Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models

Published:August 04, 2021DOI:


      • Stable cell lines producing high-titer cell culture-generated HBV of various genotypes were established.
      • HBV genotypes showed stable infectivity in both in vitro and in vivo models.
      • HBV genotypes exhibit different infectivity, antigen expression, replication and responses to treatment.
      • These model systems are valuable tools for antiviral development.

      Background & Aims

      HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment.


      Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model.


      HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock.


      Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models.

      Lay summary

      Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.

      Graphical abstract


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        • McNaughton A.L.
        • D'Arienzo V.
        • Ansari M.A.
        • Lumley S.F.
        • Littlejohn M.
        • Revill P.
        • et al.
        Insights from deep sequencing of the HBV genome-unique, tiny, and misunderstood.
        Gastroenterology. 2019; 156: 384-399
        • Revill P.A.
        • Chisari F.V.
        • Block J.M.
        • Dandri M.
        • Gehring A.J.
        • Guo H.
        • et al.
        A global scientific strategy to cure hepatitis B.
        Lancet Gastroenterol Hepatol. 2019; 4: 545-558
        • Loomba R.
        • Liang T.J.
        Hepatitis B reactivation associated with immune suppressive and biological modifier therapies: current concepts, management strategies, and future directions.
        Gastroenterology. 2017; 152: 1297-1309
        • Schweitzer A.
        • Horn J.
        • Mikolajczyk R.T.
        • Krause G.
        • Ott J.J.
        Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013.
        Lancet. 2015; 386: 1546-1555
        • Matthews P.C.
        • Geretti A.M.
        • Goulder P.J.
        • Klenerman P.
        Epidemiology and impact of HIV coinfection with hepatitis B and hepatitis C viruses in Sub-Saharan Africa.
        J Clin Virol. 2014; 61: 20-33
        • Spearman C.W.
        • Afihene M.
        • Ally R.
        • Apica B.
        • Awuku Y.
        • Cunha L.
        • et al.
        Hepatitis B in sub-Saharan Africa: strategies to achieve the 2030 elimination targets.
        Lancet Gastroenterol Hepatol. 2017; 2: 900-909
        • Zhang H.
        • Pan C.Q.
        • Pang Q.
        • Tian R.
        • Yan M.
        • Liu X.
        Telbivudine or lamivudine use in late pregnancy safely reduces perinatal transmission of hepatitis B virus in real-life practice.
        Hepatology. 2014;
        • Zou H.
        • Chen Y.
        • Duan Z.
        • Zhang H.
        • Pan C.
        Virologic factors associated with failure to passive-active immunoprophylaxis in infants born to HBsAg-positive mothers.
        J viral Hepat. 2012; 19: e18-e25
        • Xia Y.
        • Liang T.J.
        Development of direct-acting antiviral and host-targeting agents for treatment of hepatitis B virus infection.
        Gastroenterology. 2019; 156: 311-324
        • Akinyemiju T.
        • Abera S.
        • Ahmed M.
        • Alam N.
        • Alemayohu M.A.
        • Allen C.
        • et al.
        • Global Burden of Disease Liver Cancer C
        The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: results from the global burden of disease study 2015.
        JAMA Oncol. 2017; 3: 1683-1691
        • Bertuccio P.
        • Turati F.
        • Carioli G.
        • Rodriguez T.
        • La Vecchia C.
        • Malvezzi M.
        • et al.
        Global trends and predictions in hepatocellular carcinoma mortality.
        J Hepatol. 2017; 67: 302-309
        • Cornberg M.
        • Lok A.S.
        • Terrault N.A.
        • Zoulim F.
        • Faculty E-AHTEC
        Guidance for design and endpoints of clinical trials in chronic hepatitis B - Report from the 2019 EASL-AASLD HBV Treatment Endpoints Conference.
        Hepatology. 2019;
        • Rajoriya N.
        • Combet C.
        • Zoulim F.
        • Janssen H.L.A.
        How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach?.
        J Hepatol. 2017; 67: 1281-1297
        • Kramvis A.
