• Title, Summary, Keyword: HCV

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Rapid Detection of Serum HCV RNA by Combining Reverse Transcription and PCR without RNA Extraction

  • Jang, Jeong-Su;Lee, Kong-Joo
    • Archives of Pharmacal Research
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    • v.19 no.6
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    • pp.486-489
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    • 1996
  • A simple, rapid, specific and sensitive method for the detection of serum hepatitis C virus (HCV) RNA using the reverse transcription-polymerase chain reaction (RT-PCR) technique without conventional RNA extraction was developed. HCV template RNA from serum was obtained by boiling the serum at $95^{\circ}C$ for 2 min, cooling rapidly in ice and removing the proteins by cetrifugation. RT-PCR amplifications including the reverse transcription and first PCR amplification were performed in one vessel containing both of reverse transcriptase and Taq DNA polymerase. The detection of HCV RNA from $10^{-3}{\mu}l$. serum was possible with this method. The suitability of this method for clinical analysis was evaluated by assaying HCV RNA in 225 patient samples including anti-HCV antibody negatives (13 samples) and positives (212 samples) by enzyme-linked immunosorbent assay test (ELISA). Detections of HCV RNA with this method were in 4 of 13 anti-HCV antibody negative samples (30.8%) and 95 of 212 positive samples (44.8%). The present method can be completed in 1 hr and has a wide range of application for the clinical utilities to determine the viral RNAS.

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Differential Expression of HCV Core Protein from Two Different Quasispecies

  • Yu, Kyung-Lee;You, Ji-Chang
    • Biomolecules & Therapeutics
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    • v.17 no.2
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    • pp.151-155
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    • 2009
  • Hepatitis C virus (HCV) has genetic diversity like most of RNA viruses. HCV major genotypes are classified into several subtypes which are further divided into quasispecies having, genetically different but closely related variants. The HCV core that is a nucleocapsid protein located at the amino terminus of the viral polyprotein is relatively a conserved protein among the HCV isolates and thus it has been one of plausible targets for anti-HCV drug development. However, different quasispecies of HCV core gene have also been found. In this study, we compared the expression level of core protein between two different quasispecies of HCV genotype 1b. Our data demonstrate that a little differences of amino acid sequence lead to substantial difference of expression level. It might be another important reason of different pathogenesis among HCV infected patients.

Hepatitis C Virus Core Protein Activates p53 to Inhibit E6-associated Protein Expression via Promoter Hypermethylation (C형 간염바이러스 코어 단백질에 의한 p53 활성화와 프로모터 과메틸화를 통한 E6AP 발현 억제)

  • Kwak, Juri;Jang, Kyung Lib
    • Journal of Life Science
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    • v.28 no.9
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    • pp.1007-1015
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    • 2018
  • The E6-associated protein (E6AP) is known to induce the ubiquitination and proteasomal degradation of HCV core protein and thereby directly impair capsid assembly, resulting in a decline in HCV replication. To counteract this anti-viral host defense system, HCV core protein has evolved a strategy to inhibit E6AP expression via DNA methylation. In the present study, we further explored the mechanism by which HCV core protein inhibits E6AP expression. HCV core protein upregulated both the protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b to inhibit E6AP expression via promoter hypermethylation in HepG2 cells but not in Hep3B cells, which do not express p53. Interestingly, p53 overexpression alone in Hep3B cells was sufficient to activate DNMTs in the absence of HCV core protein and thereby inhibit E6AP expression via promoter hypermethylation. In addition, upregulation of p53 was absolutely required for the HCV core protein to inhibit E6AP expression via promoter hypermethylation, as evidenced by both p53 knockdown and ectopic expression experiments. Accordingly, levels of the ubiquitinated forms of HCV core protein were lower in HepG2 cells than in Hep3B cells. Based on these observations, we conclude that HCV core protein evades ubiquitin-dependent proteasomal degradation in a p53-dependent manner.

Detection of Antibody to Hepatitis C Virus in Psychiatric Inpatients (정신과(精神科) 입원(入院) 환자(患者)의 C형(型) 간염(肝炎) 항체(抗體) 양성솔(陽性率))

  • Cheon, Jin-Sook;Han, Ho-Sung
    • Korean Journal of Biological Psychiatry
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    • v.2 no.1
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    • pp.100-106
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    • 1995
  • Antibodies to hepatitis C drew attention because of high morbidity to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV was known to be transmitted by transfusion, sexual behavior and parenteral drug use. However, some kind of autoimmune mechanism was suggested to be involved in the genesis of HCV-induced liver diseases. We hypothesized the prevalence of having anti-HCV might be higher in psychiatric patients rather than general population because of the characteristic route of transmission. Using Abbott HCV BA kit, anti-HCV was detected in the sera of 113 psychiatric inpatients from early December in 1992 to late May in 1994. The Positivity of anti-HCY was significantly(P<0.05) higher among psychiatric inpatients(10.6%) than in healthy controls(3.0%). There were no disease specificity among psychiatric inpatients who had anti-HCV, though alcoholics tended to have more anti-HCV. We couldn't find any significant correlation of anti-HCV with age, seasons of birth, lymphocytes (%) and liver function.

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A Case of Vertical Transmission of Hepatitis C Virus in an Infant of a Mother Who had Hepatitis C during Pregnancy (C형 간염 산모로부터 출생한 영아에서 C형 간염 Virus의 수직 전파 1례)

  • Oh, Sang-Hyun;Kim, Kuk-Hwan;Yang, Eun-Seok;Park, Sang-Kee;Moon, Kyung-Rye
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.2 no.1
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    • pp.109-115
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    • 1999
  • Hepatitis C virus (HCV) has been identified as an important cause of posttransfusion hepatitis, but vertical transmission of chronic infected HCV RNA positive mothers has been documented in some cases. The reports of the risk of perinatal infection have been widely varied in the literature. The authors experienced one case of vertical transmission of HCV in an infant of a mother who had hepatitis C during pregnancy. At admission, HCV RNA (+), Ig G anti HCV (+) and Ig M anti HCV (+) were found in the mother. Also at admission, HCV RNA (+), Ig G anti HCV (+), Ig M anti HCV (+), elevation of liver aminotransferase level and hepatosplenomegaly on ultrasonography were found in the baby on day 31. HCV RNA (-), Ig M anti HCV (-) and normal of liver aminotransferase level were noted on day 250 in the serum of the infant. We used reverse transcriptase polymerase chain reaction (RT-PCR) technique to find a very small amount of HCV RNA in the serum. All the findings suggest vertical transmission of HCV RNA from mother to infant during 3rd trimester of pregnancy.

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Inhibition of the Replication of Hepatitis C Virus Replicon with Nuclease-Resistant RNA Aptamers

  • Shin, Kyung-Sook;Lim, Jong-Hoon;Kim, Jung-Hye;Myung, Hee-Joon;Lee, Seong-Wook
    • Journal of Microbiology and Biotechnology
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    • v.16 no.10
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    • pp.1634-1639
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    • 2006
  • Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

Prevalence of Anti-HCV among the Health-checkup Adults in Jeonbuk Province (전북 지역 건강 검진자들의 Anti-HCV 양성률 조사)

  • Kim, Yoohyun
    • Korean Journal of Clinical Laboratory Science
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    • v.42 no.1
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    • pp.32-37
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    • 2010
  • The author was performed to investigation of current status of prevalence for anti-hepatitis C virus (HCV) among the health-checkup adults in Jeonbuk province. A toal of 1,553 (male 1,046, female 507) serum samples were diagnosed by 3rd generation enzyme immunoassay (EIA) for anti-HCV. Total prevalence of anti-HCV was 0.9%, and prevalence of male and female were 0.8% and 1.2%, respectively. The prevalence of female was higher than male. According to ages group, prevalence of anti-HCV was highest in 60 age group, but it was not found in 20 age group. 14 samples with anti-HCV positive were diagnosed by EIA for hepatitis B virus surface antigen (HBs Ag), by chemiluminescence immunoassay (CLIA) for serum albumin, alanine transaminase (ALT) and asparagine transaminase (AST). Positive for HBs Ag was not found. The mean of serum albumin levels was 4.5 g/dL, and mean of ALT and AST were 34.3 IU and 31.9 IU, respectively. Through this study, I know that the prevalence of anti-HCV among adults in Jeonbuk, and suggest that the positive of anti-HCV persons who have lower serum albumin, normal to mild elevations in serum enzymes are chronic hepatitis.

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Comparative Analysis of Intracellular Trans-Splicing Ribozyme Activity Against Hepatitis C Virus Internal Ribosome Entry Site

  • Ryu Kyung-Ju;Lee Seong-Wook
    • Journal of Microbiology
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    • v.42 no.4
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    • pp.361-364
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    • 2004
  • Internal ribosome entry site (IRES) of the hepatitis C virus (HCV) is known to be essential for HCV replication and most conserved among HCV variants. Hence, IRES RNA is a good therapeutic target for RNA-based inhibitors, such as ribozymes. We previously proposed a new anti-HCV modulation strategy based on trans-splicing ribozymes, which can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. To explore this procedure, sites which are accessible to ribozymes in HCV IRES were previously determined by employing an RNA mapping method in vitro. In this study, we evaluate the intracellular accessibility of the ribozymes by comparing the trans-splicing activ­ities in cells of several ribozymes targeting different sites of the HCV IRES RNA. We assessed the intra­cellular activities of the ribozymes by monitoring their target-specific induction degree of both reporter gene activity and cytotoxin expression. The ribozyme capable of targeting the most accessible site iden­tified by the mapping studies then harbored the most active trans-splicing activity in cells. These results suggest that the target sites predicted to be accessible are truly the most accessible in the cells, and thus, could be applied to the development of various RNA-based anti-HCV therapies.

Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

  • Lee, Choongho
    • Biomolecules & Therapeutics
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    • v.21 no.2
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    • pp.97-106
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    • 2013
  • Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif ($^{79}{\underline{P}}GY{\underline{P}}WP^{84}$). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif ($^{79}{\underline{A}}GY{\underline{A}}WP^{84}$) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy.

Incidence of Active HCV infection amongst Blood Donors of Mardan District, Pakistan

  • Karim, Fawad;Nasar, Abu;Alam, Ibrar;Alam, Iftikhar;Hassan, Said;Gul, Rahmat;Ullah, Sana;Rizwan, Muhammad
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.1
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    • pp.235-238
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    • 2016
  • Hepatitis C is an ailment of liver caused by hepatitis C virus (HCV) infection. About 3% of the world population is infected by this virus. HCV infection is a leading reason for liver cirrhosis and therefore a major source of hepatocellular carcinoma. The study focused on the incidence of active HCV infection in blood donors of Mardan district of KPK, Pakistan. A total of 5318 blood donors were inspected for the presence of anti-HCV antibodies and HCV-RNA using ICT (immune-chromatographic test), ELISA and RT-PCR at Mardan Medical Complex (MMC), Mardan. Out of these, 157 (2.95%) were positive by ICT, 60 (1.12%) by ELISA and 56 (1.05%) for HCV-RNA. The frequency of active HCV infectivity amongst the blood donors from district Mardan, KPK Pakistan was 1.05 %. Application of strict measures during blood donor selection and use of proper screening assays such as ELISA in place of ICT devices can give a more accurate picture so that the incidence of this viral infection in HCV negative blood recipients can be reduced.