Hepatitis B Virus In The Original Generation Of Liver Cell Co-culture Model On The Enhancement Of Infection

Approximately two billion people have been infected with human Hepatitis B virus (HBV) worldwide with over240million people are chronically infected. Among them about600,000die of HBV-related liver diseases each year. Available treatments are inadequate or non-effective to those who are chronically infected and at high risk of cirrhosis and hepatocellular carcinoma. More than50%of liver cancers worldwide are attributable to HBV and over90%in Asia.For a long period of time, primary hepatocytes, including those from humans, chimpanzees or tree shrews are the only cell types that can support complete life cycle of HBV. However, these cells precipitously decline in viability and remain susceptible to infection for only a few days following their preparation from liver tissue. Moreover, the infection efficiency in these models usually is low, only5-10%of primary hepatocytes could be infected in vitro with regular infection dosage. In addition to the primary hepatocytes, a hepatoma cell line, HepaRG, is the only cell line that is susceptible to HBV infection. However, its susceptibility is subjected to a month long process with induction of corticoids and DMSO.In this study, we used primary hepatocytes of treeshrew as a model and examined if co-culturing hepatocytes with other nonparenchymal cells can improve human HBV infection of PTHs. We also investigated molecular mechanisms that are involved in the enhanced infection, and whether the recently identified receptor NTCP plays a role.We demonstrated the infection of both HBV and WMHBV on PTHs co-cultured with hepatic stellates cells (HSCs) from tupaia or primary tupaia fibroblasts (PTFs) was significantly enhanced. The enhancement of HBV infection in co-cultivation is direct intercellular contact dependent. Co-cultured hepatocytes exhibited enhanced hepatic functions and physiological characteristics as indicated by activity of cytochrome P450enzymes, expression patterns of multidrug resistance protein2(MRP2) and zonula occludens protein1(ZO-1). Moreover, the HBV susceptibility of PTH was found to be prolonged effectively through co-cultivation.The enhanced infection of HBV was partially attributable to the up-regulated hepatic transcription factors in the co-cultured hepatocytes, whereas the expression level of the newly identified cellular receptor sodium taurocholate cotransporting polypeptide (NTCP) for HBV was not significantly changed. Nonetheless, NTCP remains the receptor mediating viral entry into the co-cultured hepatoctyes. Together, experimental HBV infection on primary hepatocytes co-cultured with supportive cells sheds new light on HBV entry and provides a useful model to study contributing factors for the efficiency of HBV infection in vitro.