A New Cell Culture System For Hepatitis B Virus Infection

BackgroundHepatitis B virus(HBV) is a member of the Hepadnavirus family.HBV causes transient and chronic infections of the liver.Chronic infection with HBV is a leading cause of severe liver diseases,such as cirrhosis and hepatocellular carcinoma.More than 2 billion people alive today have been infected by HBV,and more than 350 million people are chronically infected.Patients chronically infected with HBV run the risk of developing cirrhosis and hepatocellular carcinoma in later life.Worldwide deaths from chronic liver diseases and hepatocellular carcinoma caused by HBV infection approximately 1 million each year.In China,more than 50%of the population has been infected by HBV,and 8%to 15%of them become chronically infected.Since HBV surface antigen was found by Blumberg in 1965 to the complete cloning and sequencing of HBV DNA genome in 1997,our knowledge on genetic constitution,antigenic structure,biology feature and life cycle of HBV have been riched.But Hepadnavirus infection is of strictly host specific and tissue specific, which hampers the establishment of suitable cell model for studying HBV infection.Currently,there are two main cell types used for the study of HBV in vitro, primary human hepatocytes and hepatoma-derived cell lines.Primary human hepatocytes for infection studies have the advantage that they retain important cellular characteristics of differentiated hepatocytes allowing natural penetration,and the full replication of HBV.However,difficulties arise with this system because within several days in culture the cells begin to dedifferentiate and lose the capability to support HBV infection and replication.In addition,these cells must be obtained from a fresh liver,and the response of viral uptake varies greatly according to condition of harvested cells.Because of the difficulties with primary cell cultures,HBV research has relied upon the development of well-differentiated human hepatoma cell lines transfected with cloned HBV DNA.One of these cell lines,HepG2.2.15,is stably transfected with the complete HBV genome,expressing HBV RNA,viral proteins, and secreting virus-like particles.The virus secreted by HepG2.2.15 has demonstrated infectivity in chimpanzees.Many important details of HBV replication and genomic organization have been discovered through the use of transfected hepatoma cell lines. However,because the genome is introduced into cells by transfection rather than infection,these cell lines cannot be used to study HBV-host cell infection processes such as viral attachment,penetration,and uncoating.Due to deficiency of an in vitro system for productive viral infection and replication,little has been known about the mechanisms of the early stages in HBV infection.Thereby,basic and clinical research on hepatitis B and chronic liver diseases correlated with HBV infection were hampered to some extent.Here,we have attempted to establish an in vitro infection cell culture system,which is susceptible to HBV infection and suitable for replication ensued.It could be a useful tool for investigating the mechanisms of the early stages in HBV infection,and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.SectionⅠEstablishing a Hybrid Cell Line HepCHLine-4【Objective】To establish a hybrid cell line susceptible for HBV infection by fused HepG2 cells with primary human hepatocytes. 【Methods】1 To establish a hybrid cell line by fused HepG2 cells with primary human hepatocytes.1.1 HepG2 cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT),were induced by EMS.1.2 Isolation and culture of human primary hepatocytes.Hepatocytes viability was defined as the ability of cells to exclude trypan blue dyeing.1.3 HGPRT^- HepG2 cells were fused with human primary hepatocytes.After being selected with HAT medium and limiting dilution assayed,the hybrid cell was monocloned.1.4 The modal chromosome numbers in hybrid cell was counted by the trypsin G-banding method.2 Since the primary hepatocytes we used for cell fusion was come from a patient chronically infected with HBV,it is necessary to analyze whether HepCHLine-4 contains HBV genome prior to HBV infection experiment.2.1 We analyzed intracellular HBV DNA and HBV DNA in cultivate supernants. HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.2.2 We analyzed intracellular HBV cccDNA by nested PCR after HepCHLine-4 cell genome digested with mung bean nuclease.2.3 We detected whether HepCHLine-4 cells expressed HBV specific antigens. HBcAg in HepCHLine-4 cell was analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants were identified by electrochemiluminescence.