• 상하수도학회지
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• 제29권6호
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• pp.633-641
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• 2015

Waterborne infectious disease is induced by several pathogenic microbes such as bacteria, viruses and protozoans, and the cases caused by viral infection is currently increasing. Water treatment process could reduce the number of virus in the water, but there were many difficulties to completely remove the virus particles from water. Therefore, the membrane separation technology which was reported to effectively remove pollutants from raw water has attracted increasing attention and demand. Since its efficiency has been introduced, demands for evaluation method toward the membrane filtration process are increasing. However, progression of the method development is slow due to the difficulties in cultivation of several waterborne viruses from animal models or cell culture system. To overcome the difficulties, we used adenovirus, one of the commonly isolated pathogenic waterborne viruses which can grow in cell culture system in vitro. The adenovirus used in this study was identified as human adenovirus C strain. The adenovirus was spiked in the raw water and passed through the microfiltration membrane produced by Econity, a Korean membrane company, and then the viral removal rate was evaluated by real-time PCR. In the results, the amount of virus in the filtered water was decreased approximately by 5 log scale. Because coagulant treatment has been known to reduce filtering function of the membrane by inducing fouling, we also investigated whether there was any interference of coagulant. In the results, we confirmed that coagulant treatment did not show significant interference on microfiltration membrane. In this study, we found that waterborne virus can be effectively removed by membrane filtration system. In particular, here we also suggest that real-time PCR method can rapidly, sensitively and quantitatively evaluate the removal rate of virus. These results may provide a standard method to qualifying membrane filtration processes.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.497-503
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• 2001

The purpose of this study was to examine the efficacy and mechanism of the cryo-precipitation, solvent/detergent (S/D) treatment, monoclonal anti-FVIIIc antibody (mAb) column chromatography, Q-Sepharose column chromatography, and lyophilization involved in the manufacture of antithemophilic factor VII(GreenMono) from human plasma, in the removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including the bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. BHV and EMCV were effectively partitioned from a factor VII during the cryo-precipitation with a log reduction factor of 2.83 and 3.24, respectively. S/D treatment using the organic solvent, tri(n-butyl) phosphate (TNBP), and the detergent, Triton X-100, was a robust and effective step in inactivating enveloped viruses. The titers of BHV and BVDV were reduced from the initial titer of 8.85 and $7.89{log_10} {TCID_50}$, respectively, reaching undetectable levels within 1 min of the S/D treatment. The mAb chromatography was the most effective step for removing nonenveloped viruses, EMCV and PPV, with the log reduction factors of 4.86 and 3.72, respectively. Q-Sepharose chromatography showed a significant efficacy for partitioning BHV, BVDV, EMCV, and PPV with the log reduction the log reduction factors of 2.32, 2.49, 2.60, and 1.33 respectively. Lyophilization was an effective step in inactivating g nonenveloped viruses rather than enveloped viruses, where the log reduction factors of BHV, BVDV, DMCV, and PPV were 1.41, 1.79, 4.76, and 2.05, respectively. The cumulative log reduction factors of BHV, BVDV, EMCV, and PPV were ${\geqq}$11.12, ${\geqq}$7.88, 15.46, and 7.10, respectively. These results indicate that the production process for GreenMono has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• 한국가축번식학회지
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• 제20권3호
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• pp.333-341
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• 1996

One of the biggest problems involved in transgenic animal production is lack of appropriate market genes. To overcome this problem, we tested whether the genes of SEAP (secreted alkaline phosphatase) and GFP (green fluorescence protein) on our retrovirus vectors can be applicable to the transgenic animal production. The main advantage of these marker genes over other generally mainpulation can be selected without sacrificing viability. The results obtained in this study are summarized as follows: 1. Removal of zona pellucida from the mouse zygotes did not affect embryo developments to blastocysts. 2. Co-culture of zona-free embryos with virus-producing cells for 6 hours also did not affect embryo developments to blastocysts. 3. Among 58 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells, SEAP expression was observed from the 6 blastocysts. 4. Expression of the GFP gene was detected from the virus- producing cells but no embryo expressing the gene was counted among 50 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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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.

• 한국가축번식학회지
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• 제19권2호
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• pp.89-93
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• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• BMB Reports
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• 제41권9호
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• pp.678-683
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• 2008

The initial step during assembly of the hepatitis A virus particle is driven by domain 2A of P1-2A, which is the precursor of the structural proteins. The proteolytic removal of 2A from particulate VP1-2A by an as yet unknown host enzyme presumably terminates viral morphogenesis. Using a genetic approach, we show that a basic amino acid residue at the C-terminus of VP1 is required for efficient particle assembly and that host proteases trypsin and cathepsin L remove 2A from hepatitis A virus particles in vitro. Analyses of insertion mutants in the C-terminus of 2A reveal that this part of 2A is important for liberation of P1-2A from the polyprotein. The data provide the first evidence that the VP1/2A junction is involved in both viral particle assembly and maturation and, therefore, seems to coordinate the first and last steps in viral morphogenesis.

• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.619-627
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• 2001

n order to increase the virus safety of a human intravenous immunoglobulin (IVIg) that was manufactured by a successive process of cold ethanol fractionation, polyethylene glycol precipitation, and pasteurization ($60^{\circ}C$ heat treatment for 10h), a low pH incubation process (pH 3.9 at $25{\circ}C$ for 14 days) was employed as the final step. The efficacy and mechanism of the fraction III cold ethanol fractionation, pasteurization, and low pH treatment steps in the removal and/or inactivation of blood-borne viruses were closely examined. A variety of experimental model viruses for human pathogenic viruses, including the Bovine herpes virus (BHV), Bovine viral diarrhoea virus (BVDV), Murine encephalomyocarditis virus (EMCV), and Porcine parvovirus (PPV), were selected for this study. The mechanism of reduction for the enveloped viruses (BHV and BVDV) during fraction III fractionation was both inactivation and partitioning, however, it was partitioning in the case of the nonenveloped viruses (EMCV and PPV). The log reduction factors achieved during fraction III fractionation were ${\geqq}$6.7 for BHV, ${\geqq}4.7$ for BVDV, 4.5 for EMCV, and 4.4 for PPV. Pasteurization was found to be a robust and effective step in inactivating all the viruses tested. The log reduction factors achieved during the pasteurization process were ${\geqq}7.5$ for BHV, ${\geqq}4.8$ for BVDV, 3.0 for EMCV, and 3.3 for PPV. A low pH incubation was very effective in inactivating the enveloped viruses as well as EMCV. The log reduction factors achieved during low pH incubation were ${\geqq}7.4$ for BHV, ${\geqq}3.9$ for BVDV, 5.2 for EMCV, and 2.0 for PPV. These results indicate that the low pH treatment successfully improved the viral safety of the final products.

• Environmental Engineering Research
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• 제20권2호
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• pp.133-140
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• 2015

The aim of this study was to investigate the bacteriophage removal in various clay minerals and clay-amended soils. Batch experiments in kaolinite, montmorillonite, and bentonite showed that kaolinite was far more effective at the MS2 removal than montmorillonite and bentonite. In kaolinite, the log removal increased from 0.046 to 2.18, with an increase in the adsorbent dose from 0.3 to $50g\;L^{-1}$, whereas the log removals in montmorillonite and bentonite increased from 0.007 to 0.40 and from 0.012 to 0.59, respectively. The MS2 removal in kaolinite-amended silt loam soils was examined at three different soil-to-solution (STS) ratios. Results indicated that the log removal of MS2 increased with an increase in the kaolinite content and the STS ratio. At the STS ratio of 1:10, the log removal of MS2 increased from 2.33 to 2.80 with an increase in the kaolinite content from 0% to 10% in kaolinite-amended soils. The log removals of MS2 at the STS ratios of 1:2 and 1:1 increased from 2.84 to 3.47 and from 3.46 to 4.76, respectively, with an increase in the kaolinite content from 0% to 10%. Results also indicated that the log removals of PhiX174 and $Q{\beta}$ in kaolinite-amended soils were similar to each other, but they were far lower than those of MS2 at all the kaolinite contents. The log removal of PhiX174 increased from 0.16 to 0.32, whereas the log removal of $Q{\beta}$ changed from 0.17 to 0.22 with an increase in the kaolinite content from 0% to 10%.

• 미생물학회지
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• 제39권4호
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• pp.242-247
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• 2003

Murine encephalomyocarditis virus (EMCV)는 혈장유래의약품의 바이러스 안전성 검증을 위해 hepatitis A virus (HAV)의 모델 바이러스로 사용되어왔다. 근래에 혈액응고인자제제에 의한 HAV 감염사례가 보고되면서 혈장유래의약품의 HAV 안전성 검증에 대한 국제적인 규제가 강화되어가고 있다. 본 연구에서는 HAV와 EMCV의 바이러스 불활화 공정에 대한 민감도를 평가하여, 혈장유래의약품 제조공정에서 HAV 불활화 공정의 검증법을 표준화하고자 하였다. HAV와 EMCV의 바이러스 불활화 공정에 대한 민감도를 평가한 결과 HAV가 60$^{\circ}C$ 열처리, low pH 처리(pH 3.9), 0.1 M NaOH 처리, 동결건조 공정 모두에서 EMCV보다 더 저항성이 큰 것을 확인할 수 있었다. EMCV는 특히 열처리와 0.1 NaOH 처리에 민감하게 불활화 되었지만, HAV는 큰 저항성을 나타내었다. 열처리의 경우 2시간 안에 EMCV는 검출한계 이하로 감소하였지만, HAV는 5시간 후에 검출한계 이하로 감소하였다. 0.1 M NaOH 처리시 EMCV는 15분 안에 검출한계 이하로 감소하였지만, HAV는 120분 정도의 처리에도 감염성 바이러스가 검출되었다. pH 3.9에서 25$^{\circ}C$로 14일 동안 항온하였을 때 HAV와 EMCV의 log 감소인수는 각각 1.63, 3.84이었다. 또한 혈액응고 8인자 제조공정의 동결건조 과정에서 HAV와 EMCV의 log 감소인수는 각각 1.21, 4.57이었다. 이와 같은 결과는 혈장유래의약품 제조공정의 HAV 불활화 또는 제거 검증시 모델 바이러스로 사용된 EMCV의 검증 결과를 해석함에 있어 보다 신중함을 가져야 한다는 것을 보여준다. 또한 보다 정확한 HAV검증 결과를 얻고자 한다면 모델 바이러스인 EMCV 보다 HAV를 사용하는 것이 보다 더 타당하다고 사료된다.

• 미생물학회지
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• 제41권3호
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• pp.216-224
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• 2005

혈장분획제제 중 혈액응공인자제제와 일부 면역글로불린제제는 혈장에 존재하는 다양한 단백질로부터 유효한 단백성분만을 선택적으로 분리 정제하기 위해 크로마토그래피 방법을 사용하여 생산된다. 효율적인 세척(cleaning) 공정이 이루어지지 않는다면 크로마토그래피는 다양한 종류의 불순물뿐만 아니라 혈액 중 내재 또는 오염 가능성이 있는 위해인자가 오염될 가능성이 있다. 본 연구에서는 혈장분획제제 제조공정에 사용되는 크로마토그래피의 세척 공정에서 혈장유래 바이러스의 제거 및 불활화 공정의 검토 강화로 혈장분획제제의 안전성을 확보하기 위해 크로마토그래피 세척 검증 시스템을 구축하고자 하였다. 크로마토그래피 세척 공정 중 바이러스 제거 검증을 위해 혈장유래 바이러스 중 물리${\cdot}$화학적 처리에 가장 큰 저항성을 갖는 human parvovirus B19의 모델 바이러스의 porcine parvovirus(PPV)를 대상으로 real-time PCR 정량법을 확립하였다. PPV에 특이적인 primer를 선별하였으며 형광염료 SYBR Green I을 사용하여 PPV DNA를 정량하였다. 세포배양법에 의한 감염 역가와 비교한 결과 PCR 민감도는 1.5 $TCID_{50}/ml$이었다. 확립된 검증법의 신뢰성(reliability)을 보증하기 위해 실험법의 특이성(specificity), 재현성(reproducibility) 등을 검증하였다. 구축된 검증시스템을 thrombin 분리${\cdot}$정제를 위한 SP-Sepharose 양이온 크로마토그래피 공정과 factor VIII 분리${\cdot}$정제를 위한 Q-Sepharose 음이온 크로마토그래피 공정에 적용하여 크로마토그래피 세척 검증을 실시하고, 세척 검증 시스템의 적합성을 확인하였다.