• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.113.1-113
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• 2003

A potexvirus, Hosta virus X (HVX-Kr), causing mosaic and mottle symptoms was isolated from hosta plants (Hosta spp.), and its entire genome RNA sequence was determined. in Korea using cDNA library and RACE methods. The genome of HVX encodes five open reading frames coding for viral replicase, triple gene block (TGB), and viral coat protein (CP) from the 5'to 3' ends, which is a typical genome structure of potexviruses. The 3-terminal region of the virus includes the TGBI (26 kDa), TGB2 (13 kDa), TGB3 (8 kDa), and 23 kDa coat protein (CP) and the 3-nontranslated region (NTR). The CP gene of the type isolate of HVX (HVX-U) was amplified by RT-PCR and its nucleotide sequence was determined. The CPs of HVX-Kr and HVX-U had 100％ and 98.9％ identical amino acids and nucleotides, respectively. Most of the regions of the genome HVX had over 50％ nucleotide identical to other sequenced potexviruses. This is the first report of complete genome sequence information of HVX and molecular evidence supporting the virus as a distinct species of the genus Potexvirus.

• 대한수의학회지
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• 제39권1호
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• pp.138-147
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• 1999

Detection and distribution of bovine viral diarrhea virus(BVDV) was studied in formalin-fixed, paraffin-embedded tissues from two naturally infected cattle by in situ hybridization with a non-radioactive biotinylated probe. A 600 base pair cDNA probe from BVDV B-25 strain was used for probe. The whole procedure of ISH to diagnose was carried out within 1~2 hours in $Microprobe^{TM}$ capillary action system. The biotin-labelled probe was demonstrated after hybridization under standard conditions by the application of streptoavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a fast red TR/naphthol phosphatase and the sections were counterstained with hematoxylin. We have obtained the result of positive reactions in digestive tract(sm1.all intestine and colon) and epidermis of tongue in the state of the intact tissues. The result suggested that in situ hybridization method can be considered as a useful diagnostic technique for detection of specific nucleic acid sequences of BVDV.

• 대한수의학회지
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• 제43권2호
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• pp.239-246
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• 2003

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4199-4202
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• 2015

Background: Hepatitis B virus infection is one of the major world health problems. Epigallocatechin-3 gallate is the major component of the polyphenolic fraction of green tea and it has an anti-viral, anti-mutagenic, anti-tumorigenic, anti-angiogenic, anti-proliferative, and/or pro-apoptotic effects on mammalian cells. In this study, our aim was to investigate the inhibition of HBV replication by epigallocatechin-3 gallate in the Hep3B2.1-7 hepatocellular carcinoma cell line. Materials and Methods: HBV-replicating Hep3B2.1-7 cells were used to investigate the preventive effects of epigallocatechin-3 gallate on HBV DNA replication. The expression levels of HBsAg and HBeAg were determined using ELISA. Quantitative real-time-PCR was applied for the determination of the expression level of HBV DNA. Results: Cytotoxicity of epigallocathechin-3-gallate was not observed in the hepatic carcinoma cell line when the dose was lower than $100{\mu}M$. The ELISA method demonstrated that epigallocatechin-3 gallate have strong effects on HBsAg and HBeAg levels. Also it was detected by real-time PCR that epigallocatechin-3 gallate could prevent HBV DNA replication. Conclusions: The obtained data pointed out that although the exact mechanism of HBV DNA replication and related diseases remains unclear, epigallocatechin-3 gallate has a potential as an effective anti-HBV agent with low toxicity.

• Journal of Pharmaceutical Investigation
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• 제40권6호
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• pp.357-362
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• 2010

To enhance therapeutic effects of insulin-sensitizing adipokine, ADN gene and potent agonists, rosiglitazone for the $PPAR{\gamma}$, cationic liposomes as non-viral vectors were formulated. The particle size and zeta potential of drug loaded and unloaded cationic liposomes were investigated. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. In vitro transfection was investigated in HepG2, HeLa, and HEK293 cells by mRNA expression of ADN. Encapsulation efficacy of rosiglitazone-loaded liposomes was determined by UV detection. Particle sizes of cationic liposomes were in the range of 110-170 nm and those of rosiglitazone-loaded cationic liposomes were in the range of 130-180 nm, respectively. Gel retardation of complexes indicated that the complex was formed at weight ratios of cationic lipid to plasmid DNA higher than 20:1. Both complexes protected plasmid DNA from DNase either drug free or drug loading. Encapsulation efficiency of rosiglitazone-loaded emulsion was increased by drug dose. The mRNA expression levels of ADN were dose-dependently increased in cells transfected with plasmid DNA. Therefore, cationic liposomes could be potential co-delivery system for drug and gene.

• 한국동물학회지
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• 제28권3호
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• pp.166-178
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• 1985

한국형 B형 간염바이러스(HBV) DNA의 표면항원 유전자를 포유동물 세포에서 발현시켜 항원의 검출과 유전자의 분자유전학적인 연구를 하기 위하여 pre-surface antigen 지역과 표면항원 유전자 그리고 poly(A) addition site가 포함된 DNA 조각을 simian virus 40(SV 40)의 DNA 복제 원점과 promoter가 포함된 유전자 운반체에 재조함 시켰다. 우선, HBV DNA가 들어있는 pHBV 107을 Bam HI으로 부분절단한뒤 self-ligation시켜 두 HBV DNA가 같은 방향으로 들어간 pHBVD 107을 만들었다. 이 plasmid를 Bgl II로 절단하였을때 pre-surface지역과 표면항원 유전자 그리고 poly(A) addition site가 함께 포함된 2.7 kb의 insert DNA 조각을 얻었다. 유전자 운반체로는 포유동물세포에서 복제할 수 있도록 하기 위하여 SV40의 DNA 복제 원점부위와 72 bp repeats(enhancer)가 포함된 pSVOE를 만든다음 이 vector의 Pvu II 절단자리에 Bam HI linker를 붙여 insert DNA가 vector의 SV40 late promoter지역 가까이에 들어갈 수 있도록 변형시킨 pSVOB를 만들었다. 이상과 같이 만들어진 pre-surface 지역-표면항원유전자-poly(A)-addition site가 포함된 2.7 kb DNA 절편을 pSVOB promoter 뒤의 Bam HI site에 삽입하여 재조합된 plasmid pSVBS를 얻었다. 예비실험으로 pSVBS를 T-antigen이 생산되는 COS cell에 이주시켰더니 $HB_sAg$가 발현됨을 보았다.

• Molecules and Cells
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• 제27권1호
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• pp.105-111
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• 2009

Gammaherpesvirus infection of the central nervous system (CNS) has been linked to various neurological diseases, including meningitis, encephalitis, and multiple sclerosis. However, little is known about the interactions between the virus and the CNS in vitro or in vivo. Murine gammaherpesvirus 68 (MHV-68 or ${\gamma}HV-68$) is genetically related and biologically similar to human gammaherpesviruses, thereby providing a tractable animal model system in which to study both viral pathogenesis and replication. In the present study, we show the successful infection of cultured neuronal cells, microglia, and astrocytes with MHV-68 to various extents. Upon intracerebroventricular injection of a recombinant virus (MHV-68/LacZ) into 4-5-week-old and 9-10-week-old mice, the 4-5-week-old mice displayed high mortality within 5-7 days, while the majority of the 9-10-week-old mice survived until the end of the experimental period. Until a peak at 3-4 days post-infection, viral DNA replication and gene expression were similar in the brains of both mouse groups, but only the 9-10-week-old mice were able to subdue viral DNA replication and gene expression after 5 days post-infection. Pro-inflammatory cytokine mRNAs of tumor necrosis factor-${\alpha}$, interleukin $1{\beta}$, and interleukin 6 were highly induced in the brains of the 4-5-week-old mice, suggesting their possible contributions as neurotoxic factors in the age-dependent control of MHV-68 replication of the CNS.