        Genotypes and genetic variability of hepatitis B virus.
        Intervirology. 2014; 57: 141-150
        • Thomas E.
        • Liang T.J.
        Experimental models of hepatitis B and C - new insights and progress.
        Nat Rev Gastroenterol Hepatol. 2016; 13: 362-374
        • Sozzi V.
        • Walsh R.
        • Littlejohn M.
        • Colledge D.
        • Jackson K.
        • Warner N.
        • et al.
        In vitro studies show that sequence variability contributes to marked variation in hepatitis B virus replication, protein expression, and function observed across genotypes.
        J Virol. 2016; 90: 10054-10064
        • Sells M.A.
        • Zelent A.Z.
        • Shvartsman M.
        • Acs G.
        Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.
        J Virol. 1988; 62: 2836-2844
        • Shen F.
        • Li Y.
        • Wang Y.
        • Sozzi V.
        • Revill P.A.
        • Liu J.
        • et al.
        Hepatitis B virus sensitivity to interferon-alpha in hepatocytes is more associated with cellular interferon response than with viral genotype.
        Hepatology. 2018; 67: 1237-1252
        • Revill P.A.
        • Tu T.
        • Netter H.J.
        • Yuen L.K.W.
        • Locarnini S.A.
        • Littlejohn M.
        The evolution and clinical impact of hepatitis B virus genome diversity.
        Nat Rev Gastroenterol Hepatol. 2020; 17: 618-634
        • Buster E.H.
        • Hansen B.E.
        • Lau G.K.
        • Piratvisuth T.
        • Zeuzem S.
        • Steyerberg E.W.
        • et al.
        Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa.
        Gastroenterology. 2009; 137: 2002-2009
        • Wong G.L.
        • Chan H.L.
        • Yiu K.K.
        • Lai J.W.
        • Chan V.K.
        • Cheung K.K.
        • et al.
        Meta-analysis: the association of hepatitis B virus genotypes and hepatocellular carcinoma.
        Aliment Pharmacol Ther. 2013; 37: 517-526
        • Ching L.K.
        • Gounder P.P.
        • Bulkow L.
        • Spradling P.R.
        • Bruce M.G.
        • Negus S.
        • et al.
        Incidence of hepatocellular carcinoma according to hepatitis B virus genotype in Alaska Native people.
        Liver Int. 2016; 36: 1507-1515
        • Janssen H.L.
        • van Zonneveld M.
        • Senturk H.
        • Zeuzem S.
        • Akarca U.S.
        • Cakaloglu Y.
        • et al.
        Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.
        Lancet. 2005; 365: 123-129
        • Wai C.T.
        • Fontana R.J.
        • Polson J.
        • Hussain M.
        • Shakil A.O.
        • Han S.H.
        • et al.
        Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States.
        J viral Hepat. 2005; 12: 192-198
        • Galibert F.
        • Mandart E.
        • Fitoussi F.
        • Tiollais P.
        • Charnay P.
        Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli.
        Nature. 1979; 281: 646-650
        • Will H.
        • Cattaneo R.
        • Koch H.G.
        • Darai G.
        • Schaller H.
        • Schellekens H.
        • et al.
        Cloned HBV DNA causes hepatitis in chimpanzees.
        Nature. 1982; 299: 740-742
        • Sozzi V.
        • Shen F.
        • Chen J.
        • Colledge D.
        • Jackson K.
        • Locarnini S.
        • et al.
        In vitro studies identify a low replication phenotype for hepatitis B virus genotype H generally associated with occult HBV and less severe liver disease.
        Virology. 2018; 519: 190-196
        • Chu C.J.
        • Hussain M.
        • Lok A.S.
        Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C.
        Gastroenterology. 2002; 122: 1756-1762
        • Terrault N.A.
        • Lok A.S.F.
        • McMahon B.J.
        • Chang K.M.
        • Hwang J.P.
        • Jonas M.M.
        • et al.
        Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
        Hepatology. 2018; 67: 1560-1599
        • Sonneveld M.J.
        • Rijckborst V.
        • Cakaloglu Y.
        • Simon K.
        • Heathcote E.J.
        • Tabak F.
        • et al.
        Durable hepatitis B surface antigen decline in hepatitis B e antigen-positive chronic hepatitis B patients treated with pegylated interferon-alpha2b: relation to response and HBV genotype.
        Antivir Ther. 2012; 17: