• 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.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1570-1576
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• 2006

An encephalomyocarditis virus (EMCV-CBNU) was isolated from an aborted swine fetus in October 2005. To investigate the genetic origin and virulence of the EMCV-CBNU strain, we determined the complete sequence of the virus and tested its virulence in mice. Genetic characterization revealed that the RNA genome was composed of 7,713 nucleotides with a single open reading frame (2,292 amino acids), coding 12 proteins. The EMCV-CBNU had the shortest poly(C) tract, consisting of 10 C's ($C_{10}$), compared with all the other EMCV strains reported in GenBank. Amino acid and phylogenetic analyses showed that EMCV-CBNU had the highest genetic identity with strain 2887A (99.7%), which was originally isolated from a fetus in a pig breeding farm that had a history of reproductive failure. Because rodents are the natural host of EMCV, we investigated the virulence of EMCV-CBNU in mice. Surprisingly, all mice inoculated with more than $1{\times}10^2\;TCID_{50}/0.1ml$ of EMCV-CBNU showed symptoms of hind limb paralysis and eventually died during 3 and 8 days postinoculation (DPI). Furthermore, when we inoculated the virus into pregnant mice, all dams and their fetuses died in 6 DPI. This is the first report on a full genomic analysis of swine EMCV in Korea, which exhibits high virulence in mice.

• Korean Journal of Veterinary Research
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• v.50 no.3
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• pp.221-229
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• 2010

Encephalomyocarditis virus (EMCV) belongs to the genus Cardiovirus within the family Picornaviridae. EMCV has been recognized either as a cause of mortality in young pigs, due to acute myocarditis, or of reproductive failure in sows. An EMCV K3 strain was isolated from the heart and brain in a mummified and aborted swine fetus in 1989. For the molecular characterization of the poly(C)-tract of EMCV Korean isolates, K3 strain, viral RNA was extracted and digested with RNase T1, and analyzed the length of the poly(C)-tract by polyacrylamide gel electrophoresis. The poly(C) regions also were amplified by RT-PCR and sequenced. The present study shows that K3 strain of EMCV had a short polymorphic poly(C) tracts (5 to 30 C's) with sequences consisting of $C_9$, $C_{10}$, $C_{13}$, $C_{14}$, $C_{16}$, $C_{20}$, $CUC_{11}$, $C_8UCUC_3UC_{10}$, $C_9UCUC_3UC_{10}$, $C_{10}UCUC_3UC_{10}$, etc. These polymorphism of poly(C)-tracts of EMCV K3 strain implies the historical information of in vivo and/or in vitro passage.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.148-158
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• 1999

The purpose of this study was to establish a rapid, reliable diagnostic method detecting Encephalomyocarditis virus(EMCV) RNA in formalin-fixed, paraffin-embedded tissues of EMCV naturally infected pigs by cDNA probe of EMC $K_3$, the EMCV strain isolated from Korea. Using a biotin-labelled nick translated probe for the cDNA marker. We made up for some defects of radiolabeled method. In sits hybridization(ISH) technique, differently from the other nucleic acid hybridization methods, is able to detect the virus genome specifically in the state of the intact shapes of cells and/or tissues. We succeeded in performing the experiment to detect the EMCV within 1~2 hours using the $MicroProbe^{TM}$ capaillary action system. In this study, we observed highly specific positive signals of red color by staining the paraffin-embedded tissue sections of naturally EMCV-infected pig organs or tissues, including brain, heart, kidney and lacrimal gland with the Fast Red TR salt/Naphtol phosphate chromogen. The results suggested that this ISH method is considered as a highly sensitive and reliable tool for molecular biologic diagnosis of the EMC viral disease.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.615-620
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• 2003

The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro ($EC_{50}$ = 26.9 g/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1,000 g/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of $1.87\;{\times}\;10^6$, and was characterized as a homopolysaccharide of galactose with uronic acid (2.96%, w/w) and sulfate groups (10.32% w/w). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful for the development of marine bioactive exopolysaccharides for use in biotechnological and pharmaceutical products.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.615-618
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• 2000

The purpose of present study was to examine the efficacy of PAB (para-amino benzamidine) affinity column chromatography, pasteurization ($60^{\circ}C$ heat treatment for 10 h), and lyophilization steps, employed in the manufacture of urokinase from human urine, in the removal and/or inactivation of urine-born viruses. Bovine herpes virus (BHV) and Murine encephalomyocarditis virus (EMCV) were selected for this study. Samples from the relevant stages of the production process were spiked with the viruses and the amount of virus in each fraction was quantified by 50% tissue culture infectious dose ($TCID_{50}$). BHV and EMCV were effectively partitioned from urokinase during PAB chromatography with the log reduction factors of 6.71 and 5.27, respectively. Pasteurization was a robust and effective step in inactivating BHV and EMCV, of which titers were reduced from initial titers of $8.65\;log_{10}\;TCID_{50}$ and $7.81\;log_{10}\;TCID_{50}$, respectively, to undetectable levels within 1 hour of treatment. The log reduction factors achieved during lyophilization were 2.06 for BHV and 4.54 for EMCV. These results indicate that the production process for urokinase has sufficient virus reducing capacity to achieve a high margin of virus safety.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.127-140
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• 2019

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.858-864
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• 2000

The purpose of the present study was to examine the efficacy and mechanism of the fraction IV cold ethanol fractionation and pasteurization ($60^{\circ}C$ heat treatment for 10h) steps, involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of blood-born 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 selected for this study. Samples from the relevant stages of the production process were spiked with the viruses, and the amount of virus in each fraction was then quantified using a 50% tissue culture infectious dose ($TCID_{50}$). The mechanism of reduction for the enveloped viruses (BHV and BVDV) during fraction IV fractionation was inactivation rather than partitioning, however, it was partitioning in the case of the non-enveloped viruses (EMCV and PPV). The log reduction factors achieved during fraction IV fractionation were ${\geq}6.9$ BHV, $\geq5.2$ for BBDV, 4.9 for EMC, and 4.0 for PPV. Pasteurization was found to be a robust and effective step in inactivating the enveloped viruses as well as EMCV. The log reduction factors achieved during pasteurization were $\geq7.0$ for BHV, $\geq6.1$ for BVDV, $\geq6.3$ for EMCV, and 1.7 for PPV. These results indicate that the production process for albumin has sufficient virus-reducing capacity to achieve a high margin for virus safety.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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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.