• Archives of Pharmacal Research
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• v.28 no.8
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• pp.956-962
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• 2005

Leishmania virus (LRV)1-4 has been reported to produce a fusion of ORF2 and ORF3 via a programmed +1 frameshift in the region where ORF2 and ORF3 overlap (Lee et a/., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (nt. 2565-2823), frameshift region of LRV1-4, and the 71 bp (nt. 2605-2678) sub-region where ORF2 and ORF3 overlap. We then predicted the frameshift site using a new computer program (Pseudoviewer), and finally identified the specific region associated with the mechanism of the LRV1-4's+1 frameshift by means of a mutational analysis based on the predicted structure of LRV1-4 RNA. The predicted structure was confirmed by biochemical analysis. In order to measure the frameshift efficiency, constructs that generate luciferase without a frameshift or with a+1 frameshift, were generated and in vitro transcription/translation analysis was performed. Measurements of the luciferase activity generated, showed that the frameshift efficiency was about $1\%$ for both the 259bp (LRV1-4 259FS) and 71 bp region (LRV1-4 71FS). Luciferase activity was strongly reduced in a mutant (LRV1-4 NH: nt. 2635-2670) with the entire hairpin deleted and in a mutant (LRV1-4 NUS: nt. 2644-2659) with the upper stem of the hairpin deleted. These results indicate that the frameshift site in LRV1-4's is in the 71 bp region where ORF2 and ORF3 overlap, and that nt. 2644-2659 (the upward hairpin stem) playa key role in generating the +1 frameshift.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.8 no.1
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• pp.91-95
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• 2005

The familial environment may also play an important role in the epidemiology of HCV infection through vertical and horizontal transmission by infected household members. However, it is still controversial whether familial clustering of HCV occurs. We experienced a case of familial clustering of hepatitis C virus. A 10-year old girl presented with nausea, vomiting and anorexia for a month was diagnosed as hepatitis C. Her mother, grandmother, a maternal aunt and her daughter had contracted with HCV. Her laboratory findings showed AST/ALT 63/122 IU/L, positive anti-HCV Ab and HCV RNA ($3.54{\times}10^5copies/mL$). Pathologic findings of the liver biopsy revealed chronic hepatitis with minimal lobular activity, mild porto-periportal activity and mild portal fibrosis. After treatment with interferon-${\alpha}$ 2b for 6 months, the clinical symptoms and laboratory findings were normalized.

• Fisheries and Aquatic Sciences
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• v.21 no.3
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• pp.5.1-5.8
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• 2018

Stimulator of interferon gene (STING) is induced by various inflammatory agents, such as lipopolysaccharide and microbial pathogens, including virus and bacteria. In this study, we obtained a full-length cDNA of a STING homolog from olive flounder using rapid amplification of cDNA ends PCR technique. The full-length cDNA of Paralichthys olivaceus STING (PoSTING) was 1442 bp in length and contained a 1209-bp open reading frame that translated into 402 amino acids. The theoretical molecular mass of the predicted protein sequence was 45.09 kDa. In the PoSTING protein, three transmembrane domains and the STING superfamily domain were identified as characteristic features. Quantitative real-time PCR revealed that PoSTING expressed in all the tissues analyzed, but showed the highest level in the spleen. Temporal expression analysis examined the significantly upregulated expression of PoSTING mRNA after viral hemorrhagic septicemia virus (VHSV) stimulation. In contrast, no significant changes in the PoSTING expression were detected in Edwardsiella tarda-challenged group compared to the un-injected control. The expression of P. olivaceus type I interferon (PoIFN-I) was also highly upregulated upon VHSV challenge. These results suggest that STING might be involved in the essential immune defense against viral infection together with the activation of IFN-I in olive flounder.

• Journal of Ginseng Research
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• v.35 no.1
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• pp.69-79
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• 2011

Hepatocellular carcinoma (HCC) is the fi fth most common malignancy in the world and complicates liver cirrhosis related to hepatitis C virus (HCV) in many cases. We evaluated the therapeutic effect of Korean red ginseng extract (KGE) in patients with chronic liver diseases. Thirty male and female patients with HCC and another thirty with liver cirrhosis were included. Each category was divided into two groups; the first was used as control group, and received medical therapy only and the second group received the medical therapy supplemented with KGE capsules. The treated group with HCC received three KGE capsules/day (900 mg) while the treated group with HCV received two KGE capsules/day (600 mg) for 11 weeks along with their medical therapy. All patients were subjected to clinical examination and laboratory investigations, including liver function tests (at baseline, after 6 weeks of treatment and at the end of the study) and abdominal ultrasonography. Patients showing focal hepatic lesions were subjected to triphasic spiral abdominal computerized tomography and alpha-fetoprotein (AFP). HCV RNA was determined quantitatively by Roche for patients in the HCV group. Results showed that the medical therapy alone failed to normalize the liver enzymes or decrease the virus concentration. KGE administration induced a significant improvement in liver function tests, decreased the tumor marker (AFP) levels, and decreased the viral titers in HCV patients. Thus, KGE demonstrated powerful therapeutic effects against HCV and liver cancer.

• Molecules and Cells
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• v.21 no.1
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• pp.153-160
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• 2006

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature ($15^{\circ}C$) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Korean Journal of Veterinary Research
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• v.47 no.2
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• pp.175-185
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• 2007

This study was carried out to investigate the pathogenesis and pathogenicity of the porcine circovirus type 2 (PCV2) Korean isolate from weaned pigs. Twenty four weaned pigs, PCV2, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) antibodies free, were allocated to 4 groups (n = 6). Six pigs were inoculated intranasally with PCV2 alone, 6 with PCV2 and PRRSV, 6 with the combined PCV2/PRRSV/PPV inoculum, and 6 were remained as a uninoculated negative control. Pigs were killed 3 and 6 weeks after inoculation and tissue samples examined for gross and microscopic lesions and for the presence of PCV2 antigens and nucleic acids. Experimentally inoculated pigs were evaluated for 3 considerations: 1. development of postweaning multisystemic wasting syndrome (PMWS), 2. distribution of viral antigens by immunohistochemistry and polymerase chain reaction (PCR), and 3. cytokine mRNA levels in lymph nodes. Pigs inoculated with PCV2/PRRSV/PPV showed typical clinical signs, gross findings, and histopathologic characteristics of PMWS. In the PCV2/PRRSV/PPV inoculated group, the PCV2 antigen was widely distributed in various parenchymal organs such as brain, spinal cord, tonsil, lymph nodes, lung, heart, liver, kidney, spleen, and peyer's patch. Lymph node mRNA expression of IL-$1{\alpha}$, IL-2R and IL-8 was determined by real-time PCR. The pigs of PCV2/PRRSV and PCV2/PRRSV/PPV inoculation group, the mRNA expression was characterized by a decrease of IL-$1{\alpha}$, IL-2R and IL-8. The decrease of cytokine mRNA represent the state of T cell immuno-suppression in pig, and nicely support the evidence for the impairment of immune system in pigs with PMWS. In conclusion, PCV2 infection and some additional infectious causes such as PRRSV and/or PPV are warranted for the presence of PMWS in weaned pigs in Korea.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.1
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• pp.49-55
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• 2007

HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5'UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5'UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes.

• Journal of Life Science
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• v.12 no.1
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• pp.96-105
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• 2002

To investigate expression of the artificial gene encoding a repeated tripeptide lysyl-glutamyl-tryptophan in tobacco plant, the plant binary vector, pART404 has been constructed, which contains the duplicated CaMV 35S promoter, an artificial gene coding for repetitive polymer (Lys-Glu-Trp)$_{64}$, and nopaline synthase (nos) terminator. The recombinant expression vector was introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated trans-formation. The transgenic calli selected by kanamycin containing medium were then regenerated to whole plants. Southern blot analysis indicated that five transgenic plants (No. 1, 7, 9, 43, 45) showed the hybridizing signals at 1.1 kb of the expected size on EcoRI digestion and each of the transgenic plants contained 1 or 3 copies of the artificial gene inserted into its genome. By northern blot analysis, the size of the hybridized total RNA was estimated to be approximately 1.2 kb and the RNA appeared generally to have the integrity. Western blot indicated that the protein was detected at the position of 33 kDa and the expression level of the polypeptide in the transgenic plant (No. 45) was measured to approximately 0.1% of the total protein.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.77-96
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• 1995

To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.