virus challenge in Muscovy ducks.

    PubMed

    Prel, Anne; Le Gall-Reculé, Ghislaine; Cherbonnel, Martine; Grasland, Béatrice; Amelot, Michel; Jestin, Véronique

    2007-03-01

    In Asia, domestic ducks have been shown to play a pivotal role in H5 high-pathogenicity avian influenza virus transmission. We have also observed that the same situation may exist for H5 low-pathogenicity avian influenza (LPAI) virus. No data are available regarding the protection afforded by commercial inactivated vaccines against H5 LPAI virus infection in ducks, and two preliminary experiments using commercial inactivated vaccines gave poor results. Virus-like particles (VLPs) have been shown to be immunogenic in different species. With regard to the influenza model, the matrix (M) protein has been shown to be necessary for the formation of VLPs. In order to attempt to develop a VLP influenza vaccine expressing hemagglutinin and neuraminidase (NA) of interest, we generated a triple recombinant baculovirus (rB) expressing three structural proteins: H5, N3, and M, derived from a recent French LPAI virus strain. Although the three proteins were successfully expressed in rB-infected cells and displayed the expected biological activity, no VLPs were observed. Despite this result, the protection afforded to ducks by rB-infected cell lysates was assessed and was compared with the protection afforded by an inactivated commercial H5N9 vaccine. For this purpose, specific-pathogen-free Muscovy ducks (15 per group) received rB-infected cell lysates (3 wk apart), while a second group received the H5N9 vaccine. Ten days after the boost, a homologous virus challenge was implemented. Both vaccines induced positive hemagglutination inhibition titers and M immune response, whereas lysates of rB-infected cells elicited NA immune response. Tracheal and cloacal sheddings were measured using M-based real-time-reverse transcription-polymerase chain reaction and were compared with the sheddings of vaccinated and unvaccinated infected controls. Lysates of rB-infected cells afforded a significant decrease of cloacal shedding and a delayed peak of tracheal shedding, whereas the

  20. Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells.

    PubMed

    Hayman, A R; Cox, T M

    1994-01-14

    The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5

  1. 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

  2. Vestigial tooth anatomy and tusk nomenclature for monodon monoceros.

    PubMed

    Nweeia, Martin T; Eichmiller, Frederick C; Hauschka, Peter V; Tyler, Ethan; Mead, James G; Potter, Charles W; Angnatsiak, David P; Richard, Pierre R; Orr, Jack R; Black, Sandie R

    2012-06-01

    Narwhal tusks, although well described and characterized within publications, are clouded by contradictory references, which refer to them as both incisors and canines. Vestigial teeth are briefly mentioned in the scientific literature with limited descriptions and no image renderings. This study first examines narwhal maxillary osteoanatomy to determine whether the erupted tusks are best described as incisiform or caniniform teeth. The study also offers evidence to support the evolutionary obsolescence of the vestigial teeth through anatomic, morphologic, and histologic descriptions. Examination of 131 skull samples, including 110 museum skull specimens and 21 harvested skulls, revealed the erupted tusks surrounded by maxillary bone over the entire length of their bone socket insertion, and are thus more accurately termed caniniform or canine teeth. The anatomy, morphology, and development of vestigial teeth in five skull samples are more fully described and documented. Vestigial tooth samples included 14 embedded pairs or individual teeth that were partially exposed or removed from the maxillary bone. Their location was posterior, ventral, and lateral to the tusks, although male vestigial teeth often exfoliate in the mouth lodging between the palatal tissue and underlying maxillary bone. Their myriad morphologies, sizes, and eruption patterns suggest that these teeth are no longer guided by function but rather by random germ cell differentiation and may eventually cease expression entirely. The conclusions reached are that the narwhal tusks are the expression of canine teeth and that vestigial teeth have no apparent functional characteristics and are following a pattern consistent with evolutionary obsolescence.

  3. Mechanisms of colour adaptation in the prawn Penaeus monodon.

    PubMed

    Wade, Nicholas M; Anderson, Mike; Sellars, Melony J; Tume, Ron K; Preston, Nigel P; Glencross, Brett D

    2012-01-15

    Exposure of prawns to dark- or light-coloured substrates is known to trigger a strong colour adaptation response through expansion or contraction of the colouration structures in the prawn hypodermis. Despite the difference in colour triggered by this adaptive response, total levels of the predominant carotenoid pigment, astaxanthin, are not modified, suggesting that another mechanism is regulating this phenomenon. Astaxanthin binds to a specific protein called crustacyanin (CRCN), and it is the interaction between the quantities of each of these compounds that produces the diverse range of colours seen in crustacean shells. In this study, we investigated the protein changes and genetic regulatory processes that occur in prawn hypodermal tissues during adaptation to black or white substrates. The amount of free astaxanthin was higher in animals adapted to dark substrate compared with those adapted to light substrate, and this difference was matched by a strong elevation of CRCN protein. However, there was no difference in the expression of CRCN genes either across the moult cycle or in response to background substrate colour. These results indicate that exposure to a dark-coloured substrate causes an accumulation of CRCN protein, bound with free astaxanthin, in the prawn hypodermis without modification of CRCN gene expression. On light-coloured substrates, levels of CRCN protein in the hypodermis are reduced, but the carotenoid is retained, undispersed in the hypodermal tissue, in an esterified form. Therefore, the abundance of CRCN protein affects the distribution of pigment in prawn hypodermal tissues, and is a crucial regulator of the colour adaptation response in prawns.

  4. Review of Vaccinia Virus and Baculovirus Viability Versus Virucides

    DTIC Science & Technology

    2008-03-01

    including 55% ethanol (w/w) with 10% (w/w) propan -1-ol, 5.9% (w/w) propan -1.2-diol, 5.7% (w/w) butan-1.3-diol and 0.75% phosphoric acid. In suspension...to UV radiation. TiO2 prevents UV degradation by reflecting light energy rather than absorbing and is commonly used in paints , foods and sunscreens...et al., 2003). * Virucidal effects of an alcohol-based formulation including 55% ethanol (w/w) with 10% (w/w) propan -1-ol, 5.9% (w/w) propan -1.2-diol

  5. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

    PubMed Central

    Eldridge, R; Li, Y; Miller, L K

    1992-01-01

    We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. Images PMID:1404603

  6. 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...

  7. 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...

  8. 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.

  9. Three-Dimensional Geometry of the Narwhal (Monodon monoceros) Flukes in Relation to Hydrodynamics

    DTIC Science & Technology

    2011-10-01

    scales to improve discrimination of high-density elements. Images were obtained in DICOM formats, and for some data, were re- constructed at 100...archived onto magneto-optical disks for all scans in order to allow additional post-scan processing as needed. Image files were archived in DICOM format...and 512 matrices on CD. The DICOM images Table 1. Morphometrics of fluke planforms. Body Fluke Planform Aspect Sweepback Tusk Specimen Sex length (m

  10. Isolation and identification of Vibrio cholerae and Vibrio parahaemolyticus from prawn (Penaeus monodon) seafood: Preservation strategies.

    PubMed

    Yaashikaa, P R; Saravanan, A; Kumar, P Senthil

    2016-10-01

    Bacterial diseases are one of the major problems which affects the production, development and expansion of aqua culture. Vibrio sp. are widespread in marine and estuarine environments. The several pathogenic species are commonly associated with outbreaks of Vibrio species and it is mainly associated with food poisonings. In this research, the occurrence of Vibrio sp. was studied by the isolation and it is confirmed by the biochemical methods. The growth rate was studied by changing the different operating parameters. Isolation studies were done by using enrichment and selective plating methods. The different biochemical test was carried out and inferred that the isolated organisms were Vibrio choleraee and Vibrio parahaemolyticus. The antibiotic study was also performed to find out the resistant and sensitivity of the Vibrio species. From the results, it was observed that this can be able to correlate the growth of vibrio species to a limited condition and other environmental parameters for which it will be able to find the remedial measures to prevent the growth and spreading of the diseases. Also the different preservation method was carried out to suppress the growth rate of Vibrio sp.

  11. Application of molecular diagnostic methods to penaeid shrimp diseases: advances of the past 10 years for control of viral diseases in farmed shrimp.

    PubMed

    Lightner, D V; Poulos, B T; Tang-Nelson, K F J; Pantoja, C R; Nunan, L M; Navarro, S A; Redman, R M; Mohney, L L

    2006-01-01

    The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.

  12. Enumeration of coliforms and Escherichia coli in frozen black tiger shrimp Penaeus monodon by conventional and rapid methods.

    PubMed

    Suwansonthichai, Sasithorn; Rengpipat, Sirirat

    2003-03-15

    Conventional (most probable number, MPN) and rapid methods-including Chromocult coliform agar (CCA), Fluorocult(R) LMX broth (LMX), and Petrifilm Escherichia coli count plates (PEC) for enumeration of coliforms and E. coli in frozen black tiger shrimp from Thailand were compared in order to assess the possibility of using one of the rapid methods for routine analysis. Enumeration of coliforms and E. coli from 18 samples of regular frozen black tiger shrimp and 156 samples of frozen black tiger shrimp experimentally contaminated with coliforms or E. coli at concentrations of approximately 10, approximately 10(2), and approximately 10(3) CFU g(-1) revealed that at the level of approximately 10 CFU g(-1), coliform numbers ranked as LMX>CCA>MPN=PEC and E. coli as MPN=LMX=PEC=CCA. At the level of approximately 10(2) CFU g(-1), coliform numbers ranked as LMX>MPN=PEC=CCA and E. coli as MPN=LMX>PEC=CCA. At the level of 10(3) CFU g(-1), coliforms ranked as LMX>MPN=CCA>PEC and E. coli as MPN>LMX>CCA>PEC. Agreements with the conventional MPN method for coliforms were LMX 108%, PEC 87.2%, and CCA 91.2% and agreements for E. coli were LMX 101%, PEC 95.7%, and CCA 96.3%. Sensitivities (%) ranked LMX>MPN>CCA=PEC for coliforms and E. coli, whereas equal specificities (100%) of all methods for coliforms and E. coli were demonstrated. Rankings for the other parameters compared were: convenience, PEC>CCA=LMX>MPN; time to detection, MPN>LMX=PEC=CCA; expense, MPN=PEC>CCA>LMX; labor, MPN>LMX=CCA>PEC; accuracy for coliforms, PEC>CCA>MPN>LMX; and accuracy for E. coli, PEC=CCA>LMX>MPN.

  13. Process optimization for high-pressure processing of black tiger shrimp (Penaeus monodon) using response surface methodology.

    PubMed

    Kaur, Barjinder Pal; Rao, P Srinivasa

    2016-10-06

    This study aims to investigate the effect of high-pressure processing on the quality of black tiger shrimp using response surface methodology. A central composite rotatable design was applied to evaluate the effects of three processing parameters, namely pressure (300-600 MPa), temperature (30-50 ℃), and time (0-15 min), on the inactivation rate of Staphylococcus aureus and physical properties (color and texture) of shrimp and to optimize the process conditions to achieve maximum bacterial inactivation with minimal changes in quality attributes. The results revealed that the processing conditions significantly affected the studied responses and the experimental data have been adequately fitted into a second-order polynomial model with multiple regression coefficients (R(2)) of 0.92, 0.92, and 0.94 for the inactivation rate of S. aureus, hardness, and color changes, respectively. The optimized conditions targeting minimum six log cycle reductions of S. aureus with moderate changes in quality attributes were obtained as: pressure, 361 MPa; time, 12 min and temperature, 46 ℃. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data.

  14. Rapid diagnosis of vibriosis and white spot syndrome (WSS) in the culture of shrimp, Penaeus monodon in Philippines.

    PubMed

    Caipang, Christopher Marlowe A; Aguana, Mary Paz N

    2010-10-01

    Viral and bacterial pathogens have raised serious concerns in the sustainability of the shrimp culture industry in the Philippines. Heavy mortality associated with luminous vibriosis and white spot syndrome virus (WSSV) infection has been the major problem besetting the industry. Using published PCR protocols for the diagnosis of vibriosis and white spot syndrome virus (WSSV) disease in shrimp, we optimized these assays that could be suited to the shrimp aquaculture setting in the Philippines. Genomic DNAs of Vibrio spp. that exhibited luminescence as well as those that grew on thiosulfate citrate bile salt sucrose agar (TCBS) were used for the PCR amplification of the ribonuclease P (RNase P) gene. There was differential amplification of the RNase P gene based on the phenotypic characters of the Vibrio spp. Similar results were also obtained using direct colony PCR of the bacterial colonies. White spot syndrome virus was also detected in the infected shrimp and there were differences in the detection frequency in relation to the tissues used for PCR amplification. Duplex PCR was also optimized that could be used for simultaneous detection of these pathogens in shrimp.

  15. Genetic diversity of the giant tiger prawn Penaeus monodon in relation to trace metal pollution at the Tanzanian coast.

    PubMed

    Rumisha, Cyrus; Leermakers, Martine; Elskens, Marc; Mdegela, Robinson H; Gwakisa, Paul; Kochzius, Marc

    2017-01-30

    The genetic diversity of giant tiger prawns in relation to trace metals (TMs) pollution was analysed using 159 individuals from eight sites at the Tanzanian coast. The seven microsatellites analysed showed high degree of polymorphism (4-44 alleles). The measured genetic diversity (Ho=0.592±0.047) was comparable to that of populations in the Western Indian Ocean. Apart from that, correlation analysis revealed significant negative associations between genetic diversity and TMs pollution (p<0.05), supporting the genetic erosion hypothesis. Limited gene flow was indicated by a significant genetic differentiation (FST=0.059, p<0.05). The Mantel test rejected the isolation-by-distance hypothesis, but revealed that gene flow along the Tanzanian coast is limited by TMs pollution. This suggests that TMs affect larvae settlement and it may account for the measured deficiency of heterozygosity. This calls for strengthened pollution control measures in order to conserve this commercially important species.

  16. 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.

  17. 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

  18. 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...

  19. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.

    PubMed

    Noe, J; Hagenbuch, B; Meier, P J; St-Pierre, M V

    2001-05-01

    The bile salt export pump (Bsep), a member of the ATP-binding cassette superfamily of transporters, mediates the ATP-dependent canalicular secretion of bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein in Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation sites for protein kinase A, protein kinase C (PKC) and Ca(2+)-calmodulin dependent kinase II, we have also tested whether mBsep undergoes phosphorylation. MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than control membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinity and is the most potent inducer of ATPase activity. Cyclosporin A, glibenclamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt-stimulated ATPase activity. MBsep exists as a phospho-protein when expressed in Sf9 cells and the immunoprecipitated mBsep complex is a substrate for the catalytic subunit of PKC. When mBsep and the alpha-isoform of mouse PKC are co-expressed in Sf9 cells, a ninefold stimulation of phosphorylation occurs. This is further increased to 18-fold after activation by phorbol ester. Given that bile salts activate selected PKC isoforms in hepatocytes, including the alpha isoform, the phosphorylation of mBsep by PKCalpha may represent a point of regulation for this transporter that is mediated by its own substrate.

  20. Functional Analysis of the Putative Fusion Domain of the Baculovirus Envelope Fusion Protein F

    PubMed Central

    Westenberg, Marcel; Veenman, Frank; Roode, Els C.; Goldbach, Rob W.; Vlak, Just M.; Zuidema, Douwe

    2004-01-01

    Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect. PMID:15194771

  1. Chapter 9. routine maintenance and storage of lepidopteran insect cell lines and baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The various methods for maintaining (a.k.a., subculturing, splitting, or passaging) established lepidopteran cell lines are described. Three procedures are presented that are appropriate for different cell lines dependent upon the growth characteristics (in particular, cell attachment properties) o...

  2. 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.

  3. 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.

  4. A novel expression system based on host-range expansion of baculovirus.

    PubMed

    Zhu, Y; Qi, Y; Liu, D; Joshua, M N; Wang, Y

    1998-12-01

    A host range expanded recombinant Autographa californica multiple-nucleocapsid nucleopolyhedrosis virus AcMNPV/r2 was obtained by cotransfection of the bacmid DNA from Escherichia coli DH10Bac along with a plasmid pBmH-M containing HindIII M fragment of Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA. A recombinant transposon vector carrying a mutant green fluorescent protein gene (GFP) and a polyhedrin gene was constructed. Transposition was carried out in both E. coli DH10Bac and E. coli DH10BmH, which contains AcMNPV/r2 and a helper plasmid. Recombinant DNAs were transfected into Sf-9 cells to generate recombinant virus AcMNPV/r3 and AcMNPV/r4 respectively. Viral stock of AcMNPV/r4 was then infected into Bombyx mori cells (BmN) and Bombyx mori larvae (silkworm). Analysis shows that GFP was highly expressed in Bombyx mori larvae. This expression system, is practicable therefore for mass production of foreign gene products.

  5. 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...

  6. 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...

  7. 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

  8. Development of Safe, Effective Vaccines for Dengue Virus Disease by Recombinant Baculovirus. Subtitle: Development of Safe, Effective Vaccines for Dengue Disease Utilizing Viral Envelope and NSI Glycoproteins Expressed by Recombinant Baculovirus

    DTIC Science & Technology

    1991-09-16

    Eckels, K.H. (1989) pp. 351-356. In R. Lerner, H. Ginsberg, R.M. Chanock, and F. Brown (ed.)., Vaccine 89: Cold Spring Harbor Laboratory, Cold Spring Harbor , N.Y...Chanock, H.S. Ginsberg, and R. Lerner (ed.) 18 Vaccines 90: Cold Spring Harbor Laboratory, Cold Spring Harbor , N.Y. 10. Hahn, Y.S., Galler, R

  9. 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.

  10. Dynamics of Baculovirus Growth and Dispersal in Mamestra brassicae L. (LepidopteraNoctuidae) Larval Populations Introduced into Small Cabbage Plots

    PubMed Central

    Evans, Hugh F.; Allaway, Graham P.

    1983-01-01

    The nuclear polyhedrosis virus of Mamestra brassicae has been studied in larval populations of the moth introduced into small plots of cabbages. Primary dispersal of virus from single foci of infected larvae resulted from enhanced movement of the larvae, which colonized new plants logarithmically. Virus growth within the host population was quantified, and infection of young larvae in the following generation was related directly to the concentration of virus produced during the primary phase. Secondary cycling of virus resulted in dispersal of inoculum from multiple foci, and a large proportion of plants were ultimately colonized by infected larvae. The dynamics of virus growth during secondary dispersal were quantified and contrasted with results from the primary phase. The significance of these results is discussed in relation to possible control of insect pests through dispersal of virus by the host insect. PMID:16346197

  11. In situ cleavage of baculovirus occlusion-derived virus receptor binding protein P74 in the peroral infectivity complex.

    PubMed

    Peng, Ke; van Lent, Jan W M; Vlak, Just M; Hu, Zhihong; van Oers, Monique M

    2011-10-01

    Proteolytic processing of viral membrane proteins is common among enveloped viruses and facilitates virus entry. The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) protein P74 is part of a complex of essential peroral infectivity factors (PIFs). Here we report that P74 is efficiently cleaved into two fragments of about equal size by an occlusion body (OB) endogenous alkaline protease during ODV release when AcMNPV OBs are derived from larvae. The cleavage is specific for P74, since the other known peroral infectivity factors in the same complex (PIF1, PIF2, and PIF3) were not cleaved under the same conditions. P74 cleavage was not observed in OBs produced in three different insect cell lines, suggesting a larval host origin of the responsible protease. P74 in OBs produced in larvae of two different host species was cleaved into fragments with the same apparent molecular mass, indicating that the virus incorporates a similar alkaline protease from different hosts. Coimmunoprecipitation analysis revealed that the two P74 subunit fragments remain associated with the recently discovered PIF complex. We propose that under in vivo ODV infection conditions, P74 undergoes two sequential cleavage events, the first one being performed by an ODV-associated host alkaline protease and the second carried out by trypsin in the host midgut.

  12. Baculovirus-encoded protein BV/ODV-E26 determines tissue tropism and virulence in lepidopteran insects.

    PubMed

    Katsuma, Susumu; Kobayashi, Jun; Koyano, Yasue; Matsuda-Imai, Noriko; Kang, WonKyung; Shimada, Toru

    2012-03-01

    Lepidopteran nucleopolyhedroviruses (NPVs) show distinct tissue tropism in host insect larvae. However, the molecular mechanism of this tropism is largely unknown. We quantitatively investigated NPV tissue tropism by measuring mRNA levels of viral genes in 16 tissues from Bombyx mori NPV (BmNPV)-infected B. mori larvae and found clear tissue tropism, i.e., BmNPV replicates poorly in the silk glands, midgut, and Malpighian tubule compared with other larval tissues. We next identified the viral genes determining tissue tropism in NPV infection by investigating the phenotypes of larvae infected with 44 BmNPV mutants in which one gene was functionally disrupted by a LacZ cassette insertion. We found that occlusion body (OB) production was markedly enhanced compared with that of the wild type in the middle silk glands (MSGs) of larvae infected with three mutants in which one of three tandemly arrayed genes (Bm7, Bm8, and Bm9) was disrupted. We generated additional mutants in which one or two genes of this gene cluster were partially deleted and showed that Bm8, also known as BV/ODV-E26, was solely required for the suppression of OB production in the MSGs of BmNPV-infected B. mori larvae. Western blotting showed that a LacZ cassette insertion in Bm7 or Bm9 resulted in aberrant expression of Bm8, presumably leading to abnormal OB production in the MSGs. Larval bioassays also revealed that disruption of Bm8 accelerated the death of B. mori larvae. These results suggest that the group I NPV-specific protein BV/ODV-E26 determines tissue tropism and virulence in host lepidopteran insects.

  13. 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...

  14. 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.

  15. 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...

  16. Inactivation of baculovirus by isoflavonoids on chickpea (Cicer arietinum) leaf surfaces reduces the efficacy of nucleopolyhedrovirus against Helicoverpa armigera.

    PubMed

    Stevenson, Philip C; D'Cunha, Reju F; Grzywacz, David

    2010-02-01

    Biological pesticides based on nucleopolyhedroviruses (NPVs) can provide an effective and environmentally benign alternative to synthetic chemicals. On some crops, however, the efficacy and persistence of NPVs is known to be reduced by plant specific factors. The present study investigated the efficacy of Helicoverpa armigera NPV (HearNPV) for control of H. armigera larvae, and showed that chickpea reduced the infectivity of virus occlusion bodies (OBs) exposed to the leaf surface of chickpea for at least 1 h. The degree of inactivation was greater on chickpea than that previously reported on cotton, and the mode of action is different from that of cotton. The effect was observed for larvae that consumed OBs on chickpea leaves, but it also occurred when OBs were removed after exposure to plants and inoculated onto artificial diet, indicating that inhibition was leaf surface-related and permanent. Despite their profuse exudation from trichomes on chickpea leaves and their low pH, organic acids-primarily oxalic and malic acid-caused no inhibition. When HearNPV was incubated with biochanin A and sissotrin, however, two minor constituents of chickpea leaf extracts, OB activity was reduced significantly. These two isoflavonoids increased in concentration by up to 3 times within 1 h of spraying the virus suspension onto the plants and also when spraying only the carrier, indicating induction was in response to spraying and not a specific response to the HearNPV. Although inactivation by the isoflavonoids did not account completely for the level of effect recorded on whole plants, this work constitutes evidence for a novel mechanism of NPV inactivation in legumes. Expanding the use of biological pesticides on legume crops will be dependent upon the development of suitable formulations for OBs to overcome plant secondary chemical effects.

  17. Acetylcholinesterase of the Sand Fly, Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression, and Biochemical Properties

    DTIC Science & Technology

    2013-01-01

    papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of...identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17...classified as specific for butyrylcholinesterase. The enzyme is strongly inhibited by paraoxon and malaoxon (oxidized forms of parathion and malathion ) and

  18. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    PubMed

    Liao, Jen-Chung

    2016-07-05

    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.

  19. 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...

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

    PubMed

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

    2015-08-01

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

  1. Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV.

    PubMed

    Ustün-Aytekin, Ozlem; Gürhan, Ismet Deliloğlu; Ohura, Kayoko; Imai, Teruko; Ongen, Gaye

    2014-01-01

    Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL(-1)) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L(-1) h(-1)).

  2. Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Solenopsis invicta virus 3 (SINV-3) is an unclassified positive stranded RNA virus whose characteristics are amenable for development as a microbial insecticide or as a classical biological control agent for the red imported fire ant, Solenopsis invicta. Bait formulations containing SINV-3 have been...

  3. Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression.

    PubMed

    Jose, S; Jayesh, P; Sudheer, N S; Poulose, G; Mohandas, A; Philip, R; Singh, I S Bright

    2012-05-01

    Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

  4. Mercury distribution in the skin of beluga (Delphinapterus leucas) and narwhal (Monodon monoceros) from the Canadian Arctic and mercury burdens and excretion by moulting.