【Results】1 We establish a hybrid cell line successfully by fused HepG2 cells with primary human hepatocytes.1.1 HepG2 cells deficient in HGPRT(HGPRT^- HepG2) were induced by EMS. HGPRT^- HepG2 cells were resistant to 6-MP but could not grow in HAT medium, indicating a complete defect of inosinic acid pyrophosphorylase(IPP).1.2 Human primary hepatocytes viability was defined≧ 90%.1.3 After cell fusion and being selected with HAT medium and limiting dilution assayed,we established a bybrid cell line,named HepCHLine-4.HepCHLine-4 was similar to HGPRT^- HepG2 cells morphologically,and can be subcultured in vitro.HepCHLine-4 cell line can be resuscitated after being freezed.1.4 The Karyotype analysis results showed that the modal chromosome numbers of wild-type HepG2,HGPRT^- HepG2 and HepCHLine-4 were 51,53 and 99, respectively,indicating that the HepCHLine-4 was a hybrid cell line between HGPRT^- HepG2 cells(53 chromosomes) and human hepatocytes(46 chromosomes),and contained all genomic factors from both human hepatocytes and HepG2 cells.2 The hybrid cell line doesn't contain HBV genome prior to HBV infection experiment.2.1 HBV DNA could not be detected in HepCHLine-4 cells or in the cultivate supernants.2.2 HBV cccDNA,the replicative intermediate of HBV,could not be detected in HepCHLine-4 cells.2.3 HepCHLine-4 cells could not express HBV specific antigens prior to HBV infection.HBcAg could not be detected in HepCHLine-4 cells.HBsAg and HBeAg could not be detected in HepCHLine-4 cultivate supernants,either.【Conclusion】1 We establish a new hybrid cell line HepCHLine-4 successfully.HepCHLine-4 inherits the characteristics of both HepG2 and human primary hepatocyte,and can be used for establishing an in vitro HBV infection cell model.2 The hybrid cell line HepCHLine-4 did not contain HBV genome prior to HBV infection experiment,so it can be a useful tool to study the process of HBV infection. SectionⅡInvestigate the Susceptibility of Hybrid Cell Line HepCHLine-4 to Serum-derived HBV Virions【Objective】In order to investigate the possibility of hybrid cell line HepCHLine-4 served as an in vitro HBV infection cell culture system,HepCHLine-4 cells were co-incubated with serum-derived HBV virions,and the susceptibility of HepCHLine-4 to HBV infection was evaluated.【Methods】1.Co-incubated HepCHLine-4 cells and HepG2 cells with serum-derived HBV virions respectively,here HepG2 cells were set as control.2.We analyzed intracellular HBV DNA in infected cells and HBV DNA in cultivate supernants of infected cells.HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.3.We analyzed intracellular HBV cccDNA in infected cells by nested PCR after cell genome digested with mung bean nuclease.4.We detected the capacity of HepCHLine-4 cells expressed HBV specific antigens post infection,HBcAg in infected cells were analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants of infected cells were identified by electrochemiluminescence.5.Examination of HBV-specific particles that infected HepCHLine-4 cells released into the supernants by electron microscopy.【Results】1.HBV DNA was detected both in HepCHLine-4 cells and in the culture media consistently between days 6 and 15 post infection,whereas could not be detected in infected HepG2 cell or its culture media.2.HBV cccDNA was detected consistently between days 5 and 10 p.i.of HepCHLine-4 cells,whereas could not be detected in infected HepG2 cells. 3.(1) The core antigen was detected throughout the cytoplasm of HepCHLine-4 cells between days 1 to 30 p.i.,and in the nucleus of HepCHLine-4 cells post infection between days 2 to 30 p.i.;whereas could not be detected within HBV-infected HepG2 cells.(2) HepCHLine-4 cells consistently secreted HBsAg between days 6 and 15 p.i.,and HBsAg in culture media was from 3.850 to 89.590 IU/mL(≥1 was considered positive).HepCHLine-4 cells consistently secreted HBeAg between days 8 and 17 p.i.,and HBeAg in culture media was from 1.000 to 1.690 S/Co(≥1 was considered positive).HBsAg and HBeAg could not be detected in culture media of infected HepG2 cells.4.Culture media of the 6^th day post infection were collected and subjected to ultracentrifugation.The pelleted viral particles were directly examined by electron microscopy after negative staining with 2%phosphotungstic acid.22-nm-diameter spherical and 22-nm-long filamentous forms HBsAg particles as well as few 42-nm-diameter double-shelled Dane-like particles were detected.However,no HBV-associated particles were found when similar samples were examined from culture media of the control,HepG2 cells.【Conclusion】The results indicated that the hybrid cell line HepCHLine-4 was susceptible to serum-derived HBV virions.HepCHLine-4 cells post infection were found to be able to initiate viral DNA replication and the production of replicative intermediate and HBV-specific protein.Furthermore,HBV-specific viral particles were produced and released into culture media.Till now,HepCHLine-4 cell has experienced more than one year,and the cell strain can be resuscitated after being freezed.HepCHLine-4 was morphologically unchanged after the 50th subculture,and maintained its susceptibility to HBV.【In conclusion】All these results combined indicate that the hybrid cell line HepCHLine-4 inherits the high susceptibility to HBV from human hepatocytes;on the other hand,it inherits the immortality of HepG2 cells,overcoming the deficiency of primary human hepatocytes as a HBV infection system.HepCHLine-4 cell line is susceptible to HBV and can initiate viral replication,produce HBV-specific antigens and HBV-specific progeny virions after HBV infection.In conclusion,HepCHLine-4 cell line is a suitable cell model for studying the early events of HBV infection and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.