title: Hepatitis B virus infection and replication in a new cell culture system established by fusing HepG2 cells with primary human hepatocytes.
abstract: BACKGROUND
  Hepatitis B virus (HBV) infection is strictly species and tissue specific, therefore none of the cell models established previously can reproduce the natural infection process of HBV in vitro. The aim of this study was to establish a new cell line that is susceptible to HBV and can support the replication of HBV.
  
  
  METHODS
  A hybrid cell line was established by fusing primary human hepatocytes with HepG2 cells. The hybrid cells were incubated with HBV-positive serum for 12 hours. HBV DNA was detected by quantitative fluorescence polymerase chain reaction (QF-PCR). HBsAg (surface antigen) and HBeAg (extracellular form of core antigen) were observed by electrochemiluminescence (ECL). HBcAg (core antigen) was detected by the indirect immunofluorescence technique. HBV covalently closed circular DNA (cccDNA) was analyzed by Southern blot hybridization and quantified using real-time PCR.
  
  
  RESULTS
  A new cell line was established and named HepCHLine-7. The extracellular HBV DNA was observed from Day 2 and the levels ranged from 9.80 (± 0.32) × 10(2) copies/mL to 3.12 (± 0.03) × 10(4) copies/mL. Intracellular HBV DNA was detected at Day 2 after infection and the levels ranged from 7.92 (± 1.08) × 10(3) copies/mL to 5.63 (± 0.11) × 10(5) copies/mL. HBsAg in the culture medium was detected from Day 4 to Day 20. HBeAg secretion was positive from Day 5 to Day 20. HBcAg constantly showed positive signals in approximately 20% (± 0.82%) of hybrid cells. Intracellular HBV cccDNA could be detected as early as 2 days postinfection and the highest level was 15.76 (± 0.26) copies/cell.
  
  
  CONCLUSIONS
  HepCHLine-7 cells were susceptible to HBV and supported the replication of HBV. They are therefore suitable for studying the complete life cycle of HBV.
authors.affiliation: Department of Infectious Diseases, Qilu Hospital, Shandong University, Wenhua Xi Road 107, Ji'nan 250012, Shandong Province, China.
authors.forename: Lin-Tao
authors.initials: LT
authors.lastname: Sai
authors.affiliation: Department of Infectious Diseases, People's Hospital of Rizhao, Tai'an Road 126, Rizhao 276800, Shandong Province, China.
authors.forename: Yong-Yuan
authors.initials: YY
authors.lastname: Yao
authors.affiliation: Department of Infectious Diseases, People's Hospital of Rizhao, Tai'an Road 126, Rizhao 276800, Shandong Province, China.
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Guan
authors.affiliation: Department of Laboratory Sciences, School of Public Health, Shandong University, Wenhua Xi Road 44, Ji'nan 250012, Shandong Province, China.
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Shao
authors.affiliation: Department of Digestive Diseases, Qianfoshan Hospital, Jingshi Road 16766, Ji'nan 250012, Shandong Province, China.
authors.forename: Rui-Ping
authors.initials: RP
authors.lastname: Ma
authors.affiliation: Department of Infectious Diseases, Qilu Hospital, Shandong University, Wenhua Xi Road 107, Ji'nan 250012, Shandong Province, China. Electronic address: Malixian1211@gmail.com.
authors.forename: Li-Xian
authors.initials: LX
authors.lastname: Ma
mesh_terms: D018929:Cell Culture Techniques; D002459:Cell Fusion / Q000379:methods*; D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D056945:Hep G2 Cells; D006512:Hepatitis B Core Antigens / Q000032:analysis; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000254:growth & development*; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology / Q000821:virology*
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens
keywords: Hepatitis B virus; Hybrid cells; Infection; Replication