Objective: By studying the replication of Hepatitis B virus (HBV) compared with that of Duck Hepatitis B virus (DHBV) in primary duck hepatocytes (PDH), we want to explore the host-specific regulating roles on the replication of HBV in hepatocytes from heterologous species.
Methods: PDH were transfected with complete HBV genome by electroporation (transfected group, 1.19 x 10(12) copies of linear HBV DNA/1 x 10(7) PDH) or infected with DHBV (infected group, 4 x 10(8) virions/1 x 10(7) PDH). 1, 3 and 5 days after transfection or infection, HBsAg, HBeAg and DHBsAg in the supernatant and lysate of PDH were measured with IMX System or ELISA. Meanwhile, replicative intermediates of HBV DNA and DHBV DNA were analyzed by Southern blotting and dot blotting. PDH electroporated only was used as control group.
Results: HBsAg in the lysates of transfected group were 15.24 (1 day), 14.55 (3 days) and 5.13 (5 days; P/N values, positive > or = 2.1), HBeAg all was negative (< 2.1), and both were negative in the supernatants of transfected group. DHBsAg in the supernatants of infected group were 14.6 (1 day), 31.53 (3 days) and 34.73(5 days; S/N values, positive > or = 2.1). Dot blotting revealed that both the total amount of HBV DNA in the transfected group and DHBV DNA in the infected group were strongly positive, whereas that of the control group was negative. Southern blot analysis of intracellular total DNA indicated that there are relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome, as the same as that of DHBV DNA in the infected group. Control groups were negative at all.
Conclusion: Our results demonstrate that expression of HBV genes and production can occur in hepatocytes from nonmammalian species and strongly support the idea that HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors could be essential for viral replication.