Cell Culture Models and Animal Models for HBV Study

  • Feng Li
  • Zhuo Wang
  • Fengyu Hu
  • Lishan SuEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1179)


Highly representative and relevant cell and mouse models are required for HBV study, including uncovering its lifecycle, investigation of the viral-host interaction, and development and evaluation of the novel antiviral therapy. During the past 40 years, both HBV cell culture models and animal models have evolved over several generations, each with significant improvement for specific purposes. In one aspect, HBV cell culture models experienced the original noninfection model including HBV plasmid DNA transfection and HBV genome integrated stable cells such as HepG2.2.15 which constitutively produces HBV virus and HepAD38 cells and its derivatives which drug-regulated HBV production. As for HBV infection models, HepaRG cells once dominated the HBV infection field for over a decade, but its complicated and labor-extensive cell differentiation procedures discouraged primary researchers from stepping in the field. The identification of human NTCP as HBV receptor evoked great enthusiasm of the whole HBV field, and its readily adaptive characteristic makes it popular in many HBV laboratories. Recombinant cccDNA (rc-cccDNA) emerged recently aiming to tackle the very basic question of how to eventually eradicate cccDNA without HBV real virus infection. In the other aspect, HBV transgenic mouse was firstly generated in the 1990s, which was helpful to decipher HBV production in vivo. However, the HBV transgenic mice were naturally immune tolerant to HBV viral products. Subsequently, a series of nonintegrated HBV mouse models were generated through plasmid hydrodynamic tail vein injection and viral vector-mediated delivery approaches, and HBV full life cycle was incomplete as cccDNA was not formed from HBV relaxed circular DNA (rcDNA). Human NTCP transgenic mouse still could not support productive HBV infection, and humanized mouse liver with human hepatocytes which supported whole HBV life cycle still dominates HBV infection in vivo, a value but expensive model until now. Other methods to empower mouse to carry HBV cccDNA were also exploited. In this chapter, we summarized the advantages and disadvantages of each model historically and provided protocols for HBV infection in HepG2-NTCP cells, HBV rc-cccDNA transfection in HepG2 cells, and HBV infection in NRG-Fah−/− liver humanized mouse.


  1. 1.
    Di Bisceglie AM (2009) Hepatitis B and hepatocellular carcinoma. Hepatology 49(5 Suppl):S56–S60PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    World Health Organization (2017) Global hepatitis report, 2017. World Health Organization, GenevaGoogle Scholar
  3. 3.
    Hutin Y, Nasrullah M, Easterbrook P, Nguimfack BD, Burrone E, Averhoff F et al (2018) Access to treatment for hepatitis B virus infection - worldwide, 2016. MMWR Morb Mortal Wkly Rep 67(28):773–777PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Nassal M (2015) HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 64(12):1972–1984PubMedCrossRefGoogle Scholar
  5. 5.
    Bukh J (2016) The history of hepatitis C virus (HCV): basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control. J Hepatol 65(1 Suppl):S2–S21PubMedCrossRefGoogle Scholar
  6. 6.
    Pradat P, Virlogeux V, Trepo E (2018) Epidemiology and elimination of HCV-related liver disease. Viruses 10(10):545PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309(5734):623–626PubMedCrossRefGoogle Scholar
  8. 8.
    Yi M, Villanueva RA, Thomas DL, Wakita T, Lemon SM (2006) Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 103(7):2310–2315PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR et al (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102(26):9294–9299PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Billich A (2001) Entecavir (Bristol-Myers Squibb). Curr Opin Investig Drugs 2(5):617–621PubMedGoogle Scholar
  11. 11.
    Mulato AS, Cherrington JM (1997) ADV and TDF anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: in vitro analyses. Antivir Res 36(2):91–97PubMedCrossRefGoogle Scholar
  12. 12.
    Rooke R, Parniak MA, Tremblay M, Soudeyns H, Li XG, Gao Q et al (1991) 3TC biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs. Antimicrob Agents Chemother 35(5):988–991PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Tsai CH, Lee PY, Stollar V, Li ML (2006) Antiviral therapy targeting viral polymerase. Curr Pharm Des 12(11):1339–1355PubMedCrossRefGoogle Scholar
  14. 14.
    Boyd A, Lacombe K, Lavocat F, Maylin S, Miailhes P, Lascoux-Combe C et al (2016) Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients. J Hepatol 65(4):683–691PubMedCrossRefGoogle Scholar
  15. 15.
    Dandri M, Petersen J (2016) Mechanism of hepatitis B virus persistence in hepatocytes and its carcinogenic potential. Clini Infect Dis 62(Suppl 4):S281–S288CrossRefGoogle Scholar
  16. 16.
    Sells MA, Zelent AZ, Shvartsman M, Acs G (1988) Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J Virol 62(8):2836–2844PubMedPubMedCentralGoogle Scholar
  17. 17.
    Kock J, Rosler C, Zhang JJ, Blum HE, Nassal M, Thoma C (2010) Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog 6(9):e1001082PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Sells MA, Chen ML, Acs G (1987) Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84(4):1005–1009PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Hu J, Lin YY, Chen PJ, Watashi K, Wakita T (2019) Cell and animal models for studying hepatitis B virus infection and drug development. Gastroenterology 156(2):338–354PubMedCrossRefGoogle Scholar
  20. 20.
    Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT et al (1997) Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41(8):1715–1720PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Cai D, Mills C, Yu W, Yan R, Aldrich CE, Saputelli JR et al (2012) Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation. Antimicrob Agents Chemother 56(8):4277–4288PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Cai D, Wang X, Yan R, Mao R, Liu Y, Ji C et al (2016) Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators. Antivir Res 132:26–37PubMedCrossRefGoogle Scholar
  23. 23.
    Gripon P, Diot C, Theze N, Fourel I, Loreal O, Brechot C et al (1988) Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 62(11):4136–4143PubMedPubMedCentralGoogle Scholar
  24. 24.
    Walter E, Keist R, Niederost B, Pult I, Blum HE (1996) Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 24(1):1–5PubMedPubMedCentralGoogle Scholar
  25. 25.
    Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I et al (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99(24):15655–15660PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Hantz O, Parent R, Durantel D, Gripon P, Guguen-Guillouzo C, Zoulim F (2009) Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J Gen Virol 90(Pt 1):127–135PubMedCrossRefGoogle Scholar
  27. 27.
    Schulze A, Mills K, Weiss TS, Urban S (2012) Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 55(2):373–383PubMedCrossRefGoogle Scholar
  28. 28.
    Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z et al (2012) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife 1:e00049PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Iwamoto M, Watashi K, Tsukuda S, Aly HH, Fukasawa M, Fujimoto A et al (2014) Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun 443(3):808–813PubMedCrossRefGoogle Scholar
  30. 30.
    Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M et al (2014) Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 146(4):1070–1083PubMedCrossRefGoogle Scholar
  31. 31.
    Guo X, Chen P, Hou X, Xu W, Wang D, Wang TY et al (2016) The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely. Sci Rep 6:25552PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Li F, Cheng L, Murphy CM, Reszka-Blanco NJ, Wu Y, Chi L et al (2016) Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs. Sci Rep 6:36483PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Yan Z, Zeng J, Yu Y, Xiang K, Hu H, Zhou X et al (2017) HBV circle: a novel tool to investigate hepatitis B virus covalently closed circular DNA. J Hepatol 66(6):1149–1157PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Qi Z, Li G, Hu H, Yang C, Zhang X, Leng Q et al (2014) Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice. J Virol 88(14):8045–8056PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Kay MA, He CY, Chen ZY (2010) A robust system for production of minicircle DNA vectors. Nat Biotechnol 28(12):1287–1289PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV (1999) Viral clearance without destruction of infected cells during acute HBV infection. Science 284(5415):825–829PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Dupinay T, Gheit T, Roques P, Cova L, Chevallier-Queyron P, Tasahsu SI et al (2013) Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island. Hepatology 58(5):1610–1620PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Kock J, Nassal M, MacNelly S, Baumert TF, Blum HE, von Weizsacker F (2001) Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus. J Virol 75(11):5084–5089PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Yan RQ, Su JJ, Huang DR, Gan YC, Yang C, Huang GH (1996) Human hepatitis B virus and hepatocellular carcinoma. I. Experimental infection of tree shrews with hepatitis B virus. J Cancer Res Clin Oncol 122(5):283–288PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Kulkarni K, Jacobson IM, Tennant BC (2007) The role of the woodchuck model in the treatment of hepatitis B virus infection. Clin Liver Dis 11(4):707–25, viiPubMedCrossRefGoogle Scholar
  41. 41.
