• The Plant Pathology Journal
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• v.25 no.1
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• pp.77-85
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• 2009

Pseudomonas fluorescens strain Gpf01, isolated from ginseng rhizosphere showed antiviral activity against Cucumber mosaic virus, when tested in a local host of CMV, Chenopodium amaranticolor. Transposon mutant library of Gpf01 was prepared using pGS9::Tn5 and the mutant Gpf01-RS19 was found to loose antiviral production. We developed primers from the flanking region of Tn5 and found a cosmid clone pAV1123, harboring 1.2 kb antiviral compound producing (avcf01) locus. When a sub-clone pPH9, which carried 9.3 kb region of pAV1123, was introduced into antivirus deficient P. fluorescens wild type strain B16, it exhibited antiviral activity. Using Tn3-gus mutagenesis and complementation analysis, it was found that the genes related to antiviral activity production resided in a 9.3 kb HindIII-HindIII fragment of pAV1123, indicating that the plasmid carries an essential genes promoting antiviral activity.

• Korean Journal Plant Pathology
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• v.2 no.2
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• pp.121-128
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• 1986

The biochemical properties of ribonucleic acid (RNA) and coat protein of the mild tobacco mosaic virus (TMV) mutant, Tw 333 are described. The molecular weight of the RNA calculated from polyacrylamide gel electrophoresis was $2.03\times10^6$ daltons. The molar ratio of the bases of the RNA was 25.4 guanine, 29.2 adenine, 17.5 cytosine and 27.9 uracil in moles. The hyperchromicity on Tw 333-RNA by thermal denaturation was $25.1\%$, indicating Tm value of $47^{\circ}C$. The virus coat protein migrated as a single component in SDS-polyacrylamide gel electrophoresis and had a molecular weight of 17,500 daltons. A total of 158 amino acid residues are present in the protein. Separation of the tryptic peptides by electrophoresis and chromatography yielded ninhydrin-positive compounds. The biochemical properties of RNA and coat protein of the mild mutant we very similar to those of wild type of TMV-OM strain, but some difference between the strains were observe in the base composition, hyperchromicity, amino acid composition and tryptic peptide map.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.537-546
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• 2015

Classical swine fever (CSF) is a highly contagious disease of pigs caused by CSF virus (CSFV). E2 is the major viral envelope protein of immune dominance that induces neutralizing antibodies and confers protection against CSFV infection. The B/C domains of E2 are variable among CSFV isolates, which could affect immunogenicity and binding to antibodies. We attempted to characterize the epitope recognized by a monoclonal antibody 2B6 (mAb-2B6) raised against the E2 B/C domains of the vaccine C-strain and to examine if mutations in the epitope region would affect antibody binding and viral neutralization. The epitope specific for mAb-2B6 recognition is linear, spanning five residues ⁷⁷⁴DGXNP⁷⁷⁸ in the B/C domains. The residue N777 is indispensable for the specificity. The epitope exists only in group 1 strains, but not in those of group 2. The recombinant viruses containing individual mutations on the epitope region lost the reactivity to mAb-2B6. The mutant virus RecC-N777S had low replication potential, about 10-fold decrease in the yield of progeny virus particles, whereas the mutant virus RecC-P778A reverted to proline upon continuous passaging. The mutations on the mAb-2B6 epitope region did not affect neutralization by anti-C-strain polyclonal sera from pigs. Deletion from aa774 covering the mAb-2B6 epitope, but not that from aa781, also affected binding with the polyclonal antibodies from vaccinated pigs, although the major binding region for the vaccinated antibodies is aa690-773.

• BMB Reports
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• v.51 no.7
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• pp.338-343
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• 2018

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.

• Korean Journal of Veterinary Service
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• v.46 no.3
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• pp.193-202
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• 2023

Porcine epidemic diarrhea is an infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV). Especially, when suckling piglets are infected, the mortality rate is close to 100%. PEDV is classified into G1 and G2 types based on genetic differences. The G2 type PEDV outbreak in the United States in 2013 was highly pathogenic and contagious, and it has spread worldwide and caused continuous economic losses. Most commercial vaccines used are G1 type vaccines, and existing vaccines do not fully protect piglets due to genetic differences. In this study, we evaluated the safety of the newly developed G2 type attenuated HSGP vaccine strain by inoculating it into piglets and testing whether the vaccine virus spreads to the non-vaccinated, negative pigs and whether the vaccine reverts to its virulence during serial passage experiments. Each experiment lasted for 7 days for each passage, and fecal viral titers, clinical symptoms, and weight gain were measured daily. After the experiment, necropsy was performed to measure intestinal virus titer and pathological evaluation. As a result of the first passage, no transmission of the vaccine virus to negative pigs co-housed with vaccinated pigs was observed. In addition, after four consecutive passage experiments, the clinical symptoms and small intestine lesions were gradually alleviated, and no virus was detected in the feces in the fourth passage experiment. Therefore, it was concluded that the vaccine was safe without virulence reversion in accordance with the guidelines of the current licensing authority. However, further studies are needed on the genetic changes and biological characteristics of the mutant virus that occur during successive passages of the attenuated vaccine since the replication and clinical symptoms of the virus increased until the third passage during successive passages of the vaccine virus. Based on this study, it was concluded that virulence reversion and safety evaluation of attenuated vaccines through serial passage in target animals can be useful to evaluate the safety of attenuated viruses.

• BMB Reports
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• v.36 no.4
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• pp.379-386
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• 2003

A mutant herpes simplex virus 1, mtHSV, was constructed by inserting the E. coli beta-galactosidase gene into the loci of icp34.5, the apoptosis-inhibiting gene of HSV. The mtHSV replicated in and lysed U251 (human glioma cells), EJ (human bladder cells), and S-180 (mice sarcoma cells), but not Wish (human amnion cells) cells. With its intact tk (thymidine kinase) gene, mtHSV exhibited susceptibility to acyclovir (ACV), which provided an approach to control viral replication. An in vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS. Tumor dimensions then were measured at serial time points, and the tumor volumes were calculated. Sarcoma growth was significantly inhibited with prolonged time and reduced tumor volume. There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs. Immunohistochemical staining revealed that virus replication was exclusively confined to the treated tumor cells. HSV-1 DNA was detected in tumors, but not in the other organs by a polymerase chain reaction analysis. From these experiments, we concluded that mtHSV should be a safe and promising oncolytic agent for cancer treatment.

• Journal of Microbiology
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• v.34 no.1
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• pp.7-14
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• 1996

Teh baculovirus gp64 glycoprotein is a major component of the envelope of budded virus (BV) and has been shown that it plays an essential role in the infection process, especially virus-cell membrane fusion. We have cloned Autographa californica Nuclear Polyhedrosis Virus (AcNPV) gp64 protein were examined for membrane fusion activity by using a synchtium formation assay under various conditions. The optimal conditions required for inducing membrane fusion are 1) form pH 4.0 to 4.8 2) 15 min exposure of cells to acidic pH 3) at least 1 .mu.g of gp64 cloned plasmid DNA per 3 * 10$^{6}$ cells 4) and an exposure of cells to acidic pH at 72 h post-transfection. In order to investigate the role of hydrophobicity of the gp64 glycoprotein for the membrane fusion, the two leucine residues (amino acid position at 229 and 230) within hydrophobic region I were substituted to alanine by PCR-derived site-directed mutagenisis and the membrane fusion activity of the mutant was anlaysed. The gp64 glycoprotein carrying double alamine substitution mutation showed no significant difference in fusion activity. This result suggested that minor changes in hydrophobicity at the amino acid position 229 and 230 does not affect the acid-induced membrane fusion activity of the gp64 glycoprotein.

• Journal of the Korean Society of Tobacco Science
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• v.23 no.2
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• pp.123-132
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• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• Clinical and Experimental Pediatrics
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• v.50 no.9
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• pp.823-834
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• 2007

Interferon (IFN) alpha has been the first line therapy of chronic hepatitis B in children, but HBeAg seroconversion occurred in 26% of treated children compared to 11% of controls in multinational randomized controlled study. Recently, lamivudine was shown to be a potent inhibitor of Hepatitis B virus (HBV) reproduction both in HBeAg positive and in HBeAg negative (the pre-core mutant form) chronic hepatitis in randomized studies worldwide. Lamivudine therapy led to considerable improvement in the seroconversion rate of HBeAg in children with chronic hepatitis B, though long-term therapy resulted in the expansion of lamivudine-resistant mutant viruses. Combination therapy with lamivudine plus alpha-IFN does not seem to improve HBe Ag seroconversion. Above all, the most effective way to prevent hepatitis B is universal HBV vaccination.

• Animal cells and systems
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• v.5 no.3
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• pp.195-198
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• 2001

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine-HCI. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a Ion- mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.