Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

The two-spotted spider mite, Tetranychus urticae Koch, is an economically important pest of crops of plant grown in the field or greenhouse worldwide. It has recently become a serious problem because of the continuous use of acaricides resulting in resistance among spider mite population. Thus, there is a need to find alternative control measures to suppress spider mite populations. In this study, we report the screening result of pathogenic fungi for the control of spider mite. Initial screenings were performed using 352 isolates of putative pathogenic fungi from Korea soils. As results, 11 strains of acaropathogenic fungi were isolated from 8 cadavers of spider mite supporting fungal conidiation. These isolated were identified as four isolates of Beauveria bassiana (6, 2R-3-3-1, 2R-4-5, 2R-4-7), two isolates of Metarhizium anisopliae (4-2, 2-2), one isolate of Clonostachys rosea 5-2, one isolate of Lecanicillium attenuatum 4-1, one isolate of Pochonia suchlasporia 2R-3-1, one isolate of Aspergillus flavus 7 and one isolate of Isaria lilacinus 2R-4-6 by microscopic examination and genetic sequencing of the ITS region. Based on the screening results, eleven isolates were tested for their virulence against adult spider mites. All fungal isolates were pathogenic to spider mite but mortality varied with isolates. These acaropathogenic fungi may be useful to develop eco-friendly acaricide to control two-spotted spider mite.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected Mamestra brassicae (Lepidoptera: Noctuidae) larvae in Korea. Restriction endonuclease fragment analysis using EcoRI, PstI, and BamHI estimated that the total genome size of MabrNPV-K1 is about 150 Kb. The full genome sequences of MabrNPV-K1 were determined, analyzed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,471 bp and had an overall G + C contents of 39.90 %. Computer-assisted analysis predicted 159 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. The gene content and arrangement in MabrNPV-K1 were most similar to those of Mamestra configurata nucleopolyhedrovirus-B (MacoNPV-B), including three polh, p10 and lef-8 gene homologues. The MabrNPV-K1 genome contains four homologous repeat regions (hr1,hr2,hr3,hr4) that account for 3.1% of the genome. The genomic positions of MabrNPV-K1 regions hr1– hr4 are conserved with the genomic positions of MacoNPV-B hr1–hr4. This indicates that the position of MabrNPV–K1 hrs is conserved with regard to both the upstream and downstream genes. Given that hrs share higher similarity within a virus strain than any hrs between species, this evidence further indicates that hrs play a fundamental role in viral life cycle and replication process appears to be tightly linked to functional conservation. The dot plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that MabrNPV-K1 is a Group II NPV that is closely related to MacoNPV but with a distinct genomic organization.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. 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. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

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.

Mamestra brassicae (cabbage moth) is a common European moth of the order Lepidoptera and the family Noctuidae. The larval stage is highly polyphagous and is known to feed on more than 70 species of host plants from 22 families, including Brassica species, lettuce, onion, potato, pea, tomato and apple. M. brassicae has become a significant pest also in Asia due to the damage caused to agriculturally and economically important Brassica crops. It is difficult to control M. brassicae using chemical insecticide because of its rapid development of resistance. The objective of our study, therefore, was the mass production and formulation of a local strain of M. brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) for the development of viral insecticide to control it. In production efficiency of MabrNPV-K1 using M. brassicae larvae, the mortality of the 3rd instar larvae was 100% when inoculated with 1.0 × 105 PIBs/larva and the yield of MabrNPV-K1 was maximal. Regarding the mortality, yield of polyhedra, inoculation doses and required time, the 1.0 × 104 PIBs/larva at 30°C was determined as optimal conditions producing polyhedra efficiently. To formulate MabrNPV-K1, feeding toxicities of various supplements including spreader and ultraviolet (UV) -protectant were determined. Tinopal UNPA-GX which is UV-protectants was effective for protection of polyhedra from UV and showed the increased mortality when added with 1% concentration. Other supplements did not influence significantly the mortality of MabrNPV-K1. Formulated MabrNPV-K1 with several supplements showed higher pathogencity than un-formulated MabrNPV-K1.

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 diamondback moth, Plutella xylostella is a one of the most important pests of various cruciferous crops and has a geographically wide ranging habitat. The heavy dependence on chemical pesticides has created severe pesticide resistance problems. In recent years, Bacillus thuringiensis product have been widely used for P. xylostella control bus genetic resistance in populations to some B. thuringiensis strains, compounded by cross-resistance to several different B. thuringiensis toxins, has also been identified. Such recent resistance problems serve to emphasize the urgent need for alternative control agents and their use within an integrated pest management approach. Baculoviruses have been used as agents for the biological control of certain insect pest species. the granuloviruses (GVs), based on the structure of the occluded virus and the occlusion body (OB). Several reports have showed P. xylostella granulovirus (PxGV) as a promise control agent for P. xylostella. However, it is very difficult to study GV because its OB, granule, has very small size and could be observed exactly under the electron microscopy (EM). This study was performed to develop rapid quantification method for granule of PxGV. After the exact quantification of granule with latex beads using EM, the universal extraction method of viral DNA was established for consistent experiment. The number of granules was calculated by the quantification of PCR products for granuline gene using spectrophotometer and densitometer. This novel calculation method for granule would be useful to study GV.

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.

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.

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.

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from M. saltuarius cadaver to control it. We collected the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius.

μ방lt emitting diodes (LED) devices are commercially introduced as an alternative for low-Ievellaser therapy (11ι，T) ， and it has several advantages over lasers such as a safe, efficient, and less-expensive altemative to treat wounds. And LED irradiation at the same biostimulatory wavelength has similar bíochemical effεαs. In the present study, to asses whether the I핑ht-emitting diode (LED) irradíation can stimulate bone regeneration, irradiated bony defects with or without grafting materials on rat calvaria were compared to corresponding nonirradiated control. Fifty male Sprague-Dawly rats weighing about 150g, were used. Factors for present study were designed as follows, 1) presence or absence of grafting materials, 2) with or without irradiation, and 3) number of irradiation. Two weeks after operation, rats were sacrifìced. Radiologic and 비stomorphologic fmdings were evaluated. Macrospically, there were no incidents of infection, dehiscence, hematoma and necrosis during study. Radiological findings showed greater radiopacity in the graft group and radiopacity increased as the number of irradiation increase. And microscopically, new bαle formation was great in the graft group and increased as the number of irradiation increase, Present study has shown that LED irradiation improved bone regeneration through radiologic and histomorphologic fmdings in rat.

본 연구에서는 SWAT 모형과 random forest를 이용하여 미래 기후변화에 따른 한강유역(34,148 km2)의 수생태계 건강성을 평가하였다. 국립 환경과학원에서 8년간(2008~2015년) 봄철(4~6월)에 모니터링한 부착돌말류 지수(TDI), 저서형 대형무척추동물지수(BMI), 어류평가지수(FAI)는 0~100점, A~E등급으로 평가되며, 이를 본 연구에서 사용하였다. 수생태 건강성에 영향을 미치는 변수로는 수질(T-N, NH4, NO3, T-P, PO4)과 수온을 선정하였으며, 수질 오염도가 낮은 경우에는 수생태계 건강성 점수가 광범위하게 분포되지만 수질 오염도가 높은 경우 수생태계 건강성 점수가 낮아지는 역상관관계를 확인하였다. 기계학습의 분류 분석 기법 중 하나인 random forest 모델을 이용한 세 개의 수생태 건강성 지수 등급 분류 결과 정밀도, 재현율, f1-score 모두 0.81 이상의 예측 정확도를 나타내었다. 기상청의 HadGEM3-RA RCP 4.5와 8.5 시나리오를 적용한 미래 SWAT 수문, 수질 결과 기저유출의 증가로 인해 질소 계열 수질 농도는 기준년도 대비 최대 43.2% 증가하였고, 지표유출 감소로 인해 인 계열 수질 오염도는 최대 18.9% 감소하는 것으로 분석되었다. 미래 FAI, BMI의 등급은 개선되는 경향을 보이지만 TDI는 등급이 악화되는 것으로 나타 났다. 이를 통해 TDI는 질소 계열 수질에 민감하고 FAI, BMI는 인 계열 수질에 더 민감하다고 판단하였다.