Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and harbors 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, 55 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells, 16 of them were found affecting viral replication since they defected in producing polyhedra. Furthermore, to verify the pathogenicity of the single ORF-truncated mutants, the remaining 39 mutants were subjected to bioassay to Spodoptera exigua 3rd instar larvae. Among them, ac9-, ac49-, ac103- and ac105-knockout mutants showed higher mortality compared to that of bAc-MK. These results suggested that these ORFs could be related to pathogenicity of AcMNPV.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

Baculovirus chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. In previous report, baculovirus ChiA can offer many interseting new opportunities for pest control. Recently, a putative chitinase gene (ChiA) was identified in the Spodopter litura nucleopolyhedorvirus (SlMNPV-K1) genome. The open reading frame (ORF) contains 1,692 nucelotides (nt) and encodes a protein of 563 amino acids (aa) with a predicted molecular weight of 62.62 kDa. To conform the insecticidal activity of ChiA from SlMNPV-K1, we constructed a baculovirus transfer vector, pBac-SlChiA, and this transfer vector was co-transfected with the bApGOZA DNA into sf9 cell to generate corresponding recombinant viru which designed Ap-SlChiA. Western blot analysis indicate that SlMNPV-K1 ChiA was successfully expressed. We found the chitinase activity of recombinant virus was enhanced 53% than wide type AcMNPV by chitinase assay, and the recombinant virus showed higher evidently insecticidal activity against 3rd instar larvae of Spodotera exigua than wide type AcMNPV (4.5 time). These results suggested that the chitinase gene from SlMNPV-K1 could be successfully applied to improve pathogenicity of bauclovirus

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

Aujeszky’s disease (AD), also called pseudorabies, is an infectious viral disease, caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. AD affects many countries and regions in the world, causing important economic losses, mainly due to international trade restrictions. In this study, to determine the characteristics of the Aujeszky’s disease virus (ADV), NYJ strain, which was isolated from the serum of an infected pig in 1987, we investigated the nucleotide sequence and expression of the glycoproteins gB, gC, and gD using the bBpGOZA system. We found that the glycoproteins gB, gC, and gD of NYJ consisted of 2751 bp, 1443 bp, and 1203 bp, respectively. Comparison of the NYJ with the other strains revealed nucleotide sequence identity ranging from 91.tito 99.0%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The NYJ strain was formed a distinct branch with high bootstrap support. The expression of glycoprotein gD in insect cells was characterized by SDS-PAGE and Western blotting with an anti-ADV polyclonal antibody. Glycoprotein gD of approximately 45 kDa was detected. The results of this study have implications for both the taxonomy of ADV and vaccine development.

Entomopathogenic fungi were isolated directly from a cadaver of adult M. saltuarius (infected with white fungi) supporting fungal sporulation, to develop biological control of pine wilt disease vector, M. saltuarius which was the most abundant in the middle to northern part of Korea and caused enormous damage to native pine tree in Korea, Japan and other regions of Asia. Pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. As the result, only one of them showed pathogenic to adults of M. diphysis, with up to 100% mortality within 13 days of inoculation. Selected fungus was named as MsW1 and identified by Beauveria bassiana using microscopic examination, B. bassiana-specific PCR primers and genetic sequencing of the ITS region analysis. Pathogenicity test were conducted with various concentration of conidial suspensions of this isolate on M. saltuarius (3rd instar larvae and adults). Mortality rates varied from 57.1% to 100.0% and from 16.7% to 100.0% of M. saltuarius (3rd instar larvae and adults), respectively at 30 days. This is the first report of natural infection of M. saltuarius by B. bassiana.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. Until recent times, most of the studies for these fungi have been based on isolation from insect cadaver or soil. However, these methods, especially isolation from soil, might cause a problem involving differential isolation of the each entomopathogenic fungi. The purpose of this study is to determine the optimal isolation medium for entomopathogenic fungi using dodine, chitin, and skim milk. The growth rates of entomopathogenic fungi and non-entomopathogenic fungi were compared on dodine agar medium. The medium for this experiment was modified Veen semiselective medium which consisted of based on SDA (Sabouraund Dextrose Agar), 100 mg/ml for chloramphenicol, 50 mg/ml for streptomycin and adjusted dodine to 40, 50, 70 and 100 mg/ml. As a result, optimal concentration of dodine for isolation of entomopathogenic fungi was 50 mg/ml and 168 positive entomopathogenic fungi were isolated in 470 soil samples and 11 cadavers of insect. In addition, the isolates had significantly greater chitinase and protease activity than non-entomopathogenic fungi. The isolation method described represents a valuable tool for rapid and simple isolation of entomopathogenic fungi. These positive entomopathogenic fungi may have potential against variety pests in agriculture.

Background: Proteolytic enzymes are involved in insect molting and metamorphosis and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compared the expression profile of B. mori cathepsins B (BmCatB) and D (BmCatD) in the fat body during the larval-pupal transformation of B. mori in the BmCatB or BmCatD RNA interference (RNAi) process. Results: BmCatB is ecdysone-induced and expressed in the fat body of B. mori during the molting, and the larval-pupal and pupal-adult transformations, and its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation and BmCatB RNAi treatment resulted in the arrest of the larval-pupal transformation. RNAi-treated BmCatB knock-down sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, BmCatD RNAi up-regulated the expression of BmCatB in the fat body of final instar larvae. Conclusion: Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis and that BmCatB and BmCatD contribute collaboratively to B. mori metamorphosis

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties such as higher insecticidal activity and recovery to wild-type baculovirus. For this, Bacillus thuringiensis crystal protein gene (cry1-5) was introduced into Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrincry1- 5-polyhedrin under the control of poyhedrin gene promoter. In the opposite direction of this fusion gene, an insect-specific neurotoxin gene (AaIT) under the control of early promoter from Cotesia plutellae bracovirus was introduced by fusion of orf603 partial fragment. Western hybridization and confocal microscopy revealed that AaIT neurotoxin and Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus and that the fusion protein occluded into the polyhedra. In addition, the fusion protein was activated as about 65 kDa of crystal protein when treated with trypsin. The NeuroBactrus showed high level of insecticidal activity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Re-recombinants derived from the NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro and in vivo. These results suggested that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 fragments.

Plasmid capture system (PCS) was developed for easy cloning and manipulation of circular double-stranded DNA from various sources. Recently, we improved PCS system (named PCS-LZ) to clone relatively large-sized DNA molecules (30-150 kb). PCS-LZ donor consists of a Mini-F replicon and a kanamycin resistance marker between Tn7L and Tn7R regions. Both replicon and marker gene of PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition and the transposed DNAs can replicate in E. coli cells by transformation. White/blue screening by LacZ expression is also available to avoid backgrounds. Up to now, we acquired various circular DNA clones from four sources such as plasmids of B. thuringiensis, bacteriophage genome isolated from B. thuringiensis, genome segments of Cotesia glomerata bracovirus, and polymorphic genomes of Autographa californica nucleopolyhedrovirus. Among them, interestingly, the genome clones of bacteriphage (Ph1-3) were screened from the PCS transposition with plasmids of B. thuringiensis 1-3 strain. The genome of Ph1-3 was fully sequenced (46517 bp) and open reading frames were analyzed. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Ph1-3 showed infectivity to B. thuringiensis type strains such as subsp. galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phage in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and their sequence similarity was more than 70%. Conclusively, we expect this PCS-LZ system would be a powerful tool for genomic analysis and mutagenesis study at the area of invertebrate pathology and further its application will be enlarged to the vertebrate pathology area.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.