• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.239-246
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• 2003

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.511-515
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• 2007

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fly epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.

• The Korean Journal of Pharmacology
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• v.28 no.2
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• pp.213-222
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• 1992

We investigated effects of several chemical carcinogens, i.e., $benzo({\alpha})pyrene$ (BP),7,12-dimethylbenz(a)anthracene (DMBA), nitrosomethyl urea (NMU), and nicotine on the replication, cytolyticity, DNA synthesis, and protein synthesis of type 1 herpes simplex virus (HSV-1) in viral infected Vero cell monolayers. We observed that the BP and DMBA did not show such activity. All chemical carcinogens did not inhibit the synthesis of viral DNA, but the expression of gamma viral proteins that are expressed from the newly synthesized progeny viral DNA was somewhat notably inhibited by BP and DMBA. However, the synthesis of alpha and beta viral proteins was not altered by the chemical carcinogens. These data indicate that the gamma viral proteins expressed from the newly synthesized DNA in the presence of chemical carcinogens in the culture medium may be defective. This is further supported by the fact that the virus fail to replicate in the presence of these chemical carcinogens, in spite of viral DNA and proteins are somewhat normally synthesized.

• Korean journal of applied entomology
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• v.19 no.2 s.43
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• pp.91-95
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• 1980

The experiment was conducted to culture callus tissue induced from foliage leaf of garlic bulb for the production of virus-free stocks and for the reduction of expenses for seeds, The following results were reached. 1. Linsmaier-Skoog basal medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) $10^{-5}M$ and benzyladenine $10^{-5}M$ showed the most effective for the induction for the induction of garlic callus. 2. The growth rate of callus was the highest in Linsmaier-Skoog basal medium containing kinetin $10^{-6}M\;and\;2,4-D\;10^{-6}M$ 3. The results of periodical assay of virus concentration in callus tissues showed that virus was almost eliminated by repeated transfer of translucent and soft tissue for eight generations. 4. When virus-free garlic callus tissues were transfered to Murashige-Skoog basal medium containing kinetin $10^{-5}M$ and naphthaleneacetic acid $5\times10^{-6}M$, the tissues were redifferentiated and formed plantlet.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.1
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• pp.25-30
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• 2001

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60$\^{C}$ heat treatment for 10h) for the removal/inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose(TCID(sub)50). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log(sub)10 TCID(sub)50 to undetectable levels. The log reduction factors achieved were $\geq$ 4.5 and $\geq$ 6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log(sub)10 TCID(sub)50 to undetectable levels. The log reduction factors achieved in this case were $\geq$ 4.9 and $\geq$ 5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.

• Korean Journal of Veterinary Research
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• v.45 no.4
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• pp.569-574
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• 2005

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to reticuloendotheliosis virus (REV) at single serum dilution was standardized. REV HI, one of the Korean field isolates, was inoculated into chicken embryo fibroblast (CEF) cells and was harvested from the culture fluids and cells after 10 to 12 days. Viruses were purified by centrifugation at the $107,000{\times}g$ for 12 hours on 20, 30, 45% (W/V) sucrose gradient. Virus specific fraction was collected and used as ELISA antigen. To standardize ELISA, the optimal concentration of coating antigen ($1{\mu}g/well$) and conjugate (1/1000) was determined by corrected OD (OD value of positive serum-OD value of negative serum) and P/N ratio (OD value of positive serum/OD value of negative serum). To calculate ELISA titer by measuring absorbance at 1/400 single serum dilution, serum titrations were carried out for various sample sera together with standard positive and negative sera. The observed titers of serum samples were plotted against sample/positive (s/p) ratios at 1/400 serum dilution. From the above data, the ELISA titers could be calculated by the equation of $log_{10}$ ELISA titer = 2.2763 ($log_{10}$ s/p) + 3.482 (r = 0.93). For evaluating the sensitivity, the standardized method were compared with conventional agar gel immunodiffusion (AGID) test method using serum samples collected from REV infected field chicken flocks. Fifty seven of 60 samples (95%) were positive for REV by ELISA, whereas only 11 (18.3%) samples were positive by AGID test. This results suggested that the ELISA tests developed in this study could be used for detection of antibodies to REV with high sensitivity.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.31-36
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• 2017

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT ($PRNT_{90}$). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and $PRNT_{90}$ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and $PRNT_{90}$ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

• International Journal of Industrial Entomology and Biomaterials
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• v.29 no.2
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.55-64
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• 2002

The envelope glycoprotein of HIV-1 forms an oligomeric complex resulting in playing a role to induce neutralizing antibody and cell-mediate immune responses. The oligomer exists as a trimer of gp120-gp41 heterodimer which mediates HIV-1 attachment and fusion. We made a cDNA clone of gp140 consisting of gp120 and ectodomain of gp41 from the primary African isolate. To express the oligomeric gp140 in mammalian cells, we adopted the Semliki Forest virus (SFV) based expression system. The oligomeric gp140 in the secretory form was expressed and purified from the cell culture supernatant and characterized. The antibody inducing activity of the purified gp140 was also examined in mice inoculation.

• Journal of Microbiology and Biotechnology
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• v.31 no.12
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• pp.1716-1721
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• 2021

Chikungunya fever is an arboviral disease caused by the Chikungunya virus (CHIKV). The disease has similar clinical manifestations with other acute febrile illnesses which complicates differential diagnosis in low-resource settings. We aimed to develop a rapid test for CHIKV detection based on the nucleic acid lateral flow immunoassay technology. The system consists of a primer set that recognizes the E1 region of the CHIKV genome and test strips in an enclosed cassette which are used to detect amplicons labeled with FITC/biotin. Amplification of the viral genome was done using open-source PCR, a low-cost open-source thermal cycler. Assay performance was evaluated using a panel of RNA isolated from patients' blood with confirmed CHIKV (n = 8) and dengue virus (n = 20) infection. The open-source PCR-NALFIA platform had a limit of detection of 10 RNA copies/ml. The assay had a sensitivity and specificity of 100% (95% CI: 67.56% - 100%) and 100% (95% CI: 83.89% - 100%), respectively, compared to reference standards of any positive virus culture on C6/36 cell lines and/or qRT-PCR. Further evaluation of its performance using a larger sample size may provide important data to extend its usefulness, especially its utilization in the peripheral healthcare facilities with scarce resources and outbreak situations.