• Asian Pacific Journal of Cancer Prevention
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• 제13권1호
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• pp.339-342
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• 2012

Background: About one third of the human population suffer cancer during their lifetime and more than 20% of total morbidity is related to neoplasia. Cervical cancer is generally the most common cancer in developing countries and the second most common in women globally. The role of human papilloma viruses viruses in its induction is clear. However, the involvement of hepres simplex virus type 2 (HSV-2) is controversial. Therefore a survey was conducted of the prevalence of HSV-2 in patients with cervical cancer and also healthy people with sensitive and quantitative Taq Man real-time PCR assay. Materials and methods: Seventy six formaldehyde fixed paraffin embedded tissue specimens from patients with histologically proven history of cervical cancer as well as 150 control blocks were sectioned for deparaffinization and DNA extraction. Results: There was no HSV-2 DNA in our patient specimens but four control samples were positive, all with a history of hysterectomy. Conclusion: Considering the absence of any positive viral HSV-2 DNA in our patients and also the presence of four positive specimens among our controls, we did not find any relationship between the presence of HSV-2 DNA and cervical cancer.

• Archives of Pharmacal Research
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• 제28권6호
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• pp.722-729
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• 2005

With the aim to improve the specificity and to reduce the cytotoxicity of polyethylenimine (PEI), we have synthesized the conjugates of the branched PEI (25 kDa) with transferrin. The trans-ferrin-PEI (TP) conjugates with five compositions were synthesized using periodate oxidation method and confirmed by FT-IR spectroscopy and gel permeation chromatography. The free amine contents of TP conjugates, which were able to condense and deliver DNA, increased as the amount of PEI increased. TP/DNA polyplexes were characterized by measuring gel elec-trophoresis, ethidium bromide fluorescence quenching, particle size and zeta potential of complexes. Complete complexation of the polyplexes was observed above the N/P ratio of 5 in TP/DNA, and above 3 in PEI/DNA, respectively. The zeta potential of the complexes decreased as the amount of transferrin in TP conjugates increased. Transfection efficiency of TP conjugates was evaluated in HeLa cell and Jurkat cell systems. Among the five compositions of TP conjugates, TP-2 system mediated a higher $\beta$-galactosidase gene expression than PEI system in Jurkat cell which was known to express elevated numbers of transferrin receptors. From the results of the cell viability based on MTT assay, TP conjugates showed lower cytotoxicity com-pared with the PEI system. We expect that the TP conjugate can be used efficiently as a non-viral gene delivery vector.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제15권1호
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• pp.38-43
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• 2012

Purpose: Epstein-Barr virus (EBV) hepatitis is a usually asymptomatic and self-limiting disease in immunocompetent patients. However, the range of severity is wide, and the serological diagnosis is typically difficult until the convalescent phase. Thus, we examined the value of plasma EBV DNA real-time quantitative polymerase chain reaction (RT-qPCR) in EBV hepatitis for the timely diagnosis and the relationship between EBV viral load and clinical severity. Methods: Sixty samples were confirmed as having EBV infection by RT-qPCR with the EBV BALF5 gene sequence. We examined the clinical characteristics of EBV hepatitis by reviewing medical records. Results: The median total duration of fever was 8 days (range: 0-13 days). The mean peak value of aspartate aminotransferase (AST) was $241{\pm}214$ U/L, and the mean peak value of alanine aminotransferase (ALT) was $298{\pm}312$ U/L. There was no correlation between the serum levels of liver enzyme and plasma EBV DNA titer ($p$=0.1) or between median total duration of fever and EBV DNA titer ($p$=0.056). The median age of the EBV VCA IgM-negative group was lower compared with the EBV VCA IgM-positive group in EBV hepatitis (2 years vs. 6 years, $p$=0.0009). Conclusion: The severity of EBV hepatitis does not correlate with circulating EBV DNA load according to our data. Furthermore, we suggest that plasma EBV PCR may be valuable in young infants in whom the results of serology test for EBV infection commonly are negative.