• Korean Journal of Veterinary Research
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• v.55 no.4
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• pp.227-232
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• 2015

Akabane and bovine ephemeral fever (BEF) viruses cause vector-borne diseases. In this study, inactivated Akabane virus (AKAV)+Bovine ephemeral fever virus (BEFV) vaccines with or without recombinant vibrio flagellin (revibFlaB) protein were expressed in a baculovirus expression system to measure their safety and immunogenicity. Blood was collected from mice, guinea pigs, sows, and cattle that had been inoculated with the vaccine twice. Inactivated AKAV+BEFV vaccine induced high virus neutralizing antibody (VNA) titer against AKAV and BEFV in mice and guinea pigs. VNA titers against AKAV were higher in mice and guinea pigs immunized with the inactivated AKAV+BEFV vaccine than in animals inoculated with vaccine containing revibFlaB protein. Inactivated AKAV+BEFV vaccine elicited slightly higher VNA titers against AKAV and BEFV than the live AKAV and live BEFV vaccines in mice and guinea pigs. In addition, the inactivated AKAV+BEFV vaccine was safe, and induced high VNA titers, ranging from 1 : 64 to 1 : 512, against both AKAV and BEFV in sows and cattle. Moreover, there were no side effects observed in any treated animals. These results indicate that the inactivated AKAV+BEFV vaccine could be used in cattle with high immunogenicity and good safety.

• The Korean Journal of Pharmacology
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• v.28 no.2
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• pp.213-222
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• 1992

We investigated effects of several chemical carcinogens, i.e., $benzo({\alpha})pyrene$ (BP),7,12-dimethylbenz(a)anthracene (DMBA), nitrosomethyl urea (NMU), and nicotine on the replication, cytolyticity, DNA synthesis, and protein synthesis of type 1 herpes simplex virus (HSV-1) in viral infected Vero cell monolayers. We observed that the BP and DMBA did not show such activity. All chemical carcinogens did not inhibit the synthesis of viral DNA, but the expression of gamma viral proteins that are expressed from the newly synthesized progeny viral DNA was somewhat notably inhibited by BP and DMBA. However, the synthesis of alpha and beta viral proteins was not altered by the chemical carcinogens. These data indicate that the gamma viral proteins expressed from the newly synthesized DNA in the presence of chemical carcinogens in the culture medium may be defective. This is further supported by the fact that the virus fail to replicate in the presence of these chemical carcinogens, in spite of viral DNA and proteins are somewhat normally synthesized.

• Molecules and Cells
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• v.37 no.2
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• pp.140-148
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• 2014

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R ${\rightarrow}$ T), 313(R ${\rightarrow}$ T), 315(R ${\rightarrow}$ P), and 329(R ${\rightarrow}$ T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R ${\rightarrow}$ T), 318(K ${\rightarrow}$ T), and 324(K ${\rightarrow}$ T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.197-207
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• 1997

To investigate the NS4 region of JEV, NS4 cDNA of K94P05 (JEV strain isolated from Korea in 1994) was amplified by RT-PCR and analyzed by sequencing PCR product. Genomic size of NS4 was 1212bp and nucleotide sequence was compared with that of other JEV strains. Nucleotide homology between JaOAr582 and K94P05 was 91.1% and that between Beijing and K94P05 was 89.8%, respectively. But the nucleotide sequence of E region of JaOAr582 and K94P05 showed 97.0% homology and that of Beijing and K94P05 did 95.8% homology. NS4 protein was expressed as a form of fusion protein by a prokaryotic expression system. The induced fusion product showed a lower molecular weight than predicted size and remained insoluble. The NS4 protein might be cleavaged by E. coli protease. Concluding above results, high hydrophobicity of the NS4 protein supported the fact that this protein played a role as a membrane component and the poor nucleotide sequence conservativity among JEV strains suggested that this region might be important to adapt each viral growth environment.

• KSBB Journal
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• v.13 no.6
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• pp.656-661
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• 1998

Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

• Journal of the Korean Chemical Society
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• v.67 no.4
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• pp.226-235
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• 2023

Chikungunya fever has a high morbidity rate in humans and is caused by chikungunya virus. There are no treatments available until now for this particular viral disease. The present study was carried out by selecting 19 flavonoids, which are available naturally in fruits, vegetables, tea, red wine and medicinal plants. The molecular docking of selected 19 flavonoids was carried out against the Chikungunya virus capsid protein using the Autodock4.2 software. Binding affinity analysis based on the Intermolecular interactions such as Hydrogen bonding and hydrophobic interactions and drug-likeness properties for all the 19 flavonoids have been carried out and it is found that the top four molecules are Chrysin, Fisetin, Naringenin and Biochanin A as they fit to the chikungunya protein and have binding energy of -8.09, -8.01, -7.6, and 7.3 kcal/mol respectively. This result opens up the possibility of applying these compounds in the inhibition of chikungunya viral protein.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.33.1-33.15
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• 2020

Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca²⁺-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.83-90
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• 1992

Monoclonal antibodies against structural proteins of bovine viral diarrhea virus(BVDV) were derived by classical hybridoma techniques. These antibodies were characterized by serum neutralization, immunoblotting and immunoprecipitation. The neutralizing monoclonal antibody reacted with the 56kd to 54kd(M.W.) viral protein in western blotting and immunoprecipitation analysis. Although there was no neutralizing activity, another monoclanal antibody reacted with the 45kd protein by immunoprecipitation and with both the 45kd and 36kd proteins in immunoblotting analysis. respectively. Densitometer scanning of purified BVDV and the immunopreipitation of whole virus particles with neutralizing monoclonal antibody revealed the presence of more than twelve viral polypeptides. Although no possible precursor form of protein was identified with the neutralizing monoclonal antibody. the presence of intact virion was detected in the infected cell supernatant immediatelty after pulse labeling, indicating rapid translational processing as well as packaging of the virus. The partial peptide mapping of 45kd and 36kd proteins with Staphylococcus aureus V 8 protease showed that these two proteins are related.

• Journal of Microbiology
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• v.37 no.4
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.