• Proceedings of the Korean Society of Sericultural Science Conference
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• 1997.06a
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• pp.73-101
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• 1997

Baculoviruses are characterized by large double-stranded circular DNA genomes and rod-shaped enveloped virions. Bombyx mori nucleopolyhedrovirus(BmNPV) is a major pathogen, which causes severe damage in sericulture. Currently, BmNPV is recogtnized as an improtant tool in molecular biology, especially for expression of useful genes in B.mori cells and silkworm larvae. Our laboratories have focused on the studies of the molecular mechanisms of BmNPV replication and the application of BmNPV to agriculture and medicine. The entire nucleotide sequence of the BmNPV genome has recently determined. The BmNPV genome possessed 135 putative genes and 7 homologous repeated sequence (hrs) regions. Relatively little space, a few to a few hundred base-pairs, was observed between the open reading frames and hrs. Termination codons often overlapped. These results showed a compactly packde BmNPV genome. Based on comparative sequence analyses, we speculated that the ancestor of BmNPV was a baculovirus similar to Autographa californica NPV(AcNPV). The function of the BmNPV genes were characterized by gene deletion analysis; p35 was found to be involved in blocking apoptosis and cysteine proteinase was found to be involved in horizontal virus transmission by degrading viral-infected larval host. By AcNPV and BmNPV coinfection experiments, we identified a BmNPV gene involved in expanding host specificity of AcNPV. The identified gene was likely encoded a DNA helicase based on the amino acid sequence analysis; a few amino acid substitutions in the putative DNA helicase gene resulted in the expansion of host range of AcNPV. These findings indicate that BmNPV evolved within a short period from an AcNPV-like ancestral virus due to rapid evolution including specific amino acid substitutions and gene deletions/insertions.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.1
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• pp.35-38
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• 2007

In this study, ORF 46 of Bombyx mod nucleopolyhedrovirus(Bm46) fused with EGFP was expressed in Bombyx mod cell lines, larvae and pupae by BmNPV Bacmid system. Bm46 and EGFP were cloned into donor plasmid pFastBacHTb, which was transformed to competent DH10B cells containing helper and BmNPV bacmid by site-specific transposition. Recombinant bacmid was used to transfected BmN-4 cells to produce the recombinant baculovirus vBm-Bm46-EGFP. Recombination virus was injected into silkworm larvae and pupae. The expression of the fusion protein was monitored by examining green fluorescence using a fluorescent microscope. Intense fluorescence in cells and silkworm was observed at 4 days post-infection, indicating the Bm46-EGFP fusion gene was expressed successfully.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.315-319
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• 2003

A novel method is developed to yield virus titers in 10 h, is easy to .perform using 96-well plates, and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immune-staining. The titer is determined by counting foci produced due to infection of virus under a fluorescent microscopy. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post infection time of 4 h. Therefore, 10 hours were enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer.

• The Korean Journal of Pesticide Science
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• v.12 no.3
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• pp.283-290
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• 2008

Baculoviruses have been used to control some serious lepidopteran pests. However, their narrow target insect spectrum and slow efficacy are main limitations to be used in various applications. This study introduces a technique to overcome these limitations by inhibiting insect immune defence to enhance the viral pathogenicity. Polydnaviruses are an insect DNA virus group and symbiotic to some ichneumonid and braconid endoparasitoids. Cotesia plutellae bracovirus (CpBV) is a braconid polydnavirus and encodes several immunosuppressive genes. We selected seven CpBV genes and recombined them to wild type Autographa California multiple nucleopolyhedrovirus (AcNPV). A bioassay of these seven recombinants indicated that most recombinants had similar or superior efficacy to wild type AcNPV against beet armyworm, Spodoptera exigua, and diamondback moth, Plutella xylostella. Recombinant AcNPV with CpBV-ELP was the most potent in terms of lethal time by shortening more than 2 days compared to wild type AcNPV. This recombinant was further proved in its dose-dependent pathogenicity and its efficacy by spray application on S. exigua infesting cabbage cultivated in pots. We discussed the efficacy of CpBV-ELP recombinant AcNPV in terms of suppressing antiviral activity of target insects.

• Korean journal of applied entomology
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• v.43 no.1
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• pp.55-60
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• 2004

This experiment was conducted to improve activity of Spodoptera litura Nucleopoly-hedrovirus (SINPV) combined with organic and functional matters. In combination of SINPV mixed with organic matters, LT$_{50}$ values of SINPV 1.0${\times}$10$^{5}$ PIBs/$m\ell$ combined with boric acid of 2,000 ppm were 4.5 days. It was 1.5 days shorter than SINPV 1.0${\times}$10$^{5}$ PIBs/$m\ell$ alone. The body weight of larva infected with SINPV 1.0${\times}$10$^{5}$ PIBs/$m\ell$ combined with boric acid of 2,000ppm was not increased, and S. litura was completely dead in 7 days after treatment. It suggested that addition of boric acid in SINPV application enhanced the pathogenicity against S. litura larvae. In laboratory experiment of combination of SINPV with functional matters, LT$_{50}$values of SINPV 1.0${\times}$10$^4$ PIBs/$m\ell$ alone were 7.3 days, but those of SINPV 1.0${\times}$10$^4$ PIBs/$m\ell$ with electrolyzed oxidizing water, pyroligneous liquor or kitosan were 10.4, 9.3 and 11.2 days, respectively. Functional matter could be suppressed the insecticidal activity of SINPV

• 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.

• 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.

• BMB Reports
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• v.35 no.6
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.

• BMB Reports
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• v.40 no.2
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• pp.232-238
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• 2007

The p13 gene is uniquely present in Group II nucleopolyhedroviruses (NPVs) and some granuloviruses, but not in Group I NPVs. p13 gene was first described by our laboratory in Leucania separatamultiple nuclear polyhedrosis virus (Ls-p13) in 1995. However, the functions of Ls-P13 and of its homologues are unknown. When Ls-p13 was inserted into Autographa californica nucleopolyhedrovirus, a Group I NPV, polyhedra yield was inhibited. However, this inhibition was prevented when the leucine zipper-like domain of Ls-p13 was mutated. To determine the cause of this marked difference between Ls-P13 and leucine zipper mutated Ls-P13 (Ls-P13mL), oligomerization and secondary structure analyses were performed. High performance liquid chromatography and yeast two-hybrid assays indicated that neither Ls-P13 nor Ls-P13mL could form oligomers. Informatics and circular dichroism spectropolarimetry results further indicated marked secondary structural differences between Ls-P13 and Ls-P13mL. The LZLD of Ls-P13 has two extended heptad repeat units which form a hydrophobic surface, but it is short of a third hydrophobic heptad repeat unit for oligomerization. However, the mutated LZLD of Ls-P13mL lacks the above hydrophobic surface, and its secondary structure is markedly different. This difference in its secondary structure may explain why Ls-P13mL is unable to inhibit polyhedra yield.