    D'Ugo E, Argentini C, Giuseppetti R, Canitano A, Catone S, Rapicetta M (2010) The woodchuck hepatitis B virus infection model for the evaluation of HBV therapies and vaccine therapies. Expert Opin Drug Discovery 5(12):1153–1162CrossRefGoogle Scholar
  42. 42.
    Sprengel R, Kuhn C, Will H, Schaller H (1985) Comparative sequence analysis of duck and human hepatitis B virus genomes. J Med Virol 15(4):323–333PubMedCrossRefGoogle Scholar
  43. 43.
    Reaiche GY, Le Mire MF, Mason WS, Jilbert AR (2010) The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis. Virology 406(2):286–292PubMedCrossRefGoogle Scholar
  44. 44.
    Yang Q, Zhao X, Zang L, Fang X, Zhao J, Yang X et al (2012) Anti-hepatitis B virus activities of alpha-DDB-FNC, a novel nucleoside-biphenyldicarboxylate compound in cells and ducks, and its anti-immunological liver injury effect in mice. Antivir Res 96(3):333–339PubMedCrossRefGoogle Scholar
  45. 45.
    Chisari FV (1995) Hepatitis B virus transgenic mice: insights into the virus and the disease. Hepatology 22(4 Pt 1):1316–1325PubMedGoogle Scholar
  46. 46.
    Uprichard SL, Boyd B, Althage A, Chisari FV (2005) Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. Proc Natl Acad Sci U S A 102(3):773–778PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Xie HY, Cheng J, Xing CY, Wang JJ, Su R, Wei XY et al (2011) Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx. Hepatobiliary Pancreat Dis Int 10(3):295–302PubMedCrossRefGoogle Scholar
  48. 48.
    Yang PL, Althage A, Chung J, Chisari FV (2002) Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci U S A 99(21):13825–13830PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Huang LR, Wu HL, Chen PJ, Chen DS (2006) An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A 103(47):17862–17867PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Suzuki T, Takehara T, Ohkawa K, Ishida H, Jinushi M, Miyagi T et al (2003) Intravenous injection of naked plasmid DNA encoding hepatitis B virus (HBV) produces HBV and induces humoral immune response in mice. Biochem Biophys Res Commun 300(3):784–788PubMedCrossRefGoogle Scholar
  51. 51.
    Peng XH, Ren XN, Chen LX, Shi BS, Xu CH, Fang Z et al (2015) High persistence rate of hepatitis B virus in a hydrodynamic injection-based transfection model in C3H/HeN mice. World J Gastroenterol: WJG 21(12):3527–3536PubMedCrossRefGoogle Scholar
  52. 52.
    Chen SH, Wu HL, Kao JH, Hwang LH (2012) Persistent hepatitis B viral replication in a FVB/N mouse model: impact of host and viral factors. PLoS One 7(5):e36984PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Huang LR, Gabel YA, Graf S, Arzberger S, Kurts C, Heikenwalder M et al (2012) Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice. Gastroenterology 142(7):1447–50 e3PubMedCrossRefGoogle Scholar
  54. 54.
    Lucifora J, Vincent IE, Berthillon P, Dupinay T, Michelet M, Protzer U et al (2010) Hepatitis B virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model. Hepatology 51(6):1954–1960PubMedCrossRefGoogle Scholar
  55. 55.
    Dion S, Bourgine M, Godon O, Levillayer F, Michel ML (2013) Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules. J Virol 87(10):5554–5563PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Yang D, Liu L, Zhu D, Peng H, Su L, Fu YX et al (2014) A mouse model for HBV immunotolerance and immunotherapy. Cell Mol Immunol 11(1):71–78PubMedCrossRefGoogle Scholar
  57. 57.
    Lucifora J, Salvetti A, Marniquet X, Mailly L, Testoni B, Fusil F et al (2017) Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector. Antivir Res 145:14–19PubMedCrossRefGoogle Scholar
  58. 58.
    Li G, Zhu Y, Shao D, Chang H, Zhang X, Zhou D et al (2018) Recombinant covalently closed circular DNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model. Hepatology 67(1):56–70PubMedCrossRefGoogle Scholar
  59. 59.
    Li H, Zhuang Q, Wang Y, Zhang T, Zhao J, Zhang Y et al (2014) HBV life cycle is restricted in mouse hepatocytes expressing human NTCP. Cell Mol Immunol 11(2):175–183PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Winer BY, Shirvani-Dastgerdi E, Bram Y, Sellau J, Low BE, Johnson H et al (2018) Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection. Sci Transl Med 10(447):pii: eaap9328CrossRefGoogle Scholar
  61. 61.
    Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A et al (2001) Hepatitis C virus replication in mice with chimeric human livers. Nat Med 7(8):927–933PubMedCrossRefGoogle Scholar
  62. 62.
    Dandri M, Burda MR, Torok E, Pollok JM, Iwanska A, Sommer G et al (2001) Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology 33(4):981–988PubMedCrossRefGoogle Scholar
  63. 63.
    Petersen J, Dandri M, Mier W, Lutgehetmann M, Volz T, von Weizsacker F et al (2008) Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat Biotechnol 26(3):335–341PubMedCrossRefGoogle Scholar
  64. 64.
    Lutgehetmann M, Mancke LV, Volz T, Helbig M, Allweiss L, Bornscheuer T et al (2012) Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation. Hepatology 55(3):685–694PubMedCrossRefGoogle Scholar
  65. 65.
    Vanwolleghem T, Libbrecht L, Hansen BE, Desombere I, Roskams T, Meuleman P et al (2010) Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice. J Hepatol 53(3):468–476PubMedCrossRefGoogle Scholar
  66. 66.
    Azuma H, Paulk N, Ranade A, Dorrell C, Al-Dhalimy M, Ellis E et al (2007) Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice. Nat Biotechnol 25(8):903–910PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Bissig KD, Wieland SF, Tran P, Isogawa M, Le TT, Chisari FV et al (2010) Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest 120(3):924–930PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    Takenaka K, Prasolava TK, Wang JC, Mortin-Toth SM, Khalouei S, Gan OI et al (2007) Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat Immunol 8(12):1313–1323PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Li F, Cowley DO, Banner D, Holle E, Zhang L, Su L (2014) Efficient genetic manipulation of the NOD-Rag1−/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology. Sci Rep 4:5290PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Hasegawa M, Kawai K, Mitsui T, Taniguchi K, Monnai M, Wakui M et al (2011) The reconstituted 'humanized liver' in TK-NOG mice is mature and functional. Biochem Biophys Res Commun 405(3):405–410PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Kosaka K, Hiraga N, Imamura M, Yoshimi S, Murakami E, Nakahara T et al (2013) A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections. Biochem Biophys Res Commun 441(1):230–235PubMedCrossRefGoogle Scholar
  72. 72.
    Nakabori T, Hikita H, Murai K, Nozaki Y, Kai Y, Makino Y et al (2016) Sodium taurocholate cotransporting polypeptide inhibition efficiently blocks hepatitis B virus spread in mice with a humanized liver. Sci Rep 6:27782PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2020

Authors and Affiliations

  1. 1.Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, School of MedicineThe University of North Carolina at Chapel HillChapel HillUSA
  2. 2.Guangzhou Eighth People’s Hospital, Guangzhou Medical UniversityGuangzhouChina

Personalised recommendations