• Korean journal of applied entomology
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• v.28 no.3
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• pp.105-112
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• 1989

The nuclear polyhedrosis viruses of Bombyx mori (BmNPV) and Hyphantria cunea (HcNPV) were characterized by electron microscopic observation, SDS-PAGE of polyhedral and virion proteins, and restriction endonuclease analysis of viral DNAs. Polyhedra of BmNPV were octadecahedral in shape with the diameter of $3 \mu\textrm{m}$, whereas those of HcNPV showed irregular appearances having the diameter of $1.5~2\mu\textrm{m}$. Under alkaline protease inactivated condition, polyhedral proteins of two NPVs were resolved into a major polypeptide, 30~31 KD, and several minor polypeptides by SDS-PAGE. Examination of virion proteins by silver staining after SDS-PAGE showed that BmNPV was composed of 47 polypeptides with M.W. range of 9.6~112 KD and HcNPV was composed of 48 polypeptides with M.W. range of 9.4~111 KD. The approximate genome size of two NPVs were determined by restriction endonuclease analysis: BmNPV and HcNPV were 114.6 Kb, respectively.

• Korean journal of applied entomology
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• v.32 no.1
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• pp.51-60
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• 1993

The polyhedrin gene-containing DNA fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) was localized by southern hybridization with Autographa california CPA EcoRI-I fragment (7.3 kb), Bombyx mori NPV PatI-F fragment (7 kb) and synthetic oligonucleotide(30-mer) as probes. the PstI-L(5.3 kb) fragment of HcNPV was cloned to E. coli and the plasmid of the fragment was named as pHcP-L(8.0 kb). The pHcP-L was physically mapped and subcloned to E. coli as pHcP-L1(4.7 kb), pHcP-L2(7.1 kb), pHcP-L3(5.3 kb), pHcP-L4(4.2 kb) and pHcP-L5(4.5 kb).

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.685-691
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• 1998

Baculovirus Hyphantrin. cunea nuclear polyhedrosis virus (HcNPV) insecticide containing the insecticidal protein (ICP) gene from Bacillus thuringiensis subsp. kurstaki HD1 was constructed using a lacZ-HcNPV system. The ICP ($\delta$-endotoxin) gene was placed under the control of the polyhedrin gene promoter of the HcNPV. A polyhedrin-negative virus was derived and named ICP-HcNPV insecticide. Then, the insertion of the ICP gene in the ICP-HcNPV genome was confirmed by Southern hybridization analysis. Polyacrylamide gel electrophoresis (PAGE) analysis of the Spodoptera frugiperda cell extracts infected with the ICP-HcNPV showed that the ICP was expressed in the insect cells as 130 kDa at 5 days post-infection. The ICP produced in the cells was present in aggregates. When extracts from the cells infected with the ICP-HcNPV were fed to 20 Bombyx mori larvae, the following mortality rate was seen; 8 larvae at 1 h, 10 larvae at 3 h, and 20 larvae at 12 h. These data indicate that the B. thuringiensis ICP gene was expressed by the baculovirus insecticide in insect cells and there was a high insecticidal activity. The biological activities of the recombinant virus ICP-HcNPV were assessed in conventional bioassay tests by feeding virus particles and ICP to the insect larvae. The initial baculovirus insecticide ICP-HcNPV was developed in our laboratory and the significance of the genetically engineered virus insecticides is discussed.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.75-82
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• 2011

To elucidate the novelty of Hyphantria cunea nucleopolyhedrovirus (HcNPV) isolated in Korea, polyhedrin and inhibitor of apoptosis (iap) gene structures were determined and analyzed. The analysis of HcNPV polyhedrin showed a little difference with 97.6% at the nucleotide level but no difference at the amino acid level when compared with that of previously reported H. cunea NPV (HycuNPV). On the other hand, iap genes showed variable differences with 89.0-99.6% nucleotide and 90.0-99.6% amino acid sequence identities. Especially, the 5' and 3' non-coding flanking sequences of iap1 gene had lower degree of identity with those of HycuNPV. Although the phylogenetic analyses using polyhedrin and iap genes showed that HcNPV is closely related with HycuNPV, we could provide that HcNPV is a novel isolate having novel gene structures.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Korean journal of applied entomology
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• v.27 no.4
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• pp.211-218
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• 1988

Polyhedral proteins and the endogenous alkaline protease associated with larval-derived polyhedra of nuclear viruses isolated from Spodoptera litura, Bombyx mori, and Hyphantria cunea were investigated. Polyhedral proteins prepared under alkaline protease heat-inactivated condition were separated as one band with 31Kd in S. litura a H. cunea NpV and 30Kd in B. mori NPV by the SDS-polyacrylamide gel electroptoresis. Whereas polyhedral proteins without heat-inactivation were degraded into smaller polypeptides with a certain pattern in alkaline solution. The results of double-immunodiffusion and western blot analysis with antisera against polyhedral proteins indicated that those three polyhedral proteins had common antigenic determinants and the degradation of polyhedral proteins by alkaline protease could be confirmed.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.541-547
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• 1999

Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.

• Journal of fish pathology
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• v.8 no.1
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• pp.37-46
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• 1995

The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

• BMB Reports
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• v.33 no.6
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• pp.483-492
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• 2000

The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.