• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.529-539
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• 2021

The beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Double-stranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

• Biomedical Science Letters
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• v.11 no.3
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• pp.327-332
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• 2005

Sf21 cells become popular as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike human glucose transporters, very little is known about the characteristics of the endogenoussugar transporter(s) in Sf21 cells. Thus, some kinetic properties of the sugar transport system were investigated, involving the uptake of 2-deoxy-D-glucose (2dG1c). In order to obtain a true measure of the initial rate of uptake, the uptake of $[^3H]2dGlc$ from both low $(100{\mu}M)$ and high (10 mM) extracellular concentrations was measured over periods ranging from 30 sec to30 min. The data obtained indicated that the uptake was linear for at least 2 min at both concentrations, suggesting that measurements made over a 1min time course would reflect initial rates of the jexpse uptake. To determine $K_m\;and\;V_{max}$ of the endogenous glucose transporter(s) in Sf21 cells, the uptake of 2dG1c was measured over a range of substrate concentrations $(50{\mu}M\~10mM)$ 2dG1c uptake by the Sf21 cells appeared to involve both saturable and non-saturable (or very low affinity) components. A saturable transport system for 2dG1c was relatively high, the $K_m$ value for uptake being < 0.45 mM. The $V_{max}$ value obtained for 2dG1c transport in the Sf21 cells was about 9.7-folds higher than that reported for Chinese hamster ovary cells, which contain a GLUT1 homologue. Thus, it appeared that the transport activity of the Sf21 cells was very high. In addition, the Sf21 glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter

• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.145-153
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• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.

• International Journal of Industrial Entomology and Biomaterials
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• v.30 no.2
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• pp.50-57
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• 2015

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has type-specific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti-His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.1
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• pp.143-151
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• 2008

A muscle-specific lipase gene of the bumblebee Bombus ignitus was cloned and characterized. This gene, which we named Bi-Lipase, consists of seven exons encoding 317 amino acid residues. Bi-Lipase possesses all the features of lipases, including GXSXG consensus motif and Ser-Asp-His catalytic triad. Expressed as a 37-kDa polypeptide in baculovirus-infected insect Sf9 cells, recombinant Bi-Lipase showed an optimal pH of 9.0 and exhibited its highest catalytic activity at $40^{\circ}C$. Furthermore, through the addition of tunicamycin to the recombinant virus-infected Sf9 cells, recombinant Bi-Lipase was found to be N-glycosylated. Northern and western blot analyses indicated that Bi-Lipase was expressed in the wing, thorax, and leg muscles. These results show that Bi-Lipase is a muscle-specific lipase, suggesting a possible role of Bi-Lipase in the utilization of lipids for muscular activity in B. ignitus.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Korean Journal of Agricultural Science
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• v.43 no.2
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• pp.194-204
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• 2016

The pear psylla, Cacopsylla pyricola, is a very small sap-feeding insect of many commercial pear varieties that could be considered the most serious insect pest of pear. Detailed information on plant penetration activities of the pear psylla is essential to study its feeding behavior used to evaluate resistant traits to chemical control. The application of the electrical penetration graph technique (EPG) could provide a relevant insight into the nature of this resistance. EPG waveforms of C. pyricola were characterized on the basis of amplitude, frequency, voltage level, and electrical origin. Feeding behaviors of C. pyricola were recorded and analyzed by EPG analysis. During EPG monitoring, waveform PA occurred at the start of stylet penetration of pear leaf epidermal cell. Waveform PB followed, in which stylet secreted saliva was observed. Waveforms PC1 and PC2 involved penetrating and sucking behaviors in parenchyma cells and vascular parenchyma, respectively. In addition, waveform PC1 represented salivation into bundle sheath cells and ingestion from parenchyma. Otherwise, behaviors of salivation into phloem and ingestion from phloem produced waveforms PE1 and PE2, respectively. On the other hand, ingestion from xylem tissues showed waveform PG. Among the feeding patterns of C. pyricola described above, phloem feeding patterns occurred most frequently, followed by xylem feeding and parenchyma penetration patterns in descending order.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.485-489
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• 1995

The medium additives such as fatty acid, lipid, mannose, folic acid, $CaCl_2$ were examined to enhance recombinant ${\beta}-galactosidase\;({\beta}-gal)$ production in T-flask and air-lift bioreactor. The addition of each component. such as cholesterol, tocopherol, tricaprylin, mannose and folic acid at AcNPV infection of Tn5B1-4 cells enhanced ${\beta}-gal$ production, whereas the addition of $CaCl_2$ did not increase ${\beta}-gal$ production. The recombinant ${\beta}-gal$ production using the infection medium supplemented with a mixture of 0.34 mM cholesterol, 2.2 mM mannose and 0.045 M folic acid was enhanced 2 fold in an air-lift bioreactor, compared to the basal medium.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.82-83
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• 2003

In previous experiment, we reported when the heterologous protein is expressed by using baculovirus expression vector system (BEVS), although the amount of intracellular protein is abundant, the amount of extracellular Protein is poor. As the link in the chain of the research, we investigated the secretory pathway, important in case of the secretory protein, of the protein expressed in insect cells using BEVS. (omitted)

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.10a
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• pp.128-129
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• 2003

Cricket (Gyilus bimacutus) is mass-bred in 6 time cycles per one year in insect farms. They are used as dry or live foods for animals, tropical fish, reptile and amphibians. Therefore, it is necessary to study the genotoxicity of whole bodies of G. bimaculatus. The aim of this present study was to evaluate the genotoxicity of the G bimaculatus extract with three methods, Ames test, chromosome aberration test in Chinese hamster ovary cells in vitro and micronucleus (MN) test in vivo which involve the different test systems (bacteria, mammalian cells and mice nuclei). (omitted)