Objective To study the correlation of replication efficiency and antigen expression of hepatitis B virus (HBV) strains in patient sera and transfected cells. Methods Quantification of five maternal serum HBV DNA was carried out using dot hybridization and polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA), hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in the sera were determined by ELISA. Full-length genomes of HBV strains cloned from five pregnant women were separately used to transfect HepG2 cells. The levels of HBsAg and HBeAg in the supernatant of transfected cells were determined with Abbott EIA kits, the replication efficiency of intracelluar replicative intermediates and extracellular HBV DNA of viral particles was analyzed by Southern blot and hybridization. Results Intracellular and extracellular HBV replication and antigen expression of cloned HBV strains showed positive correlation tendency with the HBV DNA and antigen levels in serum samples. Conclusions Virus replication efficiency and expression of HBsAg and HBeAg by full-length HBV clones are in relative coincidence with the biological characteristics of HBV strains in patients. Cell transfection can be used to study the biological characteristics of individual isolates.

BACKGROUNDTo elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy.

METHODSSera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken.

RESULTSPretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain.

CONCLUSIONSLow HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.

Amino Acid Sequence ; Antiviral Agents ; therapeutic use ; Hepacivirus ; drug effects ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; RNA, Viral ; blood ; Viral Nonstructural Proteins ; genetics