Transfection of a Hepatoma Cell Line by Cloned HBV DNA and Evaluation of Transcription and Expression of Viral-specific Markers and Interferon Stimulated Genes

Authors
H. Mirshahabi 1
M. Karimi 2
F. Behzadian 3
F. Sabahi 1
1 Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Biotechnology Research Center, Pasture Institute of Iran, Tehran, Iran
3 Department of Molecular Genetics, Research Center for Science and Biotechnology, Malek Ashtar University, Tehran, Iran
Abstract
Objective: Despite availability of an effective vaccine against hepatitis B virus (HBV), the global prevalence of this virus infection has not diminished significantly. Contrary to numerous other human viruses, HBV does not have the ability to propagate in cell culture. However, infectious virus has been produced by transfection of human hepatoma cells with plasmids that contain full length HBV genome. Generation and optimization of appropriate cell culture systems can help us in demonstrating the quality of genome replication by PCR as well as expression of surface antigen secretion. Interferon stimulating genes (ISGs) are usually produced in response to interferon and can be determined as a measure of response to IFN-therapy. Therefore, in pharmacological studies, in addition to assessing the effects of a medicine on viral determinants of replication, its’ effects on stimulation of various ISGs, as indicators of innate immune responses, can be achieved.
Methods: In this study, we transfected the Huh-7 hepatoma cell line with pCH-9/3091. HBsAg production and viral mRNA transcription were subsequently evaluated. In this system, by using ISGs-specific primers, the ISG mRNAs recognition method was optimized and utilized.
Results: Huh-7 cells supported HBV replication. The peak HBsAg secretion was observed at 72 h post-transfection. By using designed primers for the S and pg/pC regions, transcription and genome replication of the virus was shown. RT-PCR results for ISG production by transfected cells showed no role for HBV in enhancement of ISGs levels in Huh-7 cells.
Conclusion: The results indicated that this system can be used for functional studies of HBV-specific genes as well as assessment of the effects of new drugs or new vaccines. In addition, it may be used to study the mechanisms of drug resistance that have resulted in difficulties in response to HBV antivirals, including IFN-α.

Keywords

 
 
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Pathobiology Reserach
Volume 16, Issue 3
December 2013
Pages 65-79