Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA.
OBJECTIVE
To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).


METHODS
Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19 x 10(12) copies of linear HBV DNA/1 x 10(7) PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.


RESULTS
HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive > or =2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.


CONCLUSIONS
Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.
Yao
Yun-Qing
YQ
Department of Infectious Diseases of the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China. cqyaoyunqing@126.com
Zhang
Ding-Feng
DF
Tang
Ni
N
Huang
Ai-Long
AL
Zou
Xiao-Yi
XY
Xiao
Jiang-Feng
JF
Luo
Yun
Y
Zhang
Da-Zhi
DZ
Wang
Bo
B
Zhou
Wei-Ping
WP
Ren
Hong
H
Liu
Qi
Q
Guo
Shu-Hua
SH
D000818:Animals; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D004372:Ducks; D018347:Hepadnaviridae Infections / Q000821:virology*; D015611:Hepatitis B Virus, Duck / Q000235:genetics* / Q000254:growth & development*; D006524:Hepatitis, Viral, Animal / Q000821:virology*; D022781:Hepatocytes / Q000166:cytology / Q000821:virology*; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral