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Tosihiko Hukuhara - One of the best experts on this subject based on the ideXlab platform.
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enhanced infection of an Entomopoxvirus in larvae of the armyworm pseudaletia separata lepidoptera noctuidae by a granulovirus
Applied Entomology and Zoology, 2003Co-Authors: Tosihiko Hukuhara, Arman Wijonarko, Yasuko Hosokawa, Hidetoshi IwanoAbstract:To determine the interaction of Pseudaletia unipuncta granulovirus and Pseudaletia separata Entomopoxvirus, larvae of the armyworm, P. separata, were perorally administered graded doses of Entomopoxvirus occlusion bodies, spheroids, in the presence or absence of granulovirus occlusion bodies, capsules. The presence of capsules in the inocula enhanced Entomopoxvirus infection. In doubly infected larvae, the inclusion bodies and virions of the two viruses coexisted in the same fatbody but never occurred in the same cells. The granulovirus appeared to facilitate Entomopoxvirus infection at the organismal level, although the two viruses interfered with each other at the cellular level.
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A Bacterially Produced Virus Enhancing Factor from an Entomopoxvirus Enhances Nucleopolyhedrovirus Infection in Armyworm Larvae
Journal of invertebrate pathology, 2001Co-Authors: Tosihiko Hukuhara, Takahiko Hayakawa, Arman WijonarkoAbstract:Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata Entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.
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Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an Entomopoxvirus gene.
Nature biotechnology, 1999Co-Authors: Tosihiko Hukuhara, Takahiko Hayakawa, Arman WijonarkoAbstract:We have introduced an Entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.
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Detection of a virus enhancing factor in the spheroid, spindle, and virion of an Entomopoxvirus.
Journal of invertebrate pathology, 1998Co-Authors: Arman Wijonarko, Tosihiko HukuharaAbstract:Spheroids, spindles, and virions of an Entomopoxvirus (EPV) enhanced the infectivity of a nuclear polyhedrosis virus (NPV) when they were perorally administered to larvae of the armyworm, Pseudaletia separata. Spheroids and spindles at the same dose exhibited nearly the same enhancing activity. When the dose of spheroids or spindles was reduced 10 times, the median infectious dose of the NPV was increased approximately 100 times. An antiserum against an enhancing factor detected the homologous antigen in spheroids, spindles, and tissue-derived EPV virions but not in spheroid-derived virions.
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Biochemical Properties of an Enhancing Factor of an Entomopoxvirus
Journal of Invertebrate Pathology, 1994Co-Authors: Tosihiko HukuharaAbstract:Abstract Spheroids of a Pseudaletia separata Entomopoxvirus contained a factor which enhanced the infection of a nuclear polyhedrosis virus in larvae of the armyworm, P. separata . The enhancing factor (EF) was purified from the spheroid solution by two-step procedures consisting of gel filtration on a sephacryl column and adsorption chromatography on a hydroxyapatite column. The final EF preparation was about 20-fold greater in enhancing activity, based on ID 50 , than the original spheroid solution. SDS-polyacrylamide gel electrophoresis indicated that the EF was a homogeneous glycoprotein of 38 kDa. It could be stored frozen or lyophilized. Although the EF differed in molecular weight from a synergistic factor of a Pseudalelia unipuncla granulosis virus, they were considerably similar in amino acid composition and shared common features of high acidic amino acid contents.
David J. Dall - One of the best experts on this subject based on the ideXlab platform.
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Spindle bodies of Heliothis armigera Entomopoxvirus develop in structures associated with host cell endoplasmic reticulum.
Journal of invertebrate pathology, 2000Co-Authors: Joan Lai-fook, David J. DallAbstract:Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera Entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism.
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An Entomopoxvirus homologue of the vaccinia virus D13L-encoded ‘rifampicin resistance’ protein
Journal of General Virology, 1996Co-Authors: Rebecca J. Osborne, Teresa M. Symonds, Joan Lai-fook, Carol A. Fernon, Alagacone Sriskantha, David J. DallAbstract:The Heliothis armigera Entomopoxvirus (HaEPV) genome encodes a predicted 68 kDa polypeptide related to the ‘rifampicin resistance’ protein of vaccinia virus (with 30% identity), and an homologous swinepox virus protein (27% identity). We were unable to isolate an HaEPV genotypic variant encoding a predicted C-terminal truncated form of the protein, suggesting that the C terminus of the molecule may be essential to protein function, and, in turn, that this function may be essential to viral replication. HaEPV replication was substantially reduced in host cells exposed to rifampicin, but the observed cytotoxic properties of the drug made it impossible to determine the specific cause of that inhibition. We suggest that possession of a gene encoding a member of this polypeptide family might represent a defining molecular characteristic of the Poxviridae.
