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Takeshi Mikami - One of the best experts on this subject based on the ideXlab platform.
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characterization of neospora caninum surface protein ncsrs2 based on baculoVirus expression system and its application for serodiagnosis of neospora infection
Journal of Clinical Microbiology, 2001Co-Authors: Yoshifumi Nishikawa, Yuko Kousaka, Khajornsak Tragoolpua, Kozo Fujisaki, Takeshi Mikami, Levi Hakwale Makala, Xuenan Xuan, Hideyuki NagasawaAbstract:The baculoVirus expression system has proved to be a useful tool for the production of Recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the Recombinant Virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using Recombinant NcSRS2 for the serologic diagnosis of Neospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each Recombinant baculoVirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the Recombinant Virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the Recombinant baculoVirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera against Toxoplasma gondii, Hammondia heydorni, or N. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the Recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculoVirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninum infection.
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Immunisation of dogs with a canine herpesVirus vector expressing Neospora caninum surface protein, NcSRS2
International journal for parasitology, 2000Co-Authors: Yoshifumi Nishikawa, Xuenan Xuan, Hideyuki Nagasawa, Haruki Otsuka, Hiroyasu Ikeda, Shiya Fukumoto, Takeshi MikamiAbstract:In order to develop a vaccine against Neospora caninum in dogs, we constructed Recombinant canine herpesVirus (CHV) expressing N. caninum surface protein, NcSRS2. Indirect immunofluorescence indicated that the antigenic structure of the Recombinant NcSRS2 was similar to the authentic parasite protein. The dogs immunised with Recombinant Virus produced IgG antibody to N. caninum, and their sera recognised the parasite protein on Western blot. The dogs inoculated with Recombinant Virus showed no clinical symptoms and infectious CHV was not recovered from the dogs, suggesting that Recombinant CHV expressing N. caninum proteins may lead to a vaccine against neosporosis in dogs.
Hideyuki Nagasawa - One of the best experts on this subject based on the ideXlab platform.
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characterization of neospora caninum surface protein ncsrs2 based on baculoVirus expression system and its application for serodiagnosis of neospora infection
Journal of Clinical Microbiology, 2001Co-Authors: Yoshifumi Nishikawa, Yuko Kousaka, Khajornsak Tragoolpua, Kozo Fujisaki, Takeshi Mikami, Levi Hakwale Makala, Xuenan Xuan, Hideyuki NagasawaAbstract:The baculoVirus expression system has proved to be a useful tool for the production of Recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the Recombinant Virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using Recombinant NcSRS2 for the serologic diagnosis of Neospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each Recombinant baculoVirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the Recombinant Virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the Recombinant baculoVirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera against Toxoplasma gondii, Hammondia heydorni, or N. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the Recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculoVirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninum infection.
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Immunisation of dogs with a canine herpesVirus vector expressing Neospora caninum surface protein, NcSRS2
International journal for parasitology, 2000Co-Authors: Yoshifumi Nishikawa, Xuenan Xuan, Hideyuki Nagasawa, Haruki Otsuka, Hiroyasu Ikeda, Shiya Fukumoto, Takeshi MikamiAbstract:In order to develop a vaccine against Neospora caninum in dogs, we constructed Recombinant canine herpesVirus (CHV) expressing N. caninum surface protein, NcSRS2. Indirect immunofluorescence indicated that the antigenic structure of the Recombinant NcSRS2 was similar to the authentic parasite protein. The dogs immunised with Recombinant Virus produced IgG antibody to N. caninum, and their sera recognised the parasite protein on Western blot. The dogs inoculated with Recombinant Virus showed no clinical symptoms and infectious CHV was not recovered from the dogs, suggesting that Recombinant CHV expressing N. caninum proteins may lead to a vaccine against neosporosis in dogs.
Yoshifumi Nishikawa - One of the best experts on this subject based on the ideXlab platform.
