Canine distemper virus (CDV) causes serious and often fatal disease in dogs. Currently, various cells or cell lines have been used to detect or produce CDV. In order to set up the conditions, we separated two different cell lines from Madin-Darby canine kidney (MDCK) and named as MDCK-F (fibroblast-like) and MDCK-E (epithelial-like) by Na2EDDA treatment. CDV seed virus was prepared using MDCK cells and inoculated into MDCK-F and MDCK-E including MDCK with various ranges of multiplicity of infection (MOI) to confirm the optimal amount of virus inoculation. The virus titer of TCID50/ml was calculated by inoculation of serially diluted virus into 96-well plate of MDCK cells. The titer and cytopathic effect (CPE) in MDCK-E were compared to those in MDCK-F. The titer of seed CDV was 1.24×106 TCID50/ml. Optimal MOI was about 0.1 for both MDCK-F and MDCK-E to obtain highest titers of 108 TCID50/ml and 5 × 108 TCID50/ml respectively. CPE in MDCK-E was shown 4 days after inoculation whether in MNCK-F 5 6 days after inoculation. We can obtain highest titer of 5 × 108 TCID50/ml with 0.1 MOI using MDCK-E. MDCK-E was more susceptible for CDV production than MDCK-F.

During 2008 2010, 943 swine sera were collected from 45 farms located nationwide. Antibodies against porcine epidemic diarrhea virus (PEDV) were tested via serum neutralization antibody test (SNT) using PEDV-SN, which was adapted and propagated on the Vero cell monolayer with trypsin-free culture media supplemented with more than 10% fetal bovine serum (FBS). All 45 farms were shown to have at least one or more seropositive pig. Of the 943 swine sera that were tested, 931 sera were neutralizing antibody positive against PEDV. These high seroprevalence rates seemed to be due to vaccination or natural infection of PEDV. In a plaque reduction neutralization test (PRNT) using a swine serum showing SN titer of 1:32, a greater than 50% plaque reduction was observed in up to 160 times serum dilution.

Highly pathogenic avian influenza virus (HPAIV) is already panzootic in poultry and caused a considerable economic loss in poultry industry. In addition, HPAIV continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. In this study, the virucidal efficacy of Citra-Kill® composed to quaternary ammonium chloride and citric acid was investigated against avian influenza H9N2 virus (AIV). A virucidal efficacy was determined with the viability of AIV contacted with the disinfectant in the allantoic membrane of chicken embryos. Citra-Kill® and AIV was reacted on the distilled water (DW), hard water (HW) or organic matter suspension (OM) condition. On DW condition, AIV was inactivated with 2,000 fold dilutions of Citra-Kill®. When the antiviral effect on HW condition was evaluated, the antiviral activity of the disinfectant showed on 1,500 fold dilutions against AIV. With the investigation of the antiviral effect of the disinfectant on OM condition, AIV was inactivated on 500 fold dilutions of Citra-Kill®. As Citra-Kill® possesses virucidal efficacy against AIV, the disinfectant solution can be used to limit the spread of animal viral diseases.

Avian influenza (AI) and Newcastle disease (ND) distress a variety of avian species, especially domestic poultry. Severe syndromes are caused by highly virulent specific virus strains termed highly pathogenic AI and velogenic ND viruses, which are potential agrobioterrorism agents. This outbreak emphasizes the need for continuing cooperation between public health and veterinary medical communities in controlling AI and ND when it has a zoonotic potential. Up to date, the stamping out and burying system were applied for controlling methods against these highly infectious diseases in the ordinary way, however these methods had many environmental problems, including leachat and effluvium. Thus, we assessed that sterilization effect of AI and ND virus dependent on several treatment conditions, such as autoclaving time and cutting types of chicken. As a result, we found that the cutting type of chicken meat revealed a reduced HA titer (20) against both of AI and ND virus after 10 min of autoclaving, while whole chicken showed same titer after 30 and 60 min. Therefore, we propose that the conditions of treatment on infected chicken should be developed for convenient, affordable, and effective prevention methods against for AI and ND.

Rabies is a zoonotic disease that causes severe destruction to the central nerve system which is usually fatal. It is one of the most important disease around the world and particular in Asia because of the high costs of prevention and post-exposure treatment. After the recurrence of sylvatic rabies in 1993, the number of raccoon dog mediating rabies cases in Korea has maintained annually until 2011. To better understand the current rabies epidemics in Korea, Korean rabies isolate (SKRBV0601GY) from Gyeonggi province in 2006 was compared with previous isolates in Korea and with isolates originating from the North-East Asia, such as Japan, China and Russia, based on complete nucleoprotein (N) gene sequences. By comparison of the N genes among these viruses, SKRBV0601GY revealed that nucleotide similarity ranged from 97.7 to 99.7%, 96.4 to 97.5%, 91.4 to 96.3%, 89.2 to 90% and 86.1 to 88.1% with Korean isolates, "Arctic-like-2" viruses, "Arctic" viruses, Russian group C - E and Chinese isolates, respectively. Phylogenetic analyses of the isolates revealed that the Korean isolate in 2006 belonged to Korean group B. The topology of the phylogenetic tree of Korean isolates related not the species and year of isolation but the geological location of the virus isolates. All of the Korean isolates showed close relationship to the "Arctic-like-2" virus (Russian group B) more than the "Arctic" virus (Russian group A) and all of the Chinese isolates (Chinese group A, B and C). The "Arctic-like-2" virus group contains the Japanese isolate and Russian group B viruses, originating from the south of East Siberia and Far East in Russia. These molecular data demonstrated that the current rabies epizootic in Korea developed independently of Chinese groups and originated from the "arctic-like-2" viruses in detail.

Ruminant pestiviruses of bovine viral diarrhea virus (BVDV) and border disease virus (BDV) are closely related to classical swine fever virus (CSFV) and all belong to the genus of Pestiviruses. BVDV is one of the most important viral pathogen of cattle and has been recorded in most countries where cattle are raised. Natural host for BVDV is cattle, but BVDV is able to infect pigs as well. The purpose of this study was to investigate the prevalence for antibodies against BVDV in domestic pig farms in South Korea from 2009 to 2011. In this study, 2,755 pigs in 239 farms in South Korea's inland and 5,293 pigs in 613 farms in Jeju province (CSF free region) were investigated for antibodies against two pestiviruses, BVDV and CSFV by a virus neutralization test (VNT). The seroprevalences on the individual level and on herd level against BVDV were 5.3 % and 21.2 % in South Korea's inland, 5.2 % and 6.5 % in Jeju province, respectively. Based on the ratio of respective antibody titers by the comparative VNT, 273 pigs in Jeju province with BVDV infection were detected and they were distinctly negative to CSF. It is recognized that porcine infections with BVDV naturally occurred in Jeju province. Whereas, antibody titers against BVDV of South Korea's inland were cross-reactivity with CSFV.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

Successful early embryogenesis of somatic cell nuclear transfer (SCNT) embryos is very important to produce cloned animals. However, poor preimplantation development of SCNT embryos has been a major obstacle to the generation of cloned animals due to a lack of understanding of developmental events and underlying mechanism(s). In the current study, we show that production of SCNT embryos with high developmental competence is dependent on the fusion method. Electrofusion causes spontaneous egg activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodeling, whereas Sendai virus (SV)-mediated fusion greatly reduces these events. In addition, SV-SCNT increased the blastocyst development rate and trophectoderm cell number compared to electrofusion-mediated SCNT (E-SCNT). In particular, expression of ER stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos, which could be alleviated by inhibition of ER stress or by using the SV-mediated fusion method. Taken together, these results strongly suggest that SV is a useful fusion material for improvement of preimplantation development of SCNT embryos through reduction of ER stress-associated apoptosis.

Rice stripe virus (RSV), the type member of the genus Tenuivirus, causes rice stripe disease and the viral transmission is mediated through the sucking by small brown planthopper, Laodelphax striatellus. Considerations have been mainly focused on the protection of rice from RSV and/or the planthopper, rather than the interaction between RSV and the insect. To clarify the interaction, in this work, mRNA was extracted from RSV-viruliferous planthopper with non-viruliferous control, and expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing technology for comparative analysis. RSV-viruliferous planthopper had ca. 2500 isotigs, which included genes on biological process (19%), cellular component (13%), molecular function (22%) and no hits (46%) from gene ontology (GO) analysis; this structure was similar to the control. However, in the viruliferous planthopper, 109 isotigs were up-regulated and 660 isotigs were down-regulated, compared to the non-viruliferous control. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

Hz-2V, which belongs to nudiviridae, is a sexually transmitted insect virus of corn earworm, Helicoverpa zea. Hz-2V is transmitted during mating or mating attempt of infected individuals, and specifically replicates in the reproductive tissues to cause abnormal development of testes and ovary in the adult moths of next generations. The malformation of Hz-2V reproductive tissues started during the early pupal stage without clear sign of virus replication. The virus replication started at the late pupal stage to cause sterility of the emerged moths. Interestingly, the infected female moths showed a unique pathology, so called ‘waxy-plug’, which is filled with virus particles, and abnormal mating behavior while they produced 6~7 times more of pheromone than the normal female moths did. To investigate the factors of Hz-2V which control the physiological, and behavioral changes of the infected moths, the whole genome sequence of Hz-2V was determined. Bioinformatics analysis demonstrated that Hz-2V contains 113 putative ORFs including juvenile hormone esterase (JHE) and histone binding protein homologues, and a miRNA candidate which probably controls the expression of viral JHE.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

고구마 재배농가에서 무병묘를 손쉽게 자가증식할 수 있는 간편한 수경재배 시스템을 도입하기 위하여, 점적호스를 이용한 순환식 자루재배 시스템을 시설하여 배지 혼합비율 및 삽수종류에 따른 무병묘의 생육을 조사하였다. 연황미와 맛나미 모두 15일경까지는 배지 종류에 따른 생육에 유의한 차이를 보이지 않았으나, 20일경부터는 원예용상토(TKS-2)와 펄라이트 함량이 높은 혼합배지(코코피트 : 펄라이트, 3 : 7 v/v)에서 생육이 양호하였다. 품종 간에는 연황미의 생육이 맛나미보다 양호하였으며, 1마디 삽수보다 정단 삽수로 증식하는 것이 효과적이었다. 연황미와 맛나미의 줄기생육은 정단 삽식에서 1마디 삽식보다 10cm 및 2cm 이상 신장하였으며, 코코피트와 펄라이트 3 : 7(v/v) 혼합배지에서의 건물중과 생체중은 원예용 상토 또는 코코피트와 펄라이트 7 : 3(v/v) 혼합배지에서보다 유의한 증가를 보였다. 따라서 코코피트와 펄라이트(3 : 7, v/v) 혼합배지를 이용한 자루재배는 고구마 무병묘 증식에 성공적으로 적용할 수 있었으며, 고구마 종순농가에서도 큰 경제적 부담없이 적용할 수 있을 것으로 기대되었다.

Horse population has been gradually increasing with the growth of the racing industry in Republic of Korea (ROK). Approximately 22,941 horses including native ponies were raised on 1,142 farms in 2006. Great care has been taken to prevent viral, bacterial and parasitic infections in horses. However, there has been few reports on equine infectious anemia (EIA) in ROK until recently since 1987. Therefore, we conducted a serological survey of EIA in ROK from 2007 through 2009. Using the agar gel immunodiffusion test, a total of 2,601 horses were tested in Foreign Animal Disease Division of Animal, Plant, and Fisheries Quarantine and Inspection Agency. This survey found no serological evidence of EIA presence in ROK. Although our surveillance found no evidence of EIA in ROK during the surveillance period, it is possible that EIA could be introduced into ROK in the near future. Increased cooperation between the government and other agencies, such as horse-racing authorities, is important for both the early detection of the disease and the development of effective veterinary and public health strategies.

Seropositivity against Japanese encephalitis virus (JEV) was measured by hemagglutination inhibition test (HI) on sera from 323 horses raised in Jeju. Enzyme-linked immunosorbent assay (ELISA) method was applied for the confirmation of its availability as a tool of detecting anti-JEV antibodies in horses. The positive rate scored 50.4%. The positive rates were increased according to ages. The highest peak was shown at October. And Jeju Ponies showed higher positivity compared to thoses of Thoroughbred horses. When compared the results obtained by HI and ELISA, there was slight correlations between the two methods (r=0.7100). Besides, ELISA could discriminate between true and false positive sera by neutralizing serum specimen with JEV antigen.

Korea had experienced second epidemic highly pathogenic avian influenza (HPAI), and there were the seven affected farms, including two breeder duck farms, from 22nd November 2006 to 6th March 2007. Here, we reported the clinico-pathological characteristics of domestic breeder ducks farms naturally infected with HPAI virus (H5N1). Clinically, the most ducks showed various signs from depression, decreased egg production and feed consumption to even, death. The most commonly gross changes were hepatomegaly, splenomegaly, petechial and ecchymotic hemorrhage on the liver surface, a white stripe on the cardiac muscle, and severely hemorrhagic and deformed eggs. The most significant histopathological changes were necrosis of various cells such as neuron, lymphocytes, cardiac myocytes, hepatocytes, blood vessels and pancreatic acinar epithelium. The viral antigen was mainly detected in the endothelium of blood vessels of various organs and tissues, peripheral nerves and neuronal cells. Based on the above results, we identified that HPAI H5N1 induced systemic infection in the adult breeder ducks.

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. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

The silkworm-baculovirus expression system has distinct advantages, such as a high yield and safe usage in vertebrates. Here, we report a novel strategy for the large-scale production of a classical swine fever virus (CSFV) envelope glycoprotein E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedraelicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen.

The sweetpotato whitefly, Bemisia tabaci, acts as a vector of more than 100 plant viruses. B. tabaci is known to harbor a primary endosymbiont (Portiera) and 6 secondary endosymbionts (Arsenophonus, Cardinium, Fritschea, Hamiltonella, Rickettsia and Wolbachia). These endosymbionts play important roles in the acquisition and transmission of plant viruses. Using PCR analysis, we identified endosymbiotic bacteria in various B. tabaci populations collected from different places of Korea. Distribution of endosymbionts was different according to the biotype of B. tabaci. Subsequently, their relative densities of endosymbionts were compared between TYLCV-viruliferous and non-viruliferous populations of the Q biotype using quantitative realtime PCR. We found that the densities of Portiera, Cardinium and Hamiltonella are higher in viruliferous than non-viruliferous whiteflies. Our results suggest the role of endosymbiont for the TYLCV transmission of whiteflies.

The sweetpotato whitefly, Bemisia tabaci, is a vector of more than 100 plantdiseased viruses as well as a serious pest to various horticultural crops. Virus acquisition affects the vector’s development and reproduction, but its mechanism is largely unknown. Here we compared the temperature responses between non-viruliferous and TYLCV-viruliferous Q biotype of B. tabaci. When both non-viruliferous and viruliferous whiteflies were exposed for 1 and 3 h at 4, 25, and 35°C, the mortality rate of viruliferous whiteflies is higher than nonviruliferous after exposure at 4°C and 35°C, but no differences at 25°C between them. Analysis of the expression levels of heat shock protein (hsp) genes using the quantitative realtime PCR showed that viruliferous whiteflies has higher expression in hsp70, and hsp90 at both 4°C and 35°C, but no differences at 25°C. The results suggest that vector insects may not be durable to unfavorable temperature conditions when they acquisite plant viruses.