• Journal of Life Science
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• v.18 no.3
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• pp.374-380
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• 2008

Rapid detection of enterovirus (EVs) is important in the management of aseptic meningitis. We examined the relative efficiency and specificity of the real-time nucleic acid sequence-based amplification (NASBA) comparing with the established reverse transcription polymerase chain reaction (RT-PCR) and viral culture method which were used for the detection of enterovirus RNA in clinical specimens. Of the total 292 samples, 145 were found to be positive to enterovirus RNA by real-time NASBA, 101 were positive by viral culture, and 86 were positive by RT-PCR. 147 samples and 46 samples were determined to be negative and positive by all methods respectively, but 4 samples were positive only by real-time NASBA. To compare the specificity of each method, various clinical samples which were diagnosed for herpes simplex virus (HSV)-1, HSV-2, adenovirus, mumps, and rhinovirus were applied. Except one rhinovirus sample which was false positive to enterovirus RNA by RT-PCR, the other different samples were negative to all three methods. The real-time NASBA procedure can be completed within 5 hours in contrast with 9 hours for the RT-PCR and 3-14 days for the viral culture. From this study, it was suggested that the real-time NASBA assay could be a standardized, rapid, specific, and sensitive procedure for the detection of enterovirus RNA.

• Research in Plant Disease
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• v.16 no.3
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• pp.254-258
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• 2010

Melon necrotic spot virus (MNSV) is a very destructive disease to melon (Cucumis melo) plants. A MNSV was isolated from melon leaf showing necrotic spot symptoms at the plastic house in Naju, Korea in 2009. The isolate, designated as MNSV-Me, was identified and characterized by biological responses on several host plants, immuno captured RT-PCR and partial nucleotide sequencings of the genome. To evaluate MNSV-Me resistance in melon, thirty-five melon cultivars were mechanically inoculated on the cotyledon of the seedlings with the virus. MNSV-Me produced necrotic spots on the inoculated leaves of the all melon cultivars tested. Twenty-five cultivars were susceptible to the virus and they showed systemic necrotic spots on the leaves and/or necrosis longer than 3 cm in length on the stems within about forty days after inoculation. Five cultivars gave moderate resistance, no symptoms on the upper leaves but necrosis on the stem shorter than 3 cm in length. In an evaluation of MNSV-Me resistance in melon cultivars, 'Elstitan', 'Elsluxery', 'Betalichihage', 'Betalichi' and 'Womderfulhagae 1st' were found to have resistance by showing only faint necrosis on their stems.

• Korean journal of applied entomology
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• v.23 no.4 s.61
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• pp.203-208
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• 1984

Four varieties of tobacco, Nicotiana tabacum var. NC 2326. NC 95, NC 744 and Havana. and two plant species, Physalis floridana Rydb., N. repanda Wild. were used for classification of ten isolates of potato virus Y(PVY) obtained Iron potato or tabacco plants showing various symptoms. Each of the 10 isolates could be distinguished by the host reactions showing necrosis, vein banding and/or mottling, or no symptom on these hosts. Five isolates of PVY, PVY-VN, PVY-N, PVY-NSNR, PVY-Chile, and PVY-Argentina, showed necrotic symptom on NC 2326, but others showed vein handing and/or mottling symptom. On NC95, each of the isolates tested showed similar symptons as on NC2326, except necrotic symptom by the isolate PVY-MSNR. Havana showed mottling reaction to the PVY-NSNR, PVY-MSNR, PVY-MSMR, PVY-VB, and necrosis to PVY-Chile, PVY-Argentina, but showed no symptom to the others. NC744 showed mottling symptom to the PVY-MSNR and PVY-MSMR, necrosis to the PVY-Chile and PVY-Argentina, and no symptom to the others. On N. repanda, necrotic reaction to PVY-Argentina. no symptom to PVY-VN and PVY-C, and nettling to others were observed.

• Journal of Life Science
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• v.20 no.10
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• pp.1427-1432
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• 2010

This study was carried out to evaluate neutralization effects against WSSV using antiserum produced from recombinant envelop protein, rVP466 of WSSV. The VP466 gene of WSSV was cloned into pCold I expression vector and rVP466 was expressed in E. coli RIPL. The antiserum against rVP466 was produced in white rabbits (New Zealand white rabbit). The specific immunoreactivity to the antigen, rVP466, was confirmed by Western blot. The constant amounts of WSSV at $1{\times}10^4$ diluted stocks were mixed with various antiserum concentrations and then injected to the muscle of shrimp, Penaeus chinensis, for the neutralization challenge. The shrimps challenged with WSSV as a positive control and those with the mixture of WSSV and preimmune serum as a preimmune control showed 100% cumulative mortality at 17 days post challenge and 83% at 25 days post challenge, respectively. The shrimps challenged with 3 different mixtures of WSSV and rVP466 antiserum at ratios of 1:0.01, 1:0.1 and 1:1 showed 73%, 53% and 46% cumulative mortalities at 25 days post challenge, respectively. These results indicated that WSSV could be neutralized by the rVP466 antiserum. These results suggest that envelop protein VP466 is involved in the initial step of WSSV infection in shrimp.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.519-524
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• 1998

A simple and reliable method to diagnose cucumber green mottle mosaic virus of watermelon in Korea (CGMMV-WK) was determined by RT-PCR, and coat protein gene for CGMMV-WK was cloned. Comparing to a method reported by Lee et al. (1996), the method developed here showed a better RT-PCR reaction. RT-PCR was possible by one step in the PCR reaction mixture that contains 20 pmol of primer, reverse transcriptase (30 unit), RNasin (5 unit) using the crude RNA solution. RT-PCR condition for specifically diagnosing CGMMV-WK was that cDNA was synthesized at 42$^{\circ}C$ for 45 min followed by pre-denaturation at 95$^{\circ}C$ for 2 min, and then PCR reaction was carried out with a programmed condition that consisted of 36 sequential cycles at 96$^{\circ}C$ for 30 sec, 6$0^{\circ}C$ for 30 sec, and 72$^{\circ}C$ for 1 min. A gene encoding the coat protein of CGMMV-WK was cloned and characterized. Nucleotide sequence of coat protein gene of CGMMV-WK shared 98.77% and 99.38% of sequence identity with those of CGMMV-W and CGMMV-SH, respecitvely, however, all of amino acid sequences were same.

• Journal of fish pathology
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• v.26 no.1
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• pp.19-30
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• 2013

Megalocytivirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogen, little is known interaction between megalocytivirus and the fish immune system. One of the cyclooxygenase isoforms, named COX-2, is playing an important role in immune regulation, and distinct from COX-1 isoform of constitutive activity. COX-2 enzyme is induced by various inflammatory signals, including injection of lipopolysaccharide or infection by pathogenic agents. We cloned COX-2 gene in rock bream using degenerated primers designed from reported sequences of other fish species in PCR followed with 5'- and 3'-end RACE-PCR. The full length of cDNA of rbCOX2 (rock bream COX-2) gene are 2655 bp and that translates into 609 amino acids. The rbCOX-2 genomic organization are found to span 10 exons separated by 9 introns. We also studied if the experimental infection of rock bream with megalocytivirus could affect the expression of COX-2 gene. When injected with LPS, expression of the COX-2 gene was reached peak level at 1 day post injection and showed 13.10 fold increased level compared with that of control. While, when injected with megalocytivirus, we were not able to find significantly increased COX-2 gene expression different from that of control. Cloned and analyzed COX-2 gene in rock bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention of viral infection.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.159-165
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• 2007

For the development of basic genetic materials for specific and effective therapeutic approach to suppress multiplication of hepatitis C virus (HCV), HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was developed. The ribozyme targeted most effectively to +382 nucleotide (nt) site of HCV IRES RNA. The allosteric ribozyme was designed to be composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nt of HCV IRES. Noticeably, we employed in vitro selection technology to identify the most appropriate communication module sequence which can induce ribozyme activity depending on the US5B protein. We demonstrated that the ribozyme was nonfunctional either in the absence of any proteins or in the presence of control bovine serum albumin. In sharp contrast, the allosteric ribozyme can induce activity of cleavage reaction with HCV IRES RNA in the presence of the HCV NS5B protein. This allosteric ribozyme can be used as lead compound for specific and effective anti-HCV agent, tool for highthroughput screening to isolate lead chemicals for HCV therapeutics, and ligand for biosensor system for HCV diagnosis.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.186-192
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• 2008

For the development of specific and effective basic genetic materials to inhibit replication of hepatitis C virus (HCV), HCV genome-targeting trans-splicing aptazyme, which activity is allosterically regulated by a specific ligand, was developed. The aptazyme was designed to be comprised of sequence of RNA aptamer to the ligand, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding the ligand to the aptamer, and trans-splicing ribozyme targeting +199 nt of HCV IRES. Especially, when the aptamer and the communication module was inserted at both P6 and P8 catalytic domain of the specific ribozyme, allosteric activity of the aptazyme was the most induced. The aptazyme was shown to induce activity of trans-splicing reaction specifically and efficiently only in the presence of the specific ligand, but neither in the absence of any ligand nor in the presence of control ligand. This aptazyme can be used as a specific and effective genetic agent against HCV, and a tool for the isolation of anti-HCV lead compounds.

• Journal of Life Science
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• v.28 no.5
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• pp.555-562
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• 2018

Swine influenza virus (SIV) causes one of the most common diseases of the pig population, and its subtypes are determined by hemagglutinin (HA) and neuraminidase (NA). Recently, the SIV subtype diagnosis has been developed. The method using antigen-antibody reaction rather than PCR was mainly used because of the large change in the ribonucleotide sequences of SIV. Here, we have developed 10 diagnostic primer sets through multi-nucleotide sequences alignment of spreaded SIV since 2008 in Korea and then optimized the reaction of the one-step RT-PCR for rapid determination of SIV subtype. In addition, specific primers were designed to early determine the pandemic SIV by detecting unique M sequences proven in highly infectious and virulent subtypes of the influenza H1N1 (pH1N1). Here, some of the SIVs spread in Korea from 2008 to 2014 have been tested to determine the subtypes and pandemic potential of SIV. All diagnostic primer sets were found to be able to accurately determine the SIV subtype and to detect the pandemic SIV. In conclusion, it was confirmed that the optimized one-step RT-PCR analysis using these primer sets is useful for rapid diagnosis of SIV subtypes. These results can be used for development of SIV subtype diagnostic kit to early detect before virulent SIV spreads do.

• The Korean Journal of Zoology
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• v.31 no.1
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• pp.49-55
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• 1988

A few SV4O-transformed human cells such as SV8O are potentially tumorigenic but rejected by athymic hosts. However, one cell line in this group (W118IVA-2) is known to be fully tumorigenic. Two clones were obtained after the injection of W118IVA-2, of which NW1SC1-1 was tumorigenic but NW18C1-2 was not in nude mice. As examined by Southern blot analysis, NW18C1-1 appears to contain more copy number of SV40 sequences than NW18C1-2 does. However, it was unable to demonstrate that this difference elicits the tumorigenicity in NW18C1-1 but not in NW18C1-2. Therefore, the latter clone was tested if it expresses SV40 early genes to produce large T as well as small t antigens using indirect immunofluorescent assay and immunoprecipitation. In addition, mouse NIH3T3 cells were transfected with the cellular DNA of NW1SC1-2 as well as that of NW18C1-1 to examine if the viral genomes in the clones can make the nontransformed cells to acquire malignant growth potential in vivo. The transformed cells expressed large T antigen and became tumorigenic. Thus, the transforming functions of NW1SC1-2 cell appers to be intact. These results clearly suggest that the inability of NW18C1-2 cell to form tumor in nude mice is not because they are inherently nontumorigenic. However, the possibility that the interaction of SV40 with its host differs in these clones can not he ruled out.