title: High level virion production and surface antigen expression with 1.5 copies of hepatitis B viral genome.
abstract: The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2kb genome, a 5'-end redundant sequence of 1.1kb to nt715 and a 3'-end redundant sequence of 500bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24h and 48h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log(10) values of viral loads were also determined with the cell lysate having a higher value (10-11 per ml) than that of the supernatant (6-7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro. The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.
authors.affiliation: Stanley Ho Centre for Emerging Infectious Diseases, School of Public Health, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Qingming
authors.initials: Q
authors.lastname: Dong
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: Yangchao
authors.initials: Y
authors.lastname: Chen
authors.forename: Chu-Yan
authors.initials: CY
authors.lastname: Chan
authors.forename: Marie C
authors.initials: MC
authors.lastname: Lin
authors.forename: Hsiang-fu
authors.initials: HF
authors.lastname: Kung
authors.forename: Henry L Y
authors.initials: HL
authors.lastname: Chan
authors.forename: Joseph J Y
authors.initials: JJ
authors.lastname: Sung
authors.forename: Ming-Liang
authors.initials: ML
authors.lastname: He
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000821:virology; D002353:Carrier State / Q000821:virology; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis*; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis*; D006513:Hepatitis B e Antigens / Q000096:biosynthesis; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification / Q000502:physiology; D019694:Hepatitis B, Chronic / Q000821:virology; D006801:Humans; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens