• Korean Journal of Microbiology
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• v.45 no.4
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• pp.346-353
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• 2009

Viral, bacterial, and fungal infection can be transmitted from donor to recipient via transplantation of human amniotic membrane. Therefore human amniotic membrane for transplantation should be disinfected and sterilized before use. The purpose of this study was to examine the efficacy of the disinfection process and sterilization processes used at human tissue bank in the inactivation of viruses, bacteria, and fungi. A variety of experimental model viruses, bacteria, and fungus for human pathogens, including the human immunodeficiency virus type 1 (HIV-1), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), porcine parvovirus (PPV), Escherichia coli, Bacillus subtilis, and Candida albicans were all selected for this study. Enveloped viruses such as HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by 70% ethanol treatment, gamma irradiation process, and ethylene oxide (EO) gas sterilization process. Also non-enveloped viruses such as HAV and PPV were effectively inactivated to undetectable levels by gamma irradiation and EO gas treatment. However HAV and PPV showed high resistance to 70% ethanol treatment. E. coli and C. albicans were effectively inactivated to undetectable levels by 70% ethanol treatment, gamma irradiation process, and EO gas treatment. Also B. subtilis was effectively inactivated to undetectable levels by gamma irradiation process and EO gas treatment. However it showed high resistance to 70% ethanol treatment.

• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.420-427
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• 2010

A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25 kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}$13.30, ${\geq}$14.32, ${\geq}$15.22, and ${\geq}$7.57, respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.314-318
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• 2012

Most types of collagen used for biomedical applications, such as cell therapy and tissue engineering, are derived from animal tissues. Therefore, special precautions must be taken during the production of these proteins in order to assure against the possibility of the products transmitting infectious diseases to the recipients. The ability to remove and/or inactivate known and potential viral contaminants during the manufacturing process is an ever-increasingly important parameter in assessing the safety of biomedical products. The purpose of this study was to evaluate the efficacies of the 70% ethanol treatment and pepsin treatment at pH 2.0 for the inactivation of bovine viruses during the manufacture of collagen type I from bovine hides. A variety of experimental model viruses for bovine viruses including bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), and bovine parvovirus (BPV), were chosen for the evaluation of viral inactivation efficacy. BHV, BVDV, BPIV-3, and BPV were effectively inactivated to undetectable levels within 1 h of 70% ethanol treatment for 24 h, with log reduction factors of ${\geq}5.58$, ${\geq}5.32$, ${\geq}5.11$, and ${\geq}3.42$, respectively. BHV, BVDV, BPIV-3, and BPV were also effectively inactivated to undetectable levels within 5 days of pepsin treatment for 14 days, with the log reduction factors of ${\geq}7.08$, ${\geq}6.60$, ${\geq}5.60$, and ${\geq}3.59$, respectively. The cumulative virus reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}12.66$, ${\geq}11.92$, ${\geq}10.71$, and ${\geq}7.01$. These results indicate that the production process for collagen type I from bovine hides has a sufficient virus-reducing capacity to achieve a high margin of virus safety.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.242-247
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• 2003

Murine encephalomyocarditis virus (EMCV) has been used as a surrogate for hepatitis A virus (HAV) for the validation of virus removal and/or inactivation during the manufacturing process of biopharmaceuticals. Recently international regulation for the validation of HAV safety has been reinforced because of the reported cases of HAV transmission to hemophiliac patients who had received ntihemophilic factors prepared from human plasma. The purpose of the present study was to compare the resistance of HAV and EMCV to various viral inactivation processes and then to standardize the HAV validation method. HAV was more resistant than EMCV to pasteurization (60oC heat treatment for 10 hr), low pH incubation (pH 3.9 at 25oC for 14 days), 0.1 M NaOH treatment, and lyophilization. EMCV was completely inactivated to undetectable levels within 2 hr of pasteurization, however, HAV was completely inactivated to undetectable levels after 5 hr treatment. EMCV was completely inactivated to undetectable levels within 15 min of 0.1 M NaOH treatment, however, residual infectivity of HAV still remained even after 120 min of treatment. The log reduction factors achieved during low pH incubation were 1.63 for HAV and 3.84 for EMCV. Also the log reduction factors achieved during a lyophilization process of antihemophilic factor VIII were 1.21 for HAV and 4.57 for EMCV. These results indicate that HAV rather than EMCV should be used for the virus validation study and the validation results obtained using EMCV should be precisely reviewed.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.168-176
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• 2010

Acellular dermal matrix (ADM), produced by decellularization from human cadaveric skin, has been used for various biomedical applications. A manufacturing process for ADM ($SureDerm^{TM}$) using tri-n-butyl phospahate (TnBP) and deoxycholic acids as the decellularization solution has been developed. The manufacturing process for $SureDerm^{TM}$ has 70% ethanol treatment and ethylene oxide gas sterilization for inactivating infectious microorganisms. The purpose of this study was to examine the efficacy of the 70% ethanol treatment, decellularization process using 0.1% TnBP and 2% deoxycholic acids, and EO gas sterilization process in the inactivation of viruses. A variety of experimental model viruses for human pathogens, including the human immunodeficiency virus type 1 (HIV-1), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), and porcine parvovirus (PPV) were all selected for this study. Enveloped viruses such as HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by 70% ethanol treatment. However HAV and PPV showed high resistance to 70% ethanol treatment with the log reduction factors of 1.85 and 1.15, respectively. HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by decellularization process. All the viruses tested were completely inactivated to undetectable levels by EO gas treatment. The cumulative log reduction factors of HIV-1, BHV, BVDV, HAV, and PPV were $\geq12.71$, $\geq18.08$, $\geq14.92$, $\geq6.57$, and $\geq7.18$, respectively. These results indicate that the production process for $SureDerm^{TM}$ has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.377-382
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• 2009

Viral safety is an important prerequisite for clinical preparations of all biopharmaceuticals derived from plasma, cell lines, or tissues of human or animal origin. To ensure the safety, implementation of multiple viral clearance (inactivation and/or removal) steps has been highly recommended for manufacturing of biopharmaceuticals. Of the possible viral clearance strategies, Ultraviolet-C (UVC) irradiation has been known as an effective viral inactivating method. However it has been dismissed by biopharmaceutical industry as a result of the potential for protein damage and the difficulty in delivering uniform doses. Recently a continuous flow UVC reactor (UVivatec) was developed to provide highly efficient mixing and maximize virus exposure to the UV light. In order to investigate the effectiveness of UVivatec to inactivate viruses without causing significant protein damage, the feasibility of the UVC irradiation process was studied with a commercial therapeutic protein. Recovery yield in the optimized condition of $3,000\;J/m^2$ irradiation was more than 98%. The efficacy and robustness of the UVC reactor was evaluated with regard to the inactivation of human immunodeficiency virus (HIV), hepatitis A virus (HAV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), bovine parvovirus (BPV), minute virus of mice (MVM), reovirus type 3 (REO), and bovine parainfluenza virus type 3 (BPIV). Non enveloped viruses (HAV, PPV, BPV, MVM, and REO) were completely inactivated to undetectable levels by $3,000\;J/m^2$ irradiation. Enveloped viruses such as HIV, BVDV, and BPIV were completely inactivated to undetectable levels. However BHV was incompletely inactivated with slight residual infectivity remaining even after $3,000\;J/m^2$ irradiation. The log reduction factors achieved by UVC irradiation were ${\geq}3.89$ for HIV, ${\geq}5.27$ for HAV, 5.29 for BHV, ${\geq}5.96$ for BVDV, ${\geq}4.37$ for PPV, ${\geq}3.55$ for BPV, ${\geq}3.51$ for MVM, ${\geq}4.20$ for REO, and ${\geq}4.15$ for BPIV. These results indicate that UVC irradiation using UVivatec was very effective and robust in inactivating all the viruses tested.

• Journal of the korean veterinary medical association
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• v.45 no.8
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• pp.746-757
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• 2009

국립수의과학검역원과의 공동연구과제(소 아까바네병, 츄잔병, 아이노 바이러스혼합불활화 예방약 산업화)에 의해 실시한 시험을 통하여 소 아까바네병, 츄잔병, 아이노 바이러스병에 대한 예방약인(소 츄잔병, 아까바네병, 아이노병 불활화 혼합오일백신) 면역원성 시험 및 야외에서의 적용시험 등을 실시한 결과를 요약하면 다음과 같다. 1. 혼합오일백신의 기니픽에 대한 면역원성 시험에서 아까바네 바이러스, 아이노 바이러스 및 츄잔 바이러스에 대한 중화항체가는 시험백신을 3주 간격 2회 접종한 시험구가 무접종 대조구에 비하여 높게 나타났다. 2. 혼합오일백신의 야외농장 3곳의 육성우에 대한 면역원성 시험에서 시험백신을 2회 접종 후 아까바네 바이러스의 항체가는 평균 102배, 115배 및 134배이였으며, 아이노 바이러스의 항체가는 평균 8배, 24배 및 27배로 나타났고, 츄잔 바이러스의 경우에는 3곳의 농장에서 평균 30배, 46배 및 172배로 나타났다. 3. 야외농장 2곳에서 혼합오일백신을 임신우에 2회 접종하였을 때, 중화항체가는 아까바네 바이러스가 평균 64배와 93배를 나타내었고, 아이노 바이러스의 중화항체가는 평균 70배와 96배, 츄잔 바이러스의 중화항체가는 평균 20배와 20배로 나타났다. 4. 혼합오일백신은 임신우에 대한 공격접종시험에서 시험백신을 3주 간격으로 2회 접종 후 3주째의 중화항체가는 아까바네 바이러스가 128$\sim$256배이었고, 아이노 바이러스는 64$\sim$256배로 나타났고, 츄잔 바이러스는 8$\sim$32배이었다. 공격 접종 후 부검 전의 중화항체가는 아까바네 바이러스가 16$\sim$128배로 나타났고, 아이노 바이러스는 32$\sim$128배였으며, 츄잔 바이러스는 4$\sim$16배였다. 혼합오일백신을 임신우에 3주 간격 2회 접종한 뒤 공격접종하여 채혈한 혈청의 면역원성이 무접종 대조군에 비하여 방어효과가 우수하였다. 5. 3롯트의 시험백신을 선정하여 2$\sim$7$^{\circ}C$의 냉암소에 보존하면서 보존 기간별(제조당시, 6개월, 12개월, 15개월, 18개월)로 특성시험, 무균시험 및 안전시험을 실시한 결과, 전 보존기간에 마우스 및 기니픽에 대한 안전성이 인정되었으며, 보존기간별로 시험백신의 기니픽에 대한 면역원성 시험을 실시한 결과, 기니픽의 중화항체가는 15개월까지 지속되는 것으로 나타났다.

• Journal of Sericultural and Entomological Science
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• v.29 no.2
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• pp.51-57
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• 1987

The intent of this research is to acquire some basic informations about formulation of the viral pesticide, Hyphantria cunea nuclear polyhedrosis virus and its virulence under field condition. The nuclear polyhedrosis virus was formulated as wettable powder using spreader, sticker and U.V. protector. The formulated product and aqueous virus were diluted with water at the concentration of 1${\times}$106PIB/ml and sprayed on mulberry leaves in the field. The leaves were fed with 3rd instar larvae of H. cunea to determine the inactivation period of the viral pesticides. The aqueous virus was completely inactivated on 5th day after spray, while the formulated one showed a spare mortality to the larvae even on 20th day after spray. In field application test, The fromulated and aqueous virus were sprayed on individual mulberry tree and 3rd instar laevae of H. cunea were fed on the trees. The mortality of the larvae one day after spray of the formulated and aqueous virus were about 50% and 40%, respectively. The formulated virus exhibited a persistent virulence to the larvae up to 9th day after spray, which the mortality was approximately 30%. The residual virulence of the formulated and aqueous virus was extended up to 4th day and 2nd day after spray, respectively.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.140-147
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• 2016

A process for manufacturing virally-safe porcine bone hydroxyapatite (HA) has been developed to serve as advanced xenograft material for dental applications. Porcine bone pieces were defatted with successive treatments of 30% hydrogen peroxide and 80% ethyl alcohol. The defatted porcine bone pieces were heat-treated in an oxygen atmosphere box furnace at $1,300^{\circ}C$ to remove collagen and organic compounds. The bone pieces were ground with a grinder and then the bone powder was sterilized by gamma irradiation. Morphological characteristics such as SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy) images of the resulting porcine bone HA (THE Graft$^{(R)}$) were similar to those of a commercial bovine bone HA (Bio-Oss$^{(R)}$). In order to evaluate the efficacy of $1,300^{\circ}C$ heat treatment and gamma irradiation at a dose of 25 kGy for the inactivation of porcine viruses during the manufacture of porcine bone HA, a variety of experimental porcine viruses including transmissible gastroenteritis virus (TGEV), pseudorabies virus (PRV), porcine rotavirus (PRoV), and porcine parvovirus (PPV) were chosen. TGEV, PRV, PRoV, and PPV were completely inactivated to undetectable levels during the $1,300^{\circ}C$ heat treatment. The mean log reduction factors achieved were $${\geq_-}4.65$$ for TGEV, $${\geq_-}5.81$$ for PRV, $${\geq_-}6.28$$ for PRoV, and $${\geq_-}5.21$$ for PPV. Gamma irradiation was also very effective at inactivating the viruses. TGEV, PRV, PRoV, and PPV were completely inactivated to undetectable levels during the gamma irradiation. The mean log reduction factors achieved were $${\geq_-}4.65$$ for TGEV, $${\geq_-}5.87$$ for PRV, $${\geq_-}6.05$$ for PRoV, and $${\geq_-}4.89$$ for PPV. The cumulative log reduction factors achieved using the two different virus inactivation processes were $${\geq_-}9.30$$ for TGEV, $${\geq_-}11.68$$ for PRV, $${\geq_-}12.33$$ for PRoV, and $${\geq_-}10.10$$ for PPV. These results indicate that the manufacturing process for porcine bone HA from porcine-bone material has sufficient virus-reducing capacity to achieve a high margin of virus safety.

• Korean journal of applied entomology
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• v.29 no.4
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• pp.244-251
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• 1990

Three viral insecticides were differently formulated with a nuclear polyhedrosis virus isolated from Spdodoptera litura by addition of feeding attractant, anti-precipitate of polyhedra, spreading agent, and UV-protectants. Sucrose was effective for attraction of larval feeding to increase the mortality and for protection of polyhedra from inactivation by sunlight when added 1% to 5% of sucrose solution to the formulations. Contents of additives to the formulations were 0.5% in polyvinyl alcohol to prohibit the precipitation of polyhedra and 0.1% in Triton X-100 to spread and wet the formulations to the plant. Inactivation of the virus under sunlight was decreased when added 800g of white carbon to 100 L of water in the white carbon formulation and 30% of molasses to the molasses's. In the formulation of white carbon and molasses mixtures, activation of the virus was increased when mixtured 500g of the former with 10% of the latter. Three formulations were persisted their pathogenicity more than 95% of mortality at 3 days p.i. Encapsulation of the polyhedral surface was more distinctively coated with the carbon and showed more effective in the residual effects of the white carbon than others, but the molasses more attractive for larval feeding.