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characterization of neospora caninum surface protein ncsrs2 based on baculoVirus expression system and its application for serodiagnosis of neospora infection
Journal of Clinical Microbiology, 2001Co-Authors: Yoshifumi Nishikawa, Yuko Kousaka, Khajornsak Tragoolpua, Kozo Fujisaki, Takeshi Mikami, Levi Hakwale Makala, Xuenan Xuan, Hideyuki NagasawaAbstract:The baculoVirus expression system has proved to be a useful tool for the production of Recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the Recombinant Virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using Recombinant NcSRS2 for the serologic diagnosis of Neospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each Recombinant baculoVirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the Recombinant Virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the Recombinant baculoVirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera against Toxoplasma gondii, Hammondia heydorni, or N. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the Recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculoVirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninum infection.
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Immunisation of dogs with a canine herpesVirus vector expressing Neospora caninum surface protein, NcSRS2
International journal for parasitology, 2000Co-Authors: Yoshifumi Nishikawa, Xuenan Xuan, Hideyuki Nagasawa, Haruki Otsuka, Hiroyasu Ikeda, Shiya Fukumoto, Takeshi MikamiAbstract:In order to develop a vaccine against Neospora caninum in dogs, we constructed Recombinant canine herpesVirus (CHV) expressing N. caninum surface protein, NcSRS2. Indirect immunofluorescence indicated that the antigenic structure of the Recombinant NcSRS2 was similar to the authentic parasite protein. The dogs immunised with Recombinant Virus produced IgG antibody to N. caninum, and their sera recognised the parasite protein on Western blot. The dogs inoculated with Recombinant Virus showed no clinical symptoms and infectious CHV was not recovered from the dogs, suggesting that Recombinant CHV expressing N. caninum proteins may lead to a vaccine against neosporosis in dogs.
Nikolaus Osterrieder - One of the best experts on this subject based on the ideXlab platform.
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a self excisable infectious bacterial artificial chromosome clone of varicella zoster Virus allows analysis of the essential tegument protein encoded by orf9
Journal of Virology, 2007Co-Authors: Karsten B Tischer, Benedikt B Kaufer, Marvin Sommer, Felix Wussow, Ann M Arvin, Nikolaus OsterriederAbstract:In order to facilitate the generation of mutant Viruses of varicella-zoster Virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing Recombinant Virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the Recombinant Virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon Virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant Virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant Virus using trans-complementation via baculoVirus-mediated gene transfer.
Linda A King - One of the best experts on this subject based on the ideXlab platform.
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assembly of amsacta moorei entomopoxVirus spheroidin into spheroids following synthesis in insect cells using a baculoVirus vector
Journal of General Virology, 1998Co-Authors: Susan A Marlow, Louise E Wilson, Alison M Lawrie, Nicola Wilkinson, Linda A KingAbstract:The gene encoding the major occlusion body protein, spheroidin, of Amsacta moorei entomopoxVirus (AmEPV) was introduced into a baculoVirus vector under control of the polyhedrin gene promoter. A Recombinant Virus produced large, ovoid occlusion body-like structures in both Spodoptera frugiperda and Trichoplusia ni cells. These structures resembled the spheroids found in AmEPV-infected Lymantria dispar cells, except they were devoid of Virus particles and were not surrounded by a membrane- or envelope-like structure. These results were confirmed by immunofluoresence microscopy and Western blotting using a specific antipeptide antibody to spheroidin, and suggest that the supramolecular assembly of spheroids is not dependent on other EPV-encoded gene products. Transmission electron microscopy and subcellular fractionation experiments revealed that the spheroid-like structures were assembled in both the nucleus and cytoplasm of the Recombinant Virus-infected cells. This contrasts with the solely cytoplasmic localization found in AmEPV-infected cells.
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Genetic modification of an entomopoxVirus: deletion of the spheroidin gene does not affect Virus replication in vitro
Journal of General Virology, 1995Co-Authors: Christopher P. Palmer, Susan A Marlow, Louise E Wilson, Alison M Lawrie, Davin P. Miller, Linda A KingAbstract:In the late stages of an entomopoxVirus infection, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the construction of a genetically modified Amsacta moorei entomopoxVirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique BamHI site in lieu of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A Recombinant Virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The Recombinant Virus was isolated from wild-type Virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the Recombinant Virus-infected cells. The Recombinant Virus was able to replicate to high titres (107 p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication in vitro. Moderate levels of CAT were synthesized in Recombinant Virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified Virus.