• Molecules and Cells
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• v.42 no.5
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• pp.418-425
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• 2019

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

• 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.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.77-83
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• 1994

Three viral strains, causing CPE in porcine alveolar macrophage cell, were isolated from aborted fetus, serum from young pig showing blue-ear sign and lung of suspected pig, respectively. The differential diagnostic results showed no characteristics of Aujeszky's disease virus(ADV), hog cholera virus (HCV), Japanese encephalitis virus(JEV), porcine parvovirus(PPV) and encephalomyocarditis virus (EMCV). However, positive reactions were demonstrated by IFA using monospecific porcine antibodies against Lelystad virus. When the paired sera of experimentally inoculated swine with one of isolate, KPRRSV-l were tested by IPMA, the result indicated that the isolate was related to United States isolate than European LV.

• 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.