Bovine viral diarrhea virus (BVDV) of the genus Pestivirus is known as a common contaminant of cell culture-derived vaccines. Hog cholera virus (HCV), which is also of the genus Pestivirus and an important livestock disease in Korea, is recognized as a potential contaminant of vaccines produced in porcine cells. However, it is difficult for the National Biological Assays of korea to adequately detect contamination of these agents in biological products. For these reasons, we established rapid and sensitive methods for the detection of BVDV and HCV contamination in cell cultures and veterinary biologicals by using RT-PCR and nested PCR assays. We designed a Pestivirus primer amplifying 152 bp to detect both BVDV and HCV and a common primer amplifying 237 bp to detect only BVDV. Also, for the differentiation between BVDV type 1 and type 2, nested PCR was conducted using the amplified 237 bp PCR product, to amplify 179 bp in BVDV type 2 genome. The sensitivity of the PCR using common primer for the detection of BVDV was 400 TCID50/ml. All 6 strains of Korean BVDV isolates, 5 vaccines strains and the standard strain NADL could be detected. No reactions were observed when testing 5 types of viruses infecting pigs (HCV, TGEV, PEDV, JEV, PRRSV), 4 types infecting cattle (Akabane virus, BEFV, BCV, BRV) and 4 types infecting cats (FIP, FPL, FCV, FVR). Using this RT-PCR assay, commercial vaccines were tested and, 55 lots from 12 vaccine companies, were negative for BVDV contaminations. Same results were obtained by the immunoflourescence assay. The newly developed PCR or RT-PCR assays can be used as rapid, reliable, sensitive, and simple methods for the detection of BVDV (Pestivirus) in cell cultures, master seeds, and live viral vaccines.
Animals* ; Biological Assay ; Biological Products ; Cats ; Cattle ; Cell Culture Techniques ; Classical swine fever virus ; Diarrhea Virus 1, Bovine Viral ; Diarrhea Virus 2, Bovine Viral ; Diarrhea* ; Genome ; Korea ; Livestock ; Pestivirus ; Polymerase Chain Reaction* ; Swine ; Vaccines ; Viral Vaccines*

Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. The core particle of JEV consists of a multiple copies of the capsid protein. In this study, the author demonstrated that the JEV capsid protein was appeared to be localized predominantly both around the perinuclear membrane structures in the cytoplasm of the JEV-infected cells and within the nucleus by using a mouse JEV capsid-specific antiserum. The presence of the capsid protein within the nucleus was also demonstrated by expression of EGFP-capsid fusion protein from a Sindbis virus-derived expression vector in the transfected cells. Amino acid sequence comparison of several flavivirus capsid proteins similar to JEV revealed that the JEV capsid protein contains a nuclear localization motif. Thus our result showing that the JEV capsid protein is localized within the nucleus as well as in the cytoplasm provides an important clue for the role of the capsid protein in JEV replication.
Amino Acid Sequence ; Animals ; Asian Continental Ancestry Group* ; Capsid Proteins ; Cytoplasm ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Humans ; Membranes ; Mice

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most common and economically significant infectious agents in the swine industry worldwide. The virus causes mild to severe reproductive failure in sows and gilts and respiratory problems in piglets. In this study, the author expressed the full-length PRRSV Nsp1a protein as a Glutathion-S-Transferase (GST)-Nsp1a fusion protein in E. coli BL-21. The full-length Nsp1a coding region of the Korean PRRSV isolate PL97-1 was amplified by RT-PCR from the genomic RNA and the cDNA amplicons were cloned into the pGEX4T-1 E. coli expression vector. The recombinant GST-Nsp1a fusion protein was used for immunization in rabbits. After a 5-shot immunization schedule, the PRRSV Nsp1a-specific antiserum was obtained. Using the PRRSV-infected MARC-145 cell lysates, immunoblotting analyses showed that these antisera specifically reacted with ~18 kDa of the PRRSV Nsp1a protein. The rabbit antisera raised against the PRRSV Nsp1a recombinant protein will provide a useful reagent to investigate the role of this protein in PRRSV replication and to diagnose infection.
Clinical Coding ; Clone Cells ; DNA, Complementary ; Immune Sera ; Immunization ; Immunization Schedule ; Immunoblotting ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Rabbits ; RNA ; Staphylococcal Protein A ; Swine

Apodemus agrarius, which accounts for three-fourths of the wild rodents, mainly inhabits in cultivated fields of Korea. Apodemus peninsulae and Eothenomys regulus are the second and third dominant species, respectively. Soochong virus (SOOV) from A. peninsulae and Puumala-related Muju virus (MUJV) from E. regulus were isolated in 1997 and 1998 in Korea, respectively. But serological characterizations of SOOV and MUJV were not identified clearly. Thus, in order to determine the serotypic classification, simultaneous cross-indirect immunofluorescent antibody (IFA) assay and cross-plaque reduction neutralization (PRN) test against four different hantaviruses were conducted with sera from 17 A. agrarius, 19 A. peninsulae, and 8 E. regulus strains. IFA titers of sera from A. agrarius and A. peninsulae were the highest to Hantaan virus (HTNV) and SOOV, respectively. However, most sera showed similar IFA titers to Seoul virus (SEOV). Therefore it was difficult to do serotyping using the sera from A. agrarius and A. peninsulae by IFA. In case of sera of E. regulus, IFA titers to Puumala virus (PUUV) were higher than HTNV, SOOV and SEOV. Cross-PRN result of A. agrarius to HTNV, SOOV, SEOV and PUUV was 6,890, 5,120, 110 and 30, respectively. In case of A. peninsulae, the mean PRN titer was the highest to SOOV (1:6,820) and those to HTNV, SEOV and PUUV were 1,580, 100 and 30, respectively. The mean PRN titers of E. regulus to HTNV, SOOV, SEOV and PUUV were 70, 10, 80 and 640. SOOV and MUJV could be distinguished from HTNV and SEOV by cross-PRNT. These results demonstrate that SOOV and MUJV could be classified as new serotype of hantavirus.
Animals ; Classification ; Hantaan virus ; Hantavirus* ; Korea ; Murinae ; Puumala virus ; Rodentia ; Seoul virus ; Serotyping

Both interleukin (IL)-12, an important cytokine skewing the immune response towards a Th1 cytokine profiles, and tumor necrosis factor (TNF)-alpha, are thought to be critical factors in defenses against mycobacteria. In this study, we evaluated the roles of phosphatidylinositol 3-kinase (PI 3-K), and extracellular signal-regulated kinase (ERK) 1/2 pathways in the expression of IL-12 in human monocyte-derived macrophages (MDMs) after stimulation with Mycobacterium tuberculosis H37Rv (M. tbc) or the Triton X-114 solublized proteins (TSP) of M. tbc. Both M. tbc and TSP rapidly phosphorylated ERK 1/2, and Akt in human MDMs. Inhibition of PI 3-K-Akt pathway by specific inhibitors (LY294002 and wortmannin) dramatically increased M. tbc- or TSP-induced IL-12 p40 and p35 mRNA and IL-12 production. In addition, blockade of ERK 1/2 pathway by specific inhibitors (PD98059 and U0126) significantly increased the mRNA levels and cytokine production in M. tbc- or TSP-treated MDMs. On the contrary, M. tbc- or TSP-induced TNF-a production was significantly depressed in human MDMs by pretreatment with inhibitors of PI 3-K or ERK pathways. The M. tbc or TSP stimulation decreased ERK 1/2 phosphorylation by 70% in the presence of wortmannin or LY294002, suggesting that some cross-talk between the PI 3-K-Akt and mitogen-activated protein kinase kinase (MEK)-ERK pathways may be operating in human monocytes during mycobacterial infection. PI 3-K activity is partially required for the M. tbc- or TSP-induced ERK 1/2 phosphorylation. Collectively, these data suggest that the PI 3-K and ERK 1/2 pathways play a central role in the negative regulation of IL-12, but not TNF-a, production by M. tbc.
Humans* ; Interleukin-12* ; Interleukins ; Macrophages* ; MAP Kinase Signaling System ; Monocytes ; Mycobacterium tuberculosis ; Neptune ; Phosphatidylinositol 3-Kinase* ; Phosphatidylinositols* ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; RNA, Messenger ; Tumor Necrosis Factor-alpha

Mycobacterium tuberculosis is a potent inducer of cytokine production by mononuclear phagocytes, which are an important cellular component in the first line immune defence. In this study, the cell wall-associated Triton X-100 soluble protein (TSP) antigens, TSP-H37Rv, TSP-H37Ra, TSP-K, and TSP-BCG, were isolated from M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. tuberculosis K-strain, and M. bovis BCG, respectively. The monocytes were isolated from the peripheral blood mononuclear cells of healthy individuals and were co-cultured with each TSP antigens and the secretory proteins of M. tuberculosis (PPD and 30-kDa antigen) to measure the production of cytokines; tumor necrosis factor (TNF)-a, interleukin (IL)-12, IL-8 and monocyte chemotactic protein-1 (MCP-1). The TSP-H37Rv antigen- stimulated monocytes showed higher level of TNF-a and IL-12 production compared to those of other TSP antigens and PPD. Especially, IL-12 production in response to the TSP-H37Rv antigen was significantly elevated in comparison with that of PPD-stimulated monocytes (TSP-H37Rv, 255.5+/-256.9 pg/ml; PPD, 55.7+/-55.4 pg/ml). However, the 30-kDa antigen did not induce TNF-alpha expression and also showed the lowest level of cytokine and chemokine production by monocytes. MCP-1 and IL-8 production were similarly increased in response to all TSP antigens and the PPD antigen. The production of IL-12 by the TSP-H37Rv antigen stimulation was significantly increased in PPD reactors than that in the non-reactor group, while the levels of other cytokines stimulated with each TSP antigens, 30-kDa and PPD antigen were not significantly different between the tuberculin reactor and the non-reactor groups. These results suggest that the cell wall-associated TSP antigen isolated from M. tuberculosis H37Rv acts as a more potent IL-12 inducer than the PPD antigen in innate immune response and thus it could further activate the Th1-mediated immune responses effectively against M. tuberculosis infection.
Chemokine CCL2 ; Cytokines ; Humans* ; Immunity, Innate ; Interleukin-10* ; Interleukin-12* ; Interleukin-8* ; Interleukins ; Monocytes* ; Mycobacterium bovis ; Mycobacterium tuberculosis* ; Mycobacterium* ; Neptune* ; Octoxynol* ; Phagocytes ; Tuberculin ; Tuberculosis ; Tumor Necrosis Factor-alpha*

Intestinal epithelial cells are known to up-regulate the expression of several chemokines in response to bacterial toxins. Since there has been little understanding on the cellular mechanisms of C. difficile toxin A-induced mucosal inflammation, we investigated whether nuclear factor-kappa B (NF-kappaB) could regulate chemokine gene expression in HT-29 intestinal epithelial cells stimulated with C. difficile toxin A. C. difficile toxin A rapidly increased signals of NF-kappaB composed with p65 and p50 subunits in HT-29 cells, whereas it decreased the signals of IkappaBalpha. Blocking the NF-kB activation by transfection with dominant negative I kappa B alpha-containing retrovirus attenuated the upregulated expression of IL-8, GRO-alpha, and MCP-1 induced by C. difficile toxin A. These results suggest that NF-kappaB is a major regulator of chemokine gene expression in C. difficile toxin A-stimulated intestinal epithelial cells.
Bacterial Toxins ; Chemokines ; Clostridium difficile* ; Clostridium* ; Epithelial Cells* ; Gene Expression ; HT29 Cells ; Humans ; I-kappa B Proteins ; Inflammation ; Interleukin-8 ; NF-kappa B ; Retroviridae ; Transfection

The motile marine bacterium, Vibrio vulnificus has a total of six flagellins. Flagellin is a structural component of flagellar filament in various locomotive bacteria and is the ligand of Toll-like receptor 5 (TLR5). TLRs, highly expressed on various types of cells including dendritic cells (DCs), recognize invading microorganisms and finally trigger host immune responses. In this study, we prepared all of six recombinant flagellin proteins and assessed the effect of six flagellins on IL-8 activation through TLR5 recognition. Although showed different activities, five out of the six flagellins stimulated significant IL-8 activation. We also investigated the immunomodulatory roles of Vv-FlaB, the crucial building block of V. vulnificus flagellar filament, on human dendritic cells. Treatment of immature DCs with Vv-FlaB resulted in an increased expression of co-stimulatory molecules and induced strong allo-T cell proliferative activities of the DCs. These results show that the Vv-FlaB may serve an epochal immune adjuvant possessing effective immunomodulatory activities.
Bacteria ; Dendritic Cells* ; Flagellin* ; Flow Cytometry ; Humans* ; Interleukin-8 ; Toll-Like Receptor 5 ; Vibrio vulnificus* ; Vibrio*

A total of 190 ticks collected from the Chungju area of Korea was examined for the presence of Spotted Fever Group(SFG) Rickettsia using a PCR assay. Twenty-five (13.2%) Haemaphysalis ticks were found positive of the groEL gene of SFG Rickettsia. The prevalence rate of R. japonica in these 25 Haemaphysalis ticks was 72% (18 out of 25 SFG Rickettsia). The prevalence rate of R. conorii and new SFG rickettsia in these 25 Haemaphysalis ticks was 4% (1 out of 25 SFG Rickettsia) and 24% (6 out of 25 SFG Rickettsia), respectively. These results suggest that R. japonica was the highest infection frequency among in Haemaphysalis ticks SFG Rickettsia, and that R. conorii and new SFG Rickettsia are also present in the Chungju area.
Chungcheongbuk-do* ; Fever* ; Korea ; Polymerase Chain Reaction ; Prevalence* ; Rickettsia* ; Ticks*

The purpose of this study is to survey the frequency of mutans streptococci species and biotypes isolated from dental plaque in Korean adolescents and adults and the relationship between species of mutans streptococci and DMFT index. The dental plaque samples were collected from the anterior and molar teeth of both of jaws in the 47 human subjects (aged 14 to 29.3 years, average age was 20.2 years). A dental examination was performed for DMFT with the WHO caries diagnostic criteria. The mutans streptococci were cultured selectively on mitis-salivarius bacitracin (MSB) agar plates. The bacterial colonies growing on the MSB plates were then identified with biochemical tests (for biotyping) as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex) (for the determination of species). The data showed that the prevalence of Streptococcus mutans, S. sobrinus, and S. downei were 93.6%, 12.8%, and 8.5%, respectively. The biotype I (87.4%) and biotype IV (19.1%) were the most frequently detected. The DMFT scores of adolescents and adults harboring both S. mutans and S. sobrinus were significantly higher than those for S. mutans alone (p < 0.05). Above results revealed that S. mutans and biotype I were frequently detected in Korean adolescents and adults. In addition, the results suggest that subjects having plaques compared of both S. mutans and S. sobrinus may be more susceptible to caries than those having S. mutans alone.
Adolescent* ; Adult* ; Agar ; Bacitracin ; Dental Plaque* ; Dextranase ; Humans ; Jaw ; Molar ; Prevalence ; Streptococcus mutans ; Tooth