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Expression and Antibody Production of Japanese Encephalitis Virus RNA Polymerase (NS5) Protein.

Jeong Min KIM ; Sang Im YUN ; Byung Hak SONG ; Yu Jeong CHOI ; Jun Sun PARK ; Young Min LEE

Journal of Bacteriology and Virology.2009;39(1):53-60. doi:10.4167/jbv.2009.39.1.53

Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.
Animals ; Antibody Formation ; Asia, Southeastern ; Asian Continental Ancestry Group ; Australia ; Clone Cells ; DNA ; Encephalitis Virus, Japanese ; Encephalitis, Japanese ; Encephalitis, Viral ; Flavivirus ; Genome ; Humans ; Immune Sera ; Immunization ; Mice ; Proteins ; RNA ; RNA Replicase ; Sprains and Strains ; Vero Cells

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Development of RT-PCR Method to Detect Various Human Enteric Viruses.

Sung Ae OH ; Mi Suk KIM ; So Young JANG ; Sang Jong KIM ; Jae In LEE ; Gyu Cheol LEE ; Chan Hee LEE

Journal of Bacteriology and Virology.2009;39(1):41-51. doi:10.4167/jbv.2009.39.1.41

Human enteric viruses are one of the major causes of acute gastroenteritis outbreaks. A rapid and precise detection of virus is critical for prompt diagnosis. For this purpose, nucleic acid-based techniques such as reverse transcription (RT)-PCR have been developed. Although RT-PCR is a rapid, specific and sensitive method to detect virus, many steps or reactions are required, especially when various types of viruses are targeted. In this study, we developed a quick and effective method to detect human enteric viruses with a few reactions. Our candidate viruses were as follows: one DNA virus (adenovirus: AdV) and seven RNA viruses including poliovirus (PV), coxsackievirus A (CoxA) and B (CoxB), human rotavirus (HRV), hepatitis A virus (HAV), norovirus (NorV), and astrovirus (AstV). With this amount of samples, theoretically, a total of fifteen biomolecular reactions have to be performed, which include seven RT reactions and eight subsequent PCR with specific primers in each case. Specific primers, enterovirus universal primers, and random primers were applied independently to compare the outcomes of RT and PCR steps in each viral sample. We found that random 9-mer is ideal for the RT reactions of RNA viruses with negligible differences in sensitivity and specificity of viral detection except HRV. Hence, HRV cDNA generated by HRV-specific primer and AdV DNA were amplified in a single tube by duplex PCR. The cDNAs generated by RT using random 9-mers were divided into two reaction tubes without losing sensitivity: one duplex PCR detects enteroviruses (PV, CoxA, CoxB) and HAV, the other detects NorV and AstV. In conclusion, it is possible to detect eight enteric viruses with a substantially reduced number of reactions, which are composed of five reactions, two RT and three PCR reactions.
Collodion ; Disease Outbreaks ; DNA ; DNA Viruses ; DNA, Complementary ; Enterovirus ; Gastroenteritis ; Hepatitis A virus ; Hip ; Humans ; Norovirus ; Poliovirus ; Polymerase Chain Reaction ; Reverse Transcription ; RNA Viruses ; Rotavirus ; Sensitivity and Specificity ; Viruses

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Identification of Shiga Toxin-producing E. coli Isolated from Diarrhea Patients and Cattle in Gwangju Area, Korea.

Min Ji KIM ; Sun Hee KIM ; Tae Sun KIM ; Hye Young KEE ; Jin Jong SEO ; Eun Sun KIM ; Jong Tae PARK ; Jae Keun CHUNG ; Jaeil LEE

Journal of Bacteriology and Virology.2009;39(1):29-39. doi:10.4167/jbv.2009.39.1.29

Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle and cause food borne disease in human. We analyzed the isolation rate of STEC in stool specimens of patients with diarrhea and in fecal samples of cattle in Gwangju, Korea. STEC strains were detected from 33 (0.19%) out of 17,148 patients with diarrhea while there has been a progressive increase in the incidence rate from 0.07% in 2004 to 0.33% in 2008. We investigated serotypes, shiga toxin genes, and antimicrobial resistance patterns of the 44 STEC isolates from human and cattle sources. The 33 STEC isolates from human belonged to 14 O serotypes including O157, O26 and O111. The 11 isolates from cattle belonged to 11 O serotypes. PCR detection for stx genes showed that 12 (27.3%) isolates carried stx1 genes, 20 (45.5%) possessed stx2 genes, and 12 (27.3%) carried both stx1 and stx2. Of the 33 STEC isolates from human, 25 strains (76%) were resistant to one or more antibiotics. High level of resistance to tetracycline (73%) was most common, followed by ticarcillin and ampicillin (64%). But none of the 33 isolates from human were resistant to amikacin, cefazolin, cefepime, cefotetan, cefotaxime, ciprofloxacin, or imipenem. The 5 strains (45%) of the 11 isolates from cattle were resistant to at least one or three antibiotics but most of the isolates were sensitive to the 16 antibiotics employed in this survey. In conclusion, toxin types and serotypes of STEC isolated from human and cattle were diverse, and non-O157 STEC was also observed to be a greater proportion of STEC isolates. According to a specific comparison solely based on the toxin types and serotypes, most of the STEC strains isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. Therefore, laboratory surveillance is required to detect and carefully monitor the potentially hypervirulent STEC not only in human and cattle but also in other animals.
Amikacin ; Ampicillin ; Animals ; Anti-Bacterial Agents ; Bacteria ; Cattle ; Cefazolin ; Cefotaxime ; Cefotetan ; Cephalosporins ; Ciprofloxacin ; Diarrhea ; Feces ; Humans ; Imipenem ; Incidence ; Korea ; Organothiophosphorus Compounds ; Polymerase Chain Reaction ; Shiga Toxin ; Shiga-Toxigenic Escherichia coli ; Tetracycline ; Ticarcillin

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Distribution of Group A Rotavirus Genotypes Circulating in Gwangju, Korea.

Sun Hee KIM ; Doo sung CHEON ; Hye Sook JEONG ; Min Ji KIM ; Hye Young KEE ; Jin Jong SEO ; Tae Sun KIM ; Eun Sun KIM ; Jong Tae PARK ; Jae Keun CHUNG

Journal of Bacteriology and Virology.2009;39(1):21-28. doi:10.4167/jbv.2009.39.1.21

To determine the distribution of rotavirus strain genotypes in Gwangju, Korea, we performed reverse-transcription polymerase chain reaction and nucleotide sequencing analysis using the 115 rotavirus EIA positive stool specimens collected from December 2006 through April 2007. The most predominant genotype was confirmed as G1P[8] (53.9%), followed by G3P[8] (29.6%), G4P[6] (8.7%), G2P[4] (4.3%) and G9P[8] (1.7%). A special attention is drawn to the unusual findings of the genotypes G11P[25] and G12P[9] during this study period. In order to investigate the phylogenetic relationships among the same or different genotypes, the nucleotide sequences of rotavirus circulating in Korea and the foreign countries were analyzed using MegAlign and Clustal X programme. The G11P[25] strain identified in this study showed the highest nucleic acid similarity with G11 /CUK1 /2006 /Korea (99.2%) and P[25] /Dhaka /2001 /BGD (98.7%). Meanwhile, the G12P[9] strain detected in this study shared 99.4~99.5% nucleotide homology with the reference strain G12P[9] /CP1030 /2004 /Japan. This incidence of new rotavirus genotypes in our area illustrates the large diversity of rotavirus strains found worldwide. Therefore, the epidemiological surveillance of rotavirus may need to be continued in a wide geographic area.
Base Sequence ; Genotype ; Incidence ; Korea ; Polymerase Chain Reaction ; Rotavirus ; Sprains and Strains

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Isolation and Identification of Lactic Acid Bacteria Inhibiting the Proliferation of Propionibacterium acnes and Staphylococcus epidermidis.

Mi Sun KANG ; Hyun Ju OH ; Hyun Chul LEE ; Jong Suk OH

Journal of Bacteriology and Virology.2009;39(1):11-19. doi:10.4167/jbv.2009.39.1.11

Propionibacterium acnes is the most common causative agent of acne. Staphylococcus epidermidis is another major bacterial strain to be found in acne lesions. Two strains of lactic acid bacteria (LAB) were isolated from normal inhabitants of humans, which inhibited the proliferation of P. acnes and S. epidermidis. The growth of P. acnes and S. epidermidis was decreased by 4-log scales after incubation for 24 h with LAB isolates, whereas the growth rate of selected LAB isolates were not affected by these pathogenic bacteria. This antibacterial activity of LAB isolates was related to lactic acids, hydrogen peroxide and bacteriocin-like compound production. Two LAB isolates efficiently adhered to human keratinocytes HaCaT and were identified by API 50 CHL medium kit and 16S rDNA partial sequencing analysis. The similarity of 16S rDNA sequences between one isolate and Lactobacillus salivarius subsp. salicinius was 100%, which suggests that they were L. salivarius subsp. salicinius. On the other hand, 16S rDNA sequence similarity between the other isolate and Lactobacillus fermentum was 99.04%, which indicates that it was L. fermentum. In conclusion, these results demonstrate that the two LAB strains isolated from human body were identified as L. salivarius subsp. salicinius and L. fermentum, which inhibit the proliferation of P. acnes and S. epidermidis.
Acne Vulgaris ; Bacteria ; DNA, Ribosomal ; Hand ; Human Body ; Humans ; Hydrogen Peroxide ; Keratinocytes ; Lactic Acid ; Lactobacillus ; Lactobacillus fermentum ; Propionibacterium ; Propionibacterium acnes ; Pyridines ; Sprains and Strains ; Staphylococcus ; Staphylococcus epidermidis ; Thiazoles ; Weights and Measures

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Mycobacterium tuberculosis Induces the Production of Tumor Necrosis Factor-alpha, Interleukin-6, and CXCL8 in Pulmonary Epithelial Cells Through Reactive Oxygen Species-dependent Mitogen-activated Protein Kinase Activation.

Hye Mi LEE ; Dong Min SHIN ; Eun Kyeong JO

Journal of Bacteriology and Virology.2009;39(1):1-10. doi:10.4167/jbv.2009.39.1.1

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of inflammatory cytokines/chemokines. In this study, we found that Mycobacterium tuberculosis (Mtb)induced reactive oxygen species (ROS) production is essential for the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and CXC-chemokine ligand (CXCL) 8 through the activation of mitogen-activated protein kinases [MAPKs; extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK] in A549 cells representing alveolar epithelial cells. We observed that Mtb rapidly enhanced ROS production after stimulation in a toll-like receptor (TLR) 2-dependent manner. In addition, Mtb triggered ERK1/2 and p38 MAPK signaling pathways which were dependent on ROS generation in A549 cells. Moreover, Mtb stimulation significantly increased the secretion of TNF-alpha, IL-6, and CXCL8 over that in untreated controls. Pretreatment of A549 cells with the antioxidant, N-acetylcysteine and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenylene iodonium, substantially inhibited Mtb-induced production of TNF-alpha, IL-6, and CXCL8. Studies using inhibitors selective for ERK1/2 and p38 MAPK pathways showed that both pathways play an essential role in the induction of TNF-alpha, IL-6, and CXCL8 at transcriptional levels in A549 cells. Collectively, our findings indicate the critical role of TLR2-dependent ROS in the Mtb-induced inflammatory cytokine/chemokine production in alveolar epithelial cells through MAPK-dependent signaling pathways.
Acetylcysteine ; Epithelial Cells ; Interleukin-6 ; Interleukins ; Mitogen-Activated Protein Kinases ; Mycobacterium ; Mycobacterium tuberculosis ; NADP ; Onium Compounds ; Oxidoreductases ; Oxygen ; p38 Mitogen-Activated Protein Kinases ; Phosphotransferases ; Protein Kinases ; Reactive Oxygen Species ; Signal Transduction ; Toll-Like Receptors ; Tumor Necrosis Factor-alpha

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Preoperative Diagnosis and Medical Treatment of Pelvic Actinomycosis.

Hong Jue LEE ; Su Jin LEE ; Young Jae KIM ; Sung Hee KIM ; Jung Han LEE ; Seung Ryoung KIM ; Sam Hyun CHO

Journal of Bacteriology and Virology.2008;38(2):89-95. doi:10.4167/jbv.2008.38.2.89

The diagnosis of the pelvic actinomycosis is seldom made preoperatively because of no reliable or specific clinical manifestation which has tendency to mimic advanced gynecological malignancy and the relative infrequency of the disease. To explore the method for improvement of preoperative diagnosis and possibility of avoiding the surgical management of pelvic actinomycosis, we collected and summarized the data of age, parity, state of menopause, history of intrauterine device (IUD) use, symptoms, laboratory findings, radiologic findings, provisional diagnosis and treatment from 14 cases diagnosed pathologically and treated in Hanyang University Hospital from 2000 to 2007. Eleven (78.6%) of 14 cases were IUD users. Most common complaints were lower abdominal pain (71.4%) and vaginal discharge (57.1%) which were followed by fever (28.6%) and back pain (28.6%). Four cases (28.6%) were identified as pelvic actinomycosis before operation and in 3 cases (21.4%) malignancy was provisional preoperative diagnosis. Pelvic actinomycosis was suspected via abdominal computed tomography (CT) or cervicovaginal cytology and confirmed via endometrial biopsy or fine needle aspiration biopsy. Two cases that were diagnosed before operation and received only antibiotics therapy had no recurrence. It was suggested that pelvic actinomycosis could be suspected via abdominal CT and cervicovaginal cytology in IUD users, and endometrial biopsy and fine needle aspiration biopsy may help establish the diagnosis before the operation. Adequate preoperative antibiotics therapy could make extensive exploratory surgery avoided or conservative surgery feasible.
Abdominal Pain ; Actinomycosis ; Anti-Bacterial Agents ; Back Pain ; Biopsy ; Biopsy, Fine-Needle ; Female ; Fever ; Hydrazines ; Intrauterine Devices ; Menopause ; Parity ; Recurrence ; Vaginal Discharge

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Integrated Cell Culture-PCR Detection of Enteroviruses and Reoviruses in Water Sources in Gyeonggi-do.

Kyung A KIM ; Jong Chan KIM ; Hoan Uck KO ; Jung Bock LEE ; Young Sug KIM ; Yong Bae PARK ; Myung Jin LEE ; Myung Gill KIM ; Jae Kwan KIM ; Eun Mi PARK

Journal of Bacteriology and Virology.2008;38(2):77-87. doi:10.4167/jbv.2008.38.2.77

The integrated cell culture-PCR (ICC-PCR) method has been suggested as an improved method for detection of viruses in water environments. We tested 57 source waters including finished water samples in Gyeonggi-do for enteric viral contamination using total culturable virus assay (TCVA) using BGMK cells and ICC-PCR. Nineteen of the 57 source water samples (33.3%) exhibited the cytopathic effect (CPE) on BGMK cells and no finished water did exhibited CPE. Nineteen samples (33.3%) of the 57 were positive for reoviruses. For the enteroviruses, only 3 samples (5.3%) of the 57 samples showed positive results. By using ICC-PCR method, 202 flasks from source water samples were positive for enteroviruses and reoviruses. Three samples from source water were positive for both viruses. However, any flasks tested was not co-infected with two types of viruses. While the enteric viral frequencies in TCVA and ICC-PCR were similar, the viral frequency for reoviruses at first passage in two type of method was higher in ICC-PCR (94.7%) than TCVA (56.9%).
Enterovirus ; Viruses ; Water

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Usefulness of Bacteriological Tests and sspE PCR for Identification of Bacillus cereus Group.

Sung Hoon KANG ; Ki Jeong KIM ; Won Yong KIM ; Sang In CHUNG

Journal of Bacteriology and Virology.2008;38(2):61-75. doi:10.4167/jbv.2008.38.2.61

The Bacillus cereus group includes B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides. The members of B. cereus group shares strong degree of DNA sequence similarity. Even though the biochemical test and bacteriological test have been used to identify the B. cereus group, an accurate identification system of the B. cereus group is required. We have developed a highly specific PCR-based assay for the B. cereus group chromosome using a sequence motif found within a spore structural gene (sspE). Using the assay, we were able to discriminate B. anthracis from the other members of B. cereus group. We also tried to find a new system for the B. cereus group identification. Five bacteriological tests (hemolysis, motility, penicillin susceptibility, rhizoid growth, toxic crystal formation), API system (API 50CHB & API 20E), MLST and sspE PCR were performed on 28 strains of the B. cereus group. The dendrogram generated from API system and bacteriological tests revealed that B. cereus and B. thuringiensis are grouped into the same cluster. In combination of sspE PCR and bacteriological tests, the dendrogram showed that 4 strains of B. cereus clustered within the same group. B. thuringiensis formed the subgroup in the same cluster. All strains of B. mycoides were encompassed together. Another cluster only included B. anthracis. The best system was determined to be sspE PCR and bacteriological tests. It is concluded that sspE PCR and bacteriological tests could be used for rapid discrimination and identification of B. anthracis and provided an effective means of differentiation between the B. cereus group.
Bacillus ; Bacillus cereus ; Base Sequence ; Discrimination (Psychology) ; Penicillins ; Polymerase Chain Reaction ; Social Identification ; Spores ; Subacute Sclerosing Panencephalitis

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Proteomic analysis of Helicobacter pylori J99 Outer Membrane Protein by Tandem Mass Spectrometry.

Kyung Mi KIM ; Seung Gyu LEE ; Jung Soo JOO ; Young Chul KWON ; Dong Won BEA ; Jea Young SONG ; Hyung Lyun KANG ; Woo Kon LEE ; Myung Je CHO ; Kwang Ho RHEE ; Hee Shang YOUN ; Seung Chul BAIK

Journal of Bacteriology and Virology.2008;38(2):53-60. doi:10.4167/jbv.2008.38.2.53

The protein identity of sarcosine-insoluble outer membrane proteins (OMPs) of Helicobacter pylori J99 was determined with the basic study of understanding the function of proteins. A sarcosine-insoluble OMPs was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were shown in the alkaline pI regions (6.0~11.0) with molecular masses of 10 to 100 kDa. We have performed an extensive proteome analysis by quadrupole time of flight (Q-TOF) mass spectrometry (MS). Here, of 50 spots processed, 42 spots were identified, which represented 16 genes and we newly detected 8 kinds of proteins (JHP0119, JHP0388, JHP1046, JHP1405, JHP0073, JHP0551, JHP1382, JHP0552) from the sarcosin-insoluble fraction of H. pylori J99. Those may be used to elucidate the characterization of the OMPs of H. pylori J99, which will help identify new potential target proteins for vaccine development and drug therapy.
Electrophoresis ; Helicobacter ; Helicobacter pylori ; Mass Spectrometry ; Membrane Proteins ; Membranes ; Proteins ; Proteome ; Proton-Motive Force ; Tandem Mass Spectrometry

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