• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.2
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• pp.237-241
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• 2009

The objective of our study was the evaluation of pathogenicity of a local strain of Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) derived from a diseased larva of M. brassicae found in Korea. The effect of temperature and larval instar on the pathogenicity and production of MabrNPV-K1 was determined under laboratory conditions. The median lethal concentration ($LC_{50}$) values of MabrNPV-K1 for 3rd instar larvae were $3.7\times10^4$ PIBs/larva at $20^{\circ}C$, $9.9\times10^2$ PIBs/larva at $25^{\circ}C$ and $3.8\times10^2$ PIBs/larva at $30^{\circ}C$, respectively. The $LC_{50}$ for the 4th instar larvae was similar to that for the 3rd instar larvae. However, the pathogenicity to the 3rd instar larvae was higher than that to the 4th instar larvae. The median lethal time ($LT_{50}$) values of MabrNPV-K1 were 11.4 to 5.0 days at $30^{\circ}C$ and 18.3 to 5.5 days at $25^{\circ}C$ for the 3rd instar larvae. The $LT_{50}$ value was lowered as temperature went up to $30^{\circ}C$ and dependent on viral concentration. In production efficiency of MabrNPV-K1 using M. brassicae larvae, the mortality of the 3rd instar larvae was 100% when inoculated with $1.0\times10^5$ PIBs/larva and the yield of MabrNPV-K1 was maximal. Regarding the mortality, yield of polyhedra, inoculation doses and required time, the $1.0\times10^4$PIBs/larva at $30^{\circ}C$ was determined as optimal conditions producing polyhedra efficiently.

• Journal of Microbiology
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• v.44 no.1
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• pp.77-82
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• 2006

The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide for biocontrol of this insect. However only limited genetic analysis for this important virus has been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis of the occlusion body showed polyhedra of 0.7 to $1.7\;{\mu}m$ in diameter containing a single nucleocapsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment contained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rrl, polyhedrin, orfl629, pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and 29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the previously described Group II NPV. Bioassays on lethal concentration $(LC_{50}\;and\;LC_{90})$ and lethal time $(LT_{50}\;and\;LT({90})$ were conducted to test the susceptibility of E. obliqua larvae to the virus.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.53-62
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• 2012

Silkworm larvae often suffer from viral infections causing heavy losses to the economy of silk industry. Insects exhibit both humoral and cellular immune responses that are effective against various pathohens like bacteria, fungi, protozoa, etc., but no insect immune responses is effective against viral infection. To obtain genes related to insect antiviral immunity from Bombyx mori, the cDNA library was constructed from B. mori nucleopolyhedrovirus (BmNPV)-infected B. mori. From the cDNA library, we selected 411 differentially expressed clones, and the 5' ends of the inserts were sequenced to generate ESTs. In this work, 135 unigenes were generated after the assembly of 411 differentially expressed clones ESTs. Of these 135 unigenes, we selected 109 antiviral response-related candidates except 26 clones that high similarity with genes derived from BmNPV. Among 109 unigenes, a total of 80% had significant matches to genes from other organisms in the database, wheres 20% of the unigenes had not matched in the database. Functional groups of these sequences with matches in database were constructed according to their putative biological function. Three largest categories were control of cellular oraganization (52%), metabolism (20%), and protein fate (10%). The genetic information reported in this study will provide more information about antiviral-related genes in silkworms.

• International Journal of Industrial Entomology and Biomaterials
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• v.12 no.2
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• pp.63-67
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• 2006

Suppressor of cytokine signaling (SOCS) is known to playa key role as a negative feedback regulator in JAK/STAT signaling cascade in innate immunity. Our laboratory has recently been interested in elucidating the interactions between Spodoptera exigua (Se) and SeNPV. This context leads us to clone and characterize SeSOCS that may have important functions in response to SeNPV infection. Using the RT-PCR and TA cloning approach, we found a partial fragment (416 bp) of SeSOCS. Blast search and multiple alignment data showed that it has a homology to various insects such as Anopheles gambiae (78%), Aedes aegypti (75%), Drosophila melanogastar (77%), Mus musculus (69%), and Homo sapiens (69%). Temporal induction patterns of SeSOCS were analysed after being immune-challenged with either NPV or laminarin. It showed that the level of SeSOCS mRNA was strongly induced in a biphasic manner in response to SeNPV and laminarin, respectively. It seems that SOCS, a negative regulator of JAK/STAT signaling system is also present in S. exigua and may playa role in innate immunity albeit its precise role should be further elucidated at the molecular and cellular level in the early phase of SeNPV infection in larvae.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1485-1490
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• 2006

In this study, comparative replicational properties of Hyphantria cunea nucleopolyhedrovirus (HycuNPV) in Lymantria dispar (IPLB-LdElta) and Spodoptera frugiperda (IPLB-Sf21) cell lines were investigated. Our microscopic observations showed that cytopathic effects (CPEs) in LdElta cells appeared 12 h later than those in Sf21 cells. Whereas polyhedral inclusion bodies (PIBs) formed at 48 h postinfection (p.i.) in LdElta cells, it formed at 36 h p.i. in Sf21 cells. Extracellular virus production determined according to the 50% tissue culture infective dose ($TCID_{50}$) method in LdElta cells started about 12 h later when compared with Sf21 cells. Titers of extracellular virus in LdElta and Sf21 cells were calculated as $1.77{\times}10^9$ plaque forming units (PFU)/ml and $5.6{\times}10^9PFU/ml$, respectively, at 72 h p.i. We also showed that viral DNA replication began at 12 h p.i. in both cell lines. Viral protein synthesis was determined by SDS-polyacrylamide gel electrophoresis (PAGE) and polyhedrin synthesis was observed at 12 h p.i. in both cell lines. The results indicate that while the synthesis of macromolecules is 12 h later and production of extracellular virus is almost 3-fold lower in LdElta cells compared with those in Sf21 cells, the LdElta cell line is still a productive cell line for infection of HycuNPV.

• 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.

• 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.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2005.05a
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• pp.195-195
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• 2005

• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.2
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• pp.169-174
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• 2004

Bombyx mori nucleopolyhedrovirus(BmNPV) ecdysteroid UDP-glucosyltransferase gene (egt) promoter fragments of different lengths were amplified from BmNPV ZJ-8 genomic DNA by PCR. Reporter plasmids pBmegt542-luc, pBmegt309-luc and pBmegtl59-luc with luciferase (lue) driven by egt promoters were constructed. Both in vitro and in vivo expressions showed that BmNPV egt promoter activity requires the transactivation of viral factor(s), and expression of luc was detected earliest at 24 hrs post infection (pi). BmNPV ZJ-8 homologous region 3 (hr3) increased the expression of luc by over 1,600-fold. Molting hormone of 1.0 - 2.0 $\mu\textrm{g}$/$m\ell$ can dramatically down regulate expression of luc. Juvenile hormone analogue of 0.5-2.0 ${\mu}g$/$m\ell$ increased expression of luc by 145.8% to 75.7%. Deletion assay revealed that the promoter fragment of 159 bp contains the basal promoter structure; Promoter fragments of 309 bp and 542 bp showed similar but much higher transcriptional activities than that of 159 bp, suggesting that nucleotide from -159 to -309 nt upstream the translation initiation site harbors the main cis-acting elements.