1.Occult hepatitis B virus infection: clearance or disguise?.
Clinical and Molecular Hepatology 2014;20(3):249-250
No abstract available.
DNA, Circular/*analysis
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Female
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Hepatitis B/*pathology
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Hepatitis B Surface Antigens/*genetics
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Hepatitis B virus/*metabolism
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Humans
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Male
2.An in vitro model of hepatitis B virus gene replication and expression in primary rat hepatocytes transfected with circular viral DNA.
Yun Qing YAO ; Ding Feng ZHANG ; Yun LUO ; Da Zhi ZHANG ; Al Long HUANG ; Bo WANG ; Wei Ping ZHOU ; Hong REN ; Shu Hua GUO
Chinese Journal of Hepatology 2002;10(4):275-278
<b>OBJECTIVEb>To establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes.
<b>METHODSb>Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group.
<b>RESULTSb>(1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection.
<b>CONCLUSIONSb>Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.
Animals ; DNA Replication ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Gene Expression ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Hepatocytes ; virology ; Male ; Rats ; Rats, Wistar ; Transfection ; Virus Replication
3.Occult HBV infection in patients with anti-HBc positive alone.
Xiang-yan HUANG ; Xiao-di LI ; Xiang-juan HUANG ; Qian SHEN
Chinese Journal of Experimental and Clinical Virology 2010;24(3):221-223
<b>OBJECTIVEb>This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection.
<b>METHODSb>Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive.
<b>RESULTSb>DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.
<b>CONCLUSIONSb>Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.
Base Sequence ; Blood Donors ; DNA, Viral ; analysis ; Genotype ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Antibodies ; Hepatitis B Core Antigens ; isolation & purification ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Protein Precursors ; genetics
4.Detection of Intrahepatic HBV DNA in HBsAg-negative Liver Diseases.
Yun Soo KIM ; Jae Young JANG ; Soo Hoon EUN ; Young Koog CHEON ; Young Seok KIM ; Jong Ho MOON ; Young Deok CHO ; So Young JIN ; Chan Sup SHIM ; Boo Sung KIM
The Korean Journal of Hepatology 2006;12(2):201-208
BACKGROUNDS/AIMS: Occult HBV infection is characterized by the presence of HBV infection with undetectable HBsAg. This study was carried out to find out the frequency of HBV infection in HBsAg- negative patients. METHODS: Fifty-six HBsAg-negative patients including 17 anti-HCV positive patients were evaluated. Patients were grouped according to their serological status; group A (anti-HBc+, anti-HBs-, n=16), B (anti-HBc+, anti-HBs+, n=26), and C (anti-HBc-, anti-HBs+/-, n=14). DNA was extracted from frozen liver biopsy specimen, and HBV DNA level was measured with real-time PCR. RESULTS: Overall frequency of detectable intrahepatic HBV DNA was 34% (19/56). The frequency was 56% (9/16) in group A, 31% (8/26) in group B and 14% (2/14) in group C (P=0.01). Intrahepatic HBV DNA levels were as follows; 2,010 +/- 6,660 copies/mg in group A, 6,180 +/- 29,530 copies/mg in group B and 350 +/- 1,220 copies/mg in group C. The frequency of occult HBV infection was not increased in anti-HCV positive patients. CONCLUSIONS: Intrahepatic HBV DNA is frequently detected in anti-HBc positive, HBsAg-negative patients, although the concentration is low.
Middle Aged
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Male
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Liver/*virology
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Humans
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Hepatitis B virus/*genetics/isolation & purification
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Hepatitis B Surface Antigens/*analysis
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Hepatitis B/*diagnosis/immunology/virology
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Female
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DNA, Viral/*analysis
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Aged
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Adult
5.Quantitative analyses of intrahepatic HBV cccDNA and serum HBsAg in 54 patients with chronic hepatitis B.
Wei-jie LI ; Bo-an LI ; Jing-min ZHAO ; Jia-qi HAN ; Yan LIU ; Ling JIANG ; Yuan-li MAO ; Feng-min LU ; Dong-ping XU
Chinese Journal of Hepatology 2011;19(11):815-817
<b>OBJECTIVEb>To quantitatively detect intrahepatic HBV covalently closed circular DNA (cccDNA) and serum HBsAg; and to analyze the relationship between the two parameters and with serum HBV DNA level.
<b>METHODSb>Intrahepatic cccDNA (copies/cell) was quantitated by plasmid-safe ATP-dependent Danes (PSAD) digestion in combination with rolling circle amplification and gap-spanning selective real-time PCR assay using formalin fixed paraffin-embedded liver biopsy samples. HBsAg was measured by chemiluminescence's reagent manufactured by Abbott Company using sera sampled at time-point of liver biopsy.
<b>RESULTSb>Intrahepatic cccDNA level was positively correlated with serum HBsAg level (r = 0.459, P < 0.001), but not correlated with serum HBV DNA level. Serum HBsAg level was positively correlated with serum HBV DNA level (r = 0.328, P = 0.015), and reversely correlated with HBV replicative efficiency defined as the ratio of serum HBV DNA to cccDNA (r = -0.373, P = 0.005).
<b>CONCLUSIONb>In patients with chronic hepatitis B, intrahepatic cccDNA level is correlated with serum HBsAg level. The two parameters combined with serum HBV DNA may comprehensively reflect HBV replication activity and help evaluation of antiviral therapeutic efficacy.
DNA, Circular ; analysis ; DNA, Viral ; analysis ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Liver ; virology ; Male ; Viral Load
6.Quantitative analysis of HBV DNA amplified products with microtiter hybridization.
Quan ZHANG ; Jing-juan DING ; Xiao-hui MIAO
Chinese Journal of Experimental and Clinical Virology 2003;17(1):39-41
<b>BACKGROUNDb>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).
<b>METHODSb>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.
<b>RESULTSb>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.
<b>CONCLUSIONSb>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.
DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity
7.Construction and characterization of hepatitis B surface antigen "a" epitope virus-like particles.
Si-Yong CHEN ; Min-Zhuo GUO ; Feng QIU ; Yong-Liang FEI ; Yao YI ; Yu GUO ; Zhi-Yuan JIA ; Tao YU ; Sheng-Li BI
Chinese Journal of Experimental and Clinical Virology 2010;24(1):30-32
<b>OBJECTIVEb>To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.
<b>METHODSb>Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody.
<b>RESULTb>The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA.
<b>CONCLUSIONb>The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.
Animals ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; analysis ; genetics ; immunology ; Hepatitis B Core Antigens ; analysis ; genetics ; immunology ; Hepatitis B Surface Antigens ; analysis ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; ultrastructure ; Protein Engineering ; Rabbits ; Virion ; chemistry ; genetics ; immunology ; ultrastructure
8.Gene chip analysis of mutation of HBV-DNA open reading frame.
Shi-jun CHEN ; Wen-jun DU ; Yan-qin LU ; Wei XU ; Yong AN
Chinese Journal of Experimental and Clinical Virology 2004;18(4):373-376
<b>OBJECTIVEb>To detect the mutation of HBV-DNA open reading frame (ORF), provide evidences for clinical diagnosis and treatment.
<b>METHODSb>By using gene chip technique, HBV-DNA was amplified by PCR, incorporated with fluorescence, hybridized with oligonucleotide to detect the gene sequence of DNA by computer analysis and observe the natural mutation of HBV-DNA ORF.
<b>RESULTSb>The mutations of HBV-DNA ORF existed widespread. The rates of mutation on Pre-C 1896, PreC 1814, BCP 1762, BCP 1764, P 528, P 552MI, P 552MV were 23.5%, 3.9%, 55.9%, 53.9%,39.2%, 38.2%, 10.8%, respectively.
<b>CONCLUSIONb>The gene chip technique possesses extremely high sensitivity and reliability,it is one of the effective methods to detect gene mutation. The mutation of HBV-DNA has important influence on the stability and progress of the disease, and on the judgement of prognosis.
DNA, Viral ; genetics ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; genetics ; immunology ; Humans ; Mutation ; Oligonucleotide Array Sequence Analysis ; methods ; Open Reading Frames ; genetics
9.Genotyping and mutational analysis of occult hepatitis B virus infection in blood donors of Shaoxing.
Lie-Yong SANG ; Li-Qiang FU ; Fang FANG ; Pei-Fen ZHUANG
Chinese Journal of Experimental and Clinical Virology 2011;25(6):470-473
<b>OBJECTIVEb>To assess the molecular biological characteristics of occult hepatitis B virus (HBV) infected blood donors in Shaoxing.
<b>METHODSb>8692 blood donors were screened using ELISA. The occult HBV infection was determined by DNA analysis among the HBsAg negative subjects. DNA sequencing and mutational analysis were further performed in the HBV DNA positive samples. The overall situation of occult HBV infection was hereby evaluated and the possible underlying mechanisms discussed.
<b>RESULTSb>Among the 8644 HBsAg negative subjects out of 8692 blood donors, 8 were HBV DNA positive. The occult HBV infection rate was 0.92 per thousand (8/8692). Among the 8 samples, 6 were genotype C (75%) and 2 genotype B (25%). In addition, a specific mutation in "a" epitope was observed in 7 OBI virus strains by amino acid analysis.
<b>CONCLUSIONb>There were occult HBV infected among blood donors in Shaoxing, which is probably associated with the gene mutation of HBV virus.
Adolescent ; Adult ; Blood Donors ; DNA Mutational Analysis ; Female ; Genotype ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; classification ; genetics ; Humans ; Male ; Middle Aged ; Phylogeny
10.Distribution of hepatitis B virus genotypes in Korea.
Ji Hyun CHO ; Kui Hyun YOON ; Key Earn LEE ; Do Sim PARK ; Young Jin LEE ; Hyung Bae MOON ; Kyoung R LEE ; Chang Soo CHOI ; Eun Young CHO ; Haak Cheoul KIM
The Korean Journal of Hepatology 2009;15(2):140-147
BACKGROUND/AIMS: Considering the incidence of prevailing hepatitis B virus (HBV) genotypes in neighboring nations, the predominance of genotype C in Korea is exceptional and needs to be confirmed by nationwide investigation. METHODS: A total of 510 HBsAg (+) or HBeAg (+) serum samples was collected from subjects in several cities and harbors throughout the Korean peninsula for genotype (A-G)-specific multiplex PCR analysis. Another 40 serum samples from chronic HBV carriers from Iksan city were selected for sequencing of the entire HBV genome. Phylogenetic analysis was performed with 22 whole genomic sequences of Korean HBV strains enrolled in GenBank. RESULTS: An amplicon was found in 377 specimens and genotype C occupied 98.1% (370 cases); none of the other genotypes were found. A mixed pattern of genotypes B and C was seen in seven specimens (1.9%), of which five were tested using PCR targeting the X fragment; no genotype B bands were found. With the exception of 1 case, which was subgenotype A2, whole sequences of Korean HBV strains (n=62) belonged to subgenotype C2. CONCLUSIONS: The prevailing HBV genotype in Korea is C2; the other genotypes occur only rarely. Future studies should include confirmation of the detection of genotypes other than C.
Genotype
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Hepatitis B Surface Antigens/blood
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Hepatitis B e Antigens/blood
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Hepatitis B virus/classification/*genetics
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Humans
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Korea/epidemiology
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Phylogeny
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Protein Precursors/analysis/genetics
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Sequence Analysis, DNA
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Viral Envelope Proteins/analysis/genetics