• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.122-128
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• 1992

Post-translational modifications of the nucleocapsid protein of bovine coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic $[^{32}P]$orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of bovine coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by $[^{32}P]$orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of bovine coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.

• International Journal of Industrial Entomology and Biomaterials
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• v.21 no.1
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• pp.117-125
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• 2010

The tropical tasar silkworms, Antheraea mylitta (D) produce famous silk 'Kosa' in central part of India. Due to outdoor rearing it became susceptible to viral infection including cytoplasmic polyhedrosis virus (CPV). The common mode of entry of cytoplasmic polyhedrosis virus is per os and cause gresserie disease to the larvae. Histopathological studies elucidated the insect CPV virus produces infective polyhedral inclusion bodies (PIBs) in the midgut cell cytoplasm of virus infected fifth instar larvae. The PIBs multiply enormously in the cytoplasm without invading the nucleus. Ultrastructural studies confirmed the pathological effects of CPV on in midgut cell cytoplasm. The multiplication of polyhedral inclusion bodies took place into the vacuoles and form virogenic stromata in the cytoplasm of cells. However, the encapsulations of polyhedral inclusion bodies into the polyhedrin protein occurred and polyhedra were released into the lumen. At the late stage of infection, cells showed the regressed cytoplasmic organelles with large vacuoles and elongated mitochondria. Hence, the horizontal transmission of CPV causing the midgut cells disintegration in the tasar silkworm, Antheraea mylitta (D) confirmed during infection.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.18-24
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• 2001

Expression of the baculovirus major envelope glycoprotein gene(gp64) is regulated by transcription from botha early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1(BmNPV-K1) was characterized. The gp64 gene was local-ized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcorR 1-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E.coli lacZ gene was introduced onto pBm64 as a reporter gene and expressed transiently in B. mori 5(Bm 5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of $\beta$-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of $\beta$-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.

• Korean journal of applied entomology
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• v.60 no.4
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• pp.379-386
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• 2021

Asian gypsy moth, Lymantria dispar asiatica (Lepidoptera: Erebidae), which is an indigenous pest in Korea, a large outbreak has been reported in Gyeonggi-do, Chungcheong-do, and northern Gyeongsangbuk-do from 2019 in forest and nearby downtown areas, causing emotional damage to forests and city dwellers. During the indoor culture of gypsy moth eggs collected in Yecheon, Gyeongbuk in 2021 we found that 79.65% (321/403 of first instar larvae) were died due to Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) infection. Sequence analyses of 36 gypsy moths collected from 12 regions in Korea using LdMNPV late expression factor-8 (lef-8) and polyhedrin (polh) genes showed a genetic variation of 0.80% and 0.86%, respectively. Comparison to GenBank data showed that the Korean samples were most similar to LdMNPV in Japan, whereas most different to those of Turkey. These results showed a high infection rate of LdMNPV in Korea and LdMNPV is one of the important population regulators of the gypsy moth.

• Journal of Microbiology
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• v.45 no.2
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• pp.133-138
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• 2007

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via ${\beta}-galactosidase$ expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.

• Journal of Sericultural and Entomological Science
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• v.39 no.1
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• pp.36-43
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• 1997

In order to express the FLP recombinase in B. mori cultured cell line, BmN-4, transient expression system using a heat shock protein gene (hsp70) promoter of Dorosophilla melnogaster was constructed. This vector was designated as pHsSV. Activity strength of the hsp70 promoter was compared with that of immediate early gene (IE-1) and polyhedrin gene of BmNPV employing the E. coli $\beta$-galactosidase gene as a reporter gene. The result showed that the pHs $\beta$-gal plasmid vector expressed the $\beta$-galactosidase at 2nd and 3rd day after the transfer of plasmid DNA into BmN-4 cells, which was similar to that of pIE1 $\beta$-gal vector, but different from that of a recombinant virus, vBm $\beta$-gal. For the construction of FLP recombinase transient expression vector, the FLP recombinase gene was cloned by polymerase chain reaction technique. To express the FLP recombinase, this gene was inserted into pHsSV plasmid vector, under the control of the hsp70 promotor, and tranfected in BmN-4 cells. The expressed FLP recombinase was estimated at 44kDa on a 12.5% SDS-PAGE.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.72-76
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• 1996

To investigate the expression efficiency of new transfer vector of Bombyx mori nuclear polyhedrosis virus (BmNPV), Escherichia coli lacZ gene was inserted into new transfer vector pBmKSK1, under the control of polyhedrin promoter and expressed in BmN-4 cells and larvae of silkworm, Bombyx mori. The recombinant virus containing lacZ gene was isolated from BmN-4 cells coinfected with transfer vectro pBmKSK1-LacZ and wild type BmNPV genome, and analysed by Southern blotting. The expression of ${\beta}$-galactosidase was characterized by SDS-PAGE, Western blotting and ${\beta}$-galactosidase activity assay. The results showed that the level of expression in silkworm larvae was higher than that of BmN-4 cells.

• Journal of Sericultural and Entomological Science
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• v.40 no.2
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• pp.105-110
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• 1998

The baculovirus egt gene encodes an ecdysteroid UDP-glucosyltransferase(EGT) which catalyzes the transfer of glucose from UDP-glucose to the insect moltion hormone ecdysteroid resulting in a functionally inactive ecdysteroid. In baculovirus-infected insect larvae, EGT has been shown block molting and pupation. In this study, we compared the development of 4th and 5th instar silkworm, Bombyx mori, larvae injected with either wild-type bombyx mori nucleopolyhedrovirus (BmNPV) or a mutant BmNPV(BmEGTZ) in which the egt gene was disrupted by the insertion of a lacZ gene cassette. Larvae injected with BmEGTZ died roughly 12 h more rapidly compared to indentical larvae infected with BmNPV. In addition, BmEGTZ- infected larvae prematurely stopped feeding and gain less weight compared to BmNPV-infected larvae. In order to investigate why BmEGTZ-infected larvae died more rapidly than BmNPV-infected larvae, the array of hemolymph proteins in BmEGTZ-or BmNPV-infected larvae were analyzed by SDS-PAGE. The hemolymph of BmEGTZ-infected larvae showed virus-specific proteins, including polyhedrin, about 12 h earlier than the hemolymph of BmNPV-infected larvae

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.466-471
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• 2005

An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early $5^{th}$ instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were $60^{\circ}C$ and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM $Ca^{2+}$, 1 mM $Mn^{2+}$, or $0.02\%$ Triton X-100.

• Korean Journal of Veterinary Research
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• v.45 no.1
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• pp.63-70
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• 2005

African swine fever (ASF) is an infectious disease of domestic and wild pigs for which there is no vaccine in the world. A proper surveillance of viral activity and a timely response to ASF outbreaks depend upon the rapid diagnosis of ASF viral infection. Internationally prescribed enzyme-linked immunosorbent assay (ELISA) is a fast, sensitive test routinely used in the diagnosis of the ASF. However, inactivated whole ASF virus antigen used in this test is a tedious to prepare and has a risk of outside exposure of infectious virus by laboratory accident during the preparation. An ASF virus noninfectious recombinant antigen is a safe and easily produced alternative antigen for use in diagnostic assay. We have cloned the ORF O61R gene of the ASF virus to generate a recombinant baculovirus producing the p12 protein in insect cells under control of the polyhedrin promoter as non-fusion protein. When used in an indirect ELISA, the p12 antigen showed reactivity with all known ASF positive pig sera but not with negative pig sera. Our results indicated that the p12 protein would be one of alternative antigens for diagnosis of the ASF.