    PubMed

    Wagemann, R; Kozlowska, H

    2005-12-01

    Beluga and narwhal skin as a whole (in Inuktitut known as "muktuk") is considered to be a delicacy by native Canadian and Greenland people. Individual strata of the skin, and muscle from 27 beluga from the western, and 20 narwhal from the eastern Canadian Arctic, were analyzed for mercury and the thickness and density of each skin layer was measured. Mercury was not uniformly distributed in the skin, but increased outwardly with each layer. The concentration was only 0.29 and 0.16 microg/g (wet wt) in the innermost layer (dermis) of belugas and narwhal respectively, and 1.5 and 1.4 microg/g (wet wt) in the outermost layer (degenerative epidermis) of beluga and narwhal, respectively. There was a significant (alpha=0.05) association between age and mercury concentration in each skin layer, the regression coefficients progressively increasing from the inner layer (dermis) to the outer layer: 0.011-0.063 microg/g year-1; 0.034 microg/g year-1 for skin as a whole; 0.054 microg/g year-1 for muscle. The concentration of total mercury was 0.84 and 0.59 microg/g (wet wt) in skin as a whole (muktuk) of beluga and narwhal respectively, and 0.12 and 0.03 microg/g in blubber, respectively. The average, total mercury concentration in muscle tissue was 1.4 and 0.81 microg/g wet wt, in beluga and narwhal respectively, exceeding (except for blubber) the Canadian Government's Guideline (0.5 microg/g wet wt) for fish export and consumption. The skin surface area of an average-size beluga and narwhal was estimated (6.10 and 6.50 m2, respectively), as were excretions of mercury through moulting (13,861 and 6721 microg year-1; 14 and 7 mg year-1) for belugas and narwhal, respectively. The whole-body mercury burden (699,300 microg; 700 mg) for a 1000 kg beluga and its various tissues were estimated, as was the fraction of mercury excreted by moulting (2-0.42% of the whole-body burden). Annual mercury burden increments in beluga skin, muscle and the whole body were estimated (2750; 17,280; 40,00 microg year-1, respectively), using regression coefficients of age on mercury concentration. The annual gross mercury intake via food was estimated (131,400 microg), of which 70% was excreted.

  5. Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon.

    PubMed

    Patil, R; Shankar, K M; Kumar, B T N; Kulkarni, A; Patil, P; Moger, N

    2013-09-01

    A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions.

  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.

  7. Multiplex PCR (polymerase chain reaction) assay for detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in spiked shrimps (Penaeus monodon).

    PubMed

    Fakruddin, M D; Sultana, Mahmuda; Ahmed, Monzur Morshed; Chowdhury, Abhijit; Choudhury, Naiyyum

    2013-03-15

    The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.

  8. Expression of the Lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus infection.

    PubMed

    Barber, G N; Clegg, J C; Lloyd, G

    1990-01-01

    The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYM1, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

  9. Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus

    PubMed Central

    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

    2014-01-01

    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. Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

    DTIC Science & Technology

    1991-01-01

    396. experiments, and Dr. Erik Henchal for helpful sugges- 10. Rice CM, Lenches EM. Eddy SR. Shin SJ. Sheets tions concerning the experiments and the...these proteins allow the pro- lovirus vector. J Gen V’irol 68: 2607-2613. posal of a complete scheme of the proteolytic 33. Smith GE. Ju G, Erikson BL

  11. Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells.

    PubMed

    Brand, Walter; Oosterhuis, Berend; Krajcsi, Peter; Barron, Denis; Dionisi, Fabiola; van Bladeren, Peter J; Rietjens, Ivonne M C M; Williamson, Gary

    2011-12-01

    The citrus flavonoid hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of hesperetin, was studied using Sf9 membranes from cells overexpressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transporters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC(50) of 0.45 µM, suggesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRP ATPase activity at high H3'G concentrations was observed. This study provides information on the potential of hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of hesperetin.

  12. Baculovirus Expression of the Small Genome Segment of Hantaan Virus and Potential Use of the Expressed Nucleocapsid Protein as a Diagnostic Antigen

    DTIC Science & Technology

    1988-01-01

    to hantaviruses have been detected in rodents throughout most of the world, both in areas where HFRS is endemic and in regions where it has not been...evidence that the expressed nucleocapsid protein may be useful for the detection of antibodies to hantaviruses . METHODS Viruses, cells, and cDNA clones...sera (Table 1). Diagnostic potential of the expressed protein for hantaviruses other than Hantaan virus More than one hantavirus capable of causing HFRS

  13. 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.

  14. 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

    2014-10-07

    Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no lic