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Replication of Heliothis armigera Entomopoxvirus in vitro
Journal of Invertebrate Pathology, 1995Co-Authors: Carol A. Fernon, Rebecca J. Osborne, Joan Lai-fook, Angela P. Vera, Rosa Crnov, David J. DallAbstract:Abstract An Entomopoxvirus which replicates in Helicoverpa armigera larvae has been adapted to grow in cultured insect cell lines. The virus multiplies in cell lines derived from Helicoverpa zea, Spodoptera frugiperda, Trichoplusia ni, and Plutella xylostella . Twenty-nine clonal lines derived from the established H. zea cell line BCIRL-Hz-AM1 showed differences in susceptibility to infection. Serological assays revealed qualitative and quantitative variation in HaEPV replication and accumulation in different clonal lines and associated culture media. High-level nonlytic replication of the virus was observed in two clona H. zea lines.
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A gene encoding a highly expressed spindle body protein of Heliothis armigera Entomopoxvirus.
Journal of General Virology, 1993Co-Authors: David J. Dall, Alagacone Sriskantha, Joan Lai-fook, Angela P. Vera, Teresa M. SymondsAbstract:The gene encoding the most abundant protein of purified preparations of Heliothis armigera Entomopoxvirus (HaEPV) has been cloned and sequenced. The gene sequence encodes a 40.1K polypeptide with a putative N-terminal 20 amino acid leader peptide, and a single potential N-glycosylation site. Analysis of the protein, which has an apparent M r of 50K on polyacrylamide gels, confirmed post-translational loss of the leader peptide, but showed no evidence of glycosylation. The protein is related to others previously described from Choristoneura biennis EPV (63% identity) and Autographa californica nuclear polyhedrosis virus (42% identity). Polyclonal antiserum raised against a bacterial fusion protein containing the majority of the HaEPV protein specifically labelled HaEPV spindle bodies; confocal laser scanning microscopy suggests that the protein is distributed throughout those viral structures.
Arman Wijonarko - One of the best experts on this subject based on the ideXlab platform.
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enhanced infection of an Entomopoxvirus in larvae of the armyworm pseudaletia separata lepidoptera noctuidae by a granulovirus
Applied Entomology and Zoology, 2003Co-Authors: Tosihiko Hukuhara, Arman Wijonarko, Yasuko Hosokawa, Hidetoshi IwanoAbstract:To determine the interaction of Pseudaletia unipuncta granulovirus and Pseudaletia separata Entomopoxvirus, larvae of the armyworm, P. separata, were perorally administered graded doses of Entomopoxvirus occlusion bodies, spheroids, in the presence or absence of granulovirus occlusion bodies, capsules. The presence of capsules in the inocula enhanced Entomopoxvirus infection. In doubly infected larvae, the inclusion bodies and virions of the two viruses coexisted in the same fatbody but never occurred in the same cells. The granulovirus appeared to facilitate Entomopoxvirus infection at the organismal level, although the two viruses interfered with each other at the cellular level.
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A Bacterially Produced Virus Enhancing Factor from an Entomopoxvirus Enhances Nucleopolyhedrovirus Infection in Armyworm Larvae
Journal of invertebrate pathology, 2001Co-Authors: Tosihiko Hukuhara, Takahiko Hayakawa, Arman WijonarkoAbstract:Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata Entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.
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Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an Entomopoxvirus gene.
Nature biotechnology, 1999Co-Authors: Tosihiko Hukuhara, Takahiko Hayakawa, Arman WijonarkoAbstract:We have introduced an Entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.
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Detection of a virus enhancing factor in the spheroid, spindle, and virion of an Entomopoxvirus.
Journal of invertebrate pathology, 1998Co-Authors: Arman Wijonarko, Tosihiko HukuharaAbstract:Spheroids, spindles, and virions of an Entomopoxvirus (EPV) enhanced the infectivity of a nuclear polyhedrosis virus (NPV) when they were perorally administered to larvae of the armyworm, Pseudaletia separata. Spheroids and spindles at the same dose exhibited nearly the same enhancing activity. When the dose of spheroids or spindles was reduced 10 times, the median infectious dose of the NPV was increased approximately 100 times. An antiserum against an enhancing factor detected the homologous antigen in spheroids, spindles, and tissue-derived EPV virions but not in spheroid-derived virions.
Madoka Nakai - One of the best experts on this subject based on the ideXlab platform.
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Replication of Mythimna separata Entomopoxvirus in High Five™ cells and the construction of a recombinant.
Journal of invertebrate pathology, 2014Co-Authors: Jun Takatsuka, Madoka NakaiAbstract:Mythimna separata Entomopoxvirus (MySEV), of the genus BetaEntomopoxvirus, was found to replicate in High Five™ cells. The infected cells produced many occlusion bodies and were hypertrophied but did not lyse. Following infection at a multiplicity of infection of 0.1, titers of extracellular virus reached a plateau 3-4days post infection at 25°C and were estimated at ca. 3×10(5) plaque-forming units per ml in TC-100 or TMN-FH media, both of which contained fetal bovine serum (FBS). Serum free medium, Express Five® SFM, also supported virus replication in High Five™ cells, but the titers were approximately one-tenth of those grown in TC-100 or TMN-FH media containing FBS. Using High Five™ cells, a recombinant MySEV was successfully constructed using homologous recombination. This study opens an avenue to the evaluation of Entomopoxvirus gene functions using reverse genetic approaches with in vitro and in vivo hosts.
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New Insights into the Evolution of Entomopoxvirinae from the Complete Genome Sequences of Four Entomopoxviruses Infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata
Journal of Virology, 2013Co-Authors: Julien Thézé, Julie Gallais, Basil Arif, Madoka Nakai, Daniel Doucet, Jun Takatsuka, Zhen Li, Elisabeth HerniouAbstract:Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two Entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the BetaEntomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei Entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaEntomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the BetaEntomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaEntomopoxviruses so as to form a new genus.
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Effect of Entomopoxvirus infection of the smaller tea tortrix, Adoxophyes sp. on the development of the endoparasitoid, Ascogaster reticulatus
Entomologia Experimentalis et Applicata, 1997Co-Authors: Madoka Nakai, Takuro Sakai, Yasuhisa KunimiAbstract:Infection of Adoxophyes sp. (Lepidoptera: Tortricidae) larvae by an Entomopoxvirus (AsEPV) adversely affected the development of the endoparasitoid, Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae). Parasitoid larvae developing in AsEPV-infected hosts grew more slowly and spent more time in their hosts than did parasitoid larvae developing in noninfected hosts. Percentages of emergence of larval parasitoids that developed in AsEPV-infected hosts were significantly lower than those of parasitoids that developed in noninfected hosts. Parasitoid larvae in AsEPV-infected host perished when their hosts died of AsEPV infection. Significant numbers of parasitized and infected larvae exhibited apolysis to the final instar, whereas noninfected-parasitized larvae died in the penultimate instars due to emergence of parasitoids.
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Effect of Entomopoxvirus infection of the smaller tea tortrix, Adoxophyes sp. on the development of the endoparasitoid, Ascogaster reticulatus
Entomologia Experimentalis et Applicata, 1997Co-Authors: Madoka Nakai, Takuro Sakai, Yasuhisa KunimiAbstract:Infection of Adoxophyes sp. (Lepidoptera: Tortricidae) larvae by an Entomopoxvirus (AsEPV) adversely affected the development of the endoparasitoid, Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae). Parasitoid larvae developing in AsEPV-infected hosts grew more slowly and spent more time in their hosts than did parasitoid larvae developing in noninfected hosts. Percentages of emergence of larval parasitoids that developed in AsEPV-infected hosts were significantly lower than those of parasitoids that developed in noninfected hosts. Parasitoid larvae in AsEPV-infected host perished when their hosts died of AsEPV infection. Significant numbers of parasitized and infected larvae exhibited apolysis to the final instar, whereas noninfected-parasitized larvae died in the penultimate instars due to emergence of parasitoids.
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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characterization of mimivirus nad dependent dna ligase
Virology, 2006Co-Authors: Delphine Benarroch, Stewart ShumanAbstract:Abstract Mimivirus, a parasite of Acanthamoeba polyphaga , is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD + -dependent nick joining enzyme homologous to bacterial LigA and Entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD + specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix–hairpin–helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD + to form the ligase–AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The ΔBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD + -dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates Entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD + -dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.
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Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxvirus
Virology, 1997Co-Authors: Birgitte Ø. Petersen, Richard W. Moyer, Richard L. Hall, Stewart ShumanAbstract:Abstract We have identified an Amsacta moorei Entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase ( k obs = 0.08 sec −1 ; K cl = 0.22) are similar to those of the vaccinia virus enzyme.
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Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxvirus
Virology, 1997Co-Authors: Birgitte Ø. Petersen, Richard W. Moyer, Richard L. Hall, Stewart ShumanAbstract:Abstract We have identified an Amsacta moorei Entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase ( k obs = 0.08 sec −1 ; K cl = 0.22) are similar to those of the vaccinia virus enzyme.