Gene expression response to HBV infection in PHH

Hepatitis B virus (HBV) is known for its ability to interact with the host cell DNA methylation machinery. In HBV-infected hepatocytes, this interaction leads to chronic liver diseases, including hepatocellular carcinoma (HCC). We studied the extent of genomic changes induced by natural HBV infection in human primary hepatocytes. Transcriptome and methylome profiles were obtained at different time points post-infection to identify HBV-specific alterations. Although gene expression and DNA methylation do not directly correlate, they both seem to reflect the effect of cell culture and viral infection at different levels.These changes in the hepatocyte cellular program shed light on the initial events leading to HBV-associated liver diseases. Primary human hepatocytes (PHH) were prepared from adult patients undergoing lobectomy or segmental liver resection. HBV was concentrated from the supernatant of HepG2.2.15 cells using centrifugal filter devices and tittered by HBV-DNA dot blot analysis after sedimentation into a CsCl density-gradient to determine enveloped DNA-containing viral particles. PHH were isolated from surgical liver resections, cultured, and infected with HBV. Infected PHH and corresponding controls were kept for 4 hours, 8 hours, and 1, 6, and 12 days. Supernatants were obtained to validate the efficiency of infection by ELISA and qRT-PCR, and nucleic acids were extracted for expression and DNA methylation analyses.