• Proceedings of the Membrane Society of Korea Conference
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• 1996.10a
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• pp.18-21
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• 1996

Virus and DNA removal in bio-drug manufacturing processes has received a great deal of attention in recent years. Removing of a virus using a membrane process is a promising method, because inactivated virus can be removed from the bio-drug and the process can be used as an additional and security inactivation after the method of general heat-inactivation of the virus in the bio-drug. The FDA and the biopharmaceutical industry have recently announced strict guidelines for impurities of virus and DNA contamination. The regulatory guidelines on residual amounts of DNA in mammalian cell culture products require DNA contamination of less than 100 pg/dose. Therefore, permeation and rejection of DNA through the porous membranes have become important in the application of DNA removal in bio-drug manufacturing using membrane technology. In this study, the permeation of DNA and chromatin through regenerated cellulose hollow fibers that have a mean pore diameter of 15 nm was investigated.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.52 no.6
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• pp.644-649
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• 2019

Killed vaccines, developed by inactivation with formalin, have been investigated for many fish viruses. In this study, the inactivation of viral hemorrhagic septicemia virus (VHSV) by formalin was investigated based on the infectivity titer. When viral cell culture supernatants were used, the infectivity titer decreased 1,000-fold at 1 d after treatment with 0.1% (v/v) formalin, but was below the detection limit at 7 and 14 d. Moreover, neither the N nor G gene were detectable by RT-PCR immediately after formalin treatment. In western blot analysis, N protein was not detected by rabbit antiserum against VHSV KR-9225 from 2 d after formalin treatment. On the other hand, when we used a virus that was purified and concentrated ~100 times, the infectivity titer was maintained at 10^6.05 TCID₅₀/mL, even at 14 d after formalin treatment, and no change in the viral structural proteins was observed. This study provides important data on the production and use of formalin-inactivated vaccines.

• Journal of Microbiology
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• v.45 no.1
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• pp.48-52
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• 2007

A new procedure for the concentration of nonoviruses from water samples has been developed. This procedure (calcium flocculation-citrate dissolution method) uses the following steps: virus flocculation formed by treatment with 1 M $CaCl_2$ and 1 M $Na_2HPO_4$, virus release by sodium citrate dissolution (0.3 M Na citrate, pH 3.5), and virus re-concentration by ultrafiltration. When reverse transcription (RT)-PCR was performed after the procedure, the overall detection sensitivity for seeded noroviruses in a one liter drinking water sample was as low as 1 RT-PCR unit, which is equal to a $10^{-6}$ dilution of the virus sample. This approach showed at least a 5-fold-higher sensitivity than the current method with its three steps of adsorption-elution-concentration. The newly developed procedure was used to test different brands of bottled drinking water from China for putative contamination with noroviruses. A total of 144 samples were analyzed; all of the samples were negative for norovirus specific nucleic acids.

• Molecules and Cells
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• v.44 no.12
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• pp.911-919
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• 2021

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M₁ progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1576-1586
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• 2023

Vaccination is the most effective method for preventing the spread of the influenza virus. Cell-based influenza vaccines have been developed to overcome the disadvantages of egg-based vaccines and their production efficiency has been previously discussed. In this study, we investigated whether treatment with forskolin (FSK), an adenylyl cyclase activator, affected the output of a cell-based influenza vaccine. We found that FSK increased the propagation of three influenza virus subtypes (A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97) in Madin-Darby canine kidney (MDCK) cells. Interestingly, FSK suppressed the growth of MDCK cells. This effect could be a result of protein kinase A (PKA)-Src axis activation, which downregulates extracellular signal-regulated kinase (ERK)1/2 activity and delays cell cycle progression from G1 to S. This delay in cell growth might benefit the binding and entry of the influenza virus in the early stages of viral replication. In contrast, FSK dramatically upregulated ERK1/2 activity via the cAMP-PKA-Raf-1 axis at a late stage of viral replication. Thus, increased ERK1/2 activity might contribute to increased viral ribonucleoprotein export and influenza virus propagation. The increase in viral titer induced by FSK could be explained by the action of cAMP in assisting the entry and binding of the influenza virus. Therefore, FSK addition to cell culture systems could help increase the production efficiency of cell-based vaccines against the influenza virus.

• Journal of Microbiology and Biotechnology
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• v.34 no.8
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• pp.1592-1598
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• 2024

Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.

• Korean Journal of Plant Tissue Culture
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• v.28 no.6
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• pp.335-339
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• 2001

Grapevine leafroll-associated 3 closterovirus (GLRaV-3) is phloem limited virus, and one of the most severe viral diseases found in Korea. However, nonhomogenous distribution and low concentration and seasonal variations of GLRaV-3 in grapevines remain as main problems which prevent the introduction and molecular biology or serology experiments. Virus-infected plantlet in vitro was obtained from node tissue cuttings, which was GLRaV-3 infected 'kyoho' vines. The amount of purified virus was highest in vitro plantlet. Moreover, viruses seem to be relatively homogeneously distributed in all organs including leaf, stem and callus derived from in vitro plantlets. RT-PCR detection using in vitro plantlet tissue as template was most effective. When comparing ELISA to RT-PCR, RT-PCR detection was 1,000 times as effective as ELISA. These results can be explained by improved quality such as tenderness or less tannins in plantlet in vitro. In conclusion, until infected herbaceous host will be available, tissue culture can be usefully adopted as a technique for a good source of GLRaV-3 closterovirus for further studies.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.118-122
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• 2000

A comlementary call line CR2 is curretly used to propagte the Disabled Infectious Single Cycle Herpes Simplex Virus Typee2 (DISC HSV-2) on a small Iaboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrierier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up out production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production time were factors which were taken into consideration whils developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the amall scale study for an envisaged manufacturing process.

• Journal of the Korean Society of Tobacco Science
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• v.26 no.2 s.52
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• pp.79-84
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• 2004

During the screening of antiviral substances having inhibitory effects on tobacco mosaic virus (TMV) infection to tobacco plants, we found a bacterial isolate KTGBP10, which was identified as a Stenotrophomonas sp., strongly inhibited the infection of TMV. When the culture filtrate from KTGBP10 was applied on the upper surface of leaves of Xanthi-nc tobacco plants at the same time or 24 hours before TMV inoculation, almost complete inhibition of TMV infection was achieved. And $40\%$ inhibition was shown with application of the culture filtrate to the under surface of leaves. In field trials, transmission of TMV from diseased seedlings to the healthy ones during transplanting work was reduced by $87.1\~92.6\%$ when the culture filtrate or cell suspension was sprayed onto the tobacco seedlings, cv. NC82, 24 hours before transplanting. No toxic effect was observed on the tobacco plants. When the broth filtrate of KTGBP10 was supplied by soaking through the cut-leaves before and/or after virus inoculation, the TMV infection was also inhibited by $50.4\~65.3\%$.

• The Journal of Korean Medicine
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• v.22 no.1
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• pp.53-62
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• 2001

Objective : The aim of this study was to evaluate the efficacy of Injinchunggan-tang on Hepatitis C virus infection, and to clarify the mechanism of treatment by indentifying the effect of Injinchunggan-tang on cytokine secretion. Methods : In vitro model of HCV infection in MOLT 4 cell was used. The effect of Injinchunggan-tang on the attachment of HCV on MOLT 4 cell was studied by PCR method. The change of cytokine secretion according to Injinchunggan-tang treatment was investigated by ELISA. Results : Injinchunggan-tang inhibited the attachment of HCV on MOLT 4 in the concentration of $10-2{\mu\textrm{g}}/\mu\textrm{\ell}$ and $10-1{\mu\textrm{g}}/\mu\textrm{\ell}$. In cytokine assay, Injinchunggan-tang increased the secretion of IL-4 of mouse splenocytes and PBMC in 48 hour culture as well as the secretion of IL-12 of mouse splenocytes and PBMC in 48 hour culture, whereas it decreased the secretion of $IFN-{\gamma}$ of mouse splenocytes in 24 and 48 hour culture. Conclusion : The results of this study show that Injinchunggan-tang has an inhibitory effect on the attachment on HCV on Mo1t4 Cell, and that it increases the secretion of IL-4 and IL-12 of mouse splenocyte and PBMC.