• 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.3 no.1
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• pp.75-81
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• 2001

We have constructed a novel recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) producing the green fluorescent polyhedra. For the production of the fluorescent polyhedra, partial polyhedrin gene containing KRKK as nuclear localization site from the BmNPV polyhedrin gene and the green fluorescent protein (gfp) gene were introduced under the control of p10 promoter of BmNPV. The recombinant BmNPV was stably produced fluorescent polyhedra in the infected Bm5 cells and the morphology of the fluorescent polyhedra was similar to that of wild-type BmNPV. The fluorescent polyhedra had 32 kDa native polyhedrin and 41 kDa fusion protein. From these data, we have further developed a novel BmNPV p10-based transfer vector producing recombinant polyhedra with foreign gene Product. The novel BmNPV P10-based transfer vector is composed of partial polyhedrin gene, factor Xa, and multiple cloning sites.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.1
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• pp.19-23
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• 2000

A novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (ACNPV) producing the green fluorescent polyhedra was constructed and characterized. The recombinant virus was stably produced fluorescent polyhedra in the infected cells and the morphology of the polyhedra was nearly similar to that of wild-type AcNPV. For the production of the fluorescent polyhedral the green fluorescent protein (GFP) gene was introduced under the control of polyhedrin gene promoter of AcNPV by translational fusion in the front and back of intact polyhedrin gene. The recombinant baculovirus was named as CXEP, As expected, the 93 kDa fusion protein was expressed in the CXEP-infected cells. Interestingly, however, the cells infected with CXEP also showed a 33 kDa protein band as cells infected with wild-type AcNPV. The results of Southern blot analysis and plaque assay suggested that two types of baculoviruses expressing the GFP fusion protein or only native polyhedrin were formed through homologous recombination between two polyhedrin genes in the same orientation. Thus, this system can be applied for the production of recombinant polyhedra with foreign gene product of diverse interest.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.36-41
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• 1996

Recombinant baculoviruses having expanded host range were selected by coinfection of Autographa california NPV and Bombyx mori NPV into Sf-9 and BmN-4 insect cell lines. In order to determine the polyhedra morhplogy of RecS-A6, one of a recombinant baculovirus, polyhedra of RecS-A6 produced in insect cells were observed by phase contrast microscope and scanning electron microscope. The results revealed that the recombinant baculovirus had a various polyhedra morphology which was different from its parental viruses, suggesting that the various morhpology of recombinant baculovirus with an expanded host range was due to the genetic recombination of viral genome. To analyze the genomic recombinantion of the recombinant baculoviruses, genomic DNAs of two parent viruses and RecS-A6 were digested with restriction endonuclease and subjected to agarose gel electrophoresis. Southern blot analysis revealed that RecS-A6 has two polyhedrin gene of AcNPV and BmNPV in a viral genome. Polyhedral protein of recombinant baculovirus was analysed by SDS-PAGE. The result showed that molecular weight of polyhedral protein of RecS-A6 containing two polyhedrin gene of AcNPV and BmNPV was as the 31 kDa band of AcNPV and 30 kDa band of BmNPV.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.1
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• pp.13-18
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• 2001

The role of polyhedrin in the polyhedra production in baculovirus Autograha californica Nucelopolyhedro-sisvirus (AcNPV) was studied by over-expression of AcNPV polyhedrin or heterologous polyhedrin from Bombyx mori (Bm) NPV or Spodoptera exigua (Se) NPV. The transfer vectors containing additional polyhedrin from AcNPV, BmNPV, or SeNPV were constructed and cotransfected with bacmid bApGOZA into Sf9 cells. The resulting recombinants, designated as vApAcPol, vApBmPol, and vApSePol were tonstructed, and the polyhedra production of the recombinant was characterized. All of the recombinants produced polyhedra in the nucleus, and the polyhedrin was over-expressed. Among three recombinants, vApAcPol and vApBmPol were discriminated by their larger polyhedra size than that of wild type AcNPV, and vApSePol also produced larger polyhedra than wild type SeNPV polyhedra.

• Korean journal of applied entomology
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• v.38 no.2
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• pp.139-144
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• 1999

We have now constructed a novel recombinant baculovirus producing fusion protein with Autogrqha c.uliforrzica nuclear polyhedrosis virus (AcNPV) polyhedrin and green fluorescent protein (GFP). The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The GFP gene was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front or back of intact polyhedrin gene. The recombinant baculoviruses were named as Ac-GFPPOL or Ac-POLGFP. respectively. As expected, the 56 kDa fusion protein was expressed in the recombinant virus-infected cells. Interestingly. however, the fluorescence of GFP in the cells infected with Ac- POLGFP was only detected within the nuclei. and that was observed as polyhedra-like granular particles. In the microscopy of cells infected with Ac-GFPPOL, furthermore, GFP was detected in both cytoplasm and nuclei although most of GFP were present within the nuclei. However, fusion protein produced by recombinant virus did not form polyhedra although the fusion protein was fused with polyhedrin and GFP. It is suggested that difference of GFP location in the infected cells appear to be involved in the region of polyhedrin in the fusion protein, and the polyhedrin in the fusion protein might be responsible for the polyhedra-like granular particles present within nuclei.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.710-715
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• 2005

A novel recombinant baculovirus, Bactrus, was constructed by the insertion of the Bacillus thuringiensis cry1Ac gene between two polyhedrin genes of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of the polyhedrin gene promoter. Polyhedra produced by Bactrus in insect cells were incorporated with 130 kDa of polyhedrin-Cry1Ac-polyhedrin fusion protein, and 30 kDa of intact polyhedrin, resulting from a homologous recombination between two polyhedrin genes, was also expressed. The insecticidal activity of Bactrus against Spodoptera exigua larvae was similar to that of AcNPV, but it showed significantly higher toxicity towards Plutella xylostella larvae in comparison with that of AcNPV. The expression level of fusion protein and the insecticidal activity of recombinant polyhedra produced by the Bactrus against P. xylostella larvae were decreased after serial passages. In conclusion, the Bactrus had improved insecticidal activity and returned to wild-type AcNPV after several passages.

• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.359-366
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• 1997

The usefulness of host range expanded recombinant viruses for economical viral insecticide and expression vector system has been studied. Host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV), and a host range expanded AcNPV recombinant, Ac-BH, constructed by substitution of the 0.6Kb fragment of the BmNPV helicase gene were compared. The restriction enzyme digestion patterns showed that RecS-B6 and RecB-8 had expanded host ranges by genomic recombination and were more similar to genome of AcNPV than that of BmNPV. SDS-PAGE and PCR analysis showed that the polyhedrin gene of RecS-B6 and RecB-8 was derived from BmNPV genomic DNA. The morphology of polyhedra of recombinant viruses showed a slight difference between the two host cells, Sf and BmN cells, indicating that the morphology of polyhedra was influenced by host cells. The bioassay data for insect larvae showed that Ac-BH, compared to wild type viruses, had superior pathogenicity against Bombyx mori larvae but inferior pathogenicity against Spodoptera exigua larvae. Although the pathogenicity was lower than that of wild type viruses in both larvae, RecS-B6 showed the pathogenicity in both larvae. These results suggested that Ac-BH was a less useful economical insecticide than random genomic recombinant virus RecS-B6.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2004.05a
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• pp.103-103
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• 2004

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2004.05a
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• pp.173-173
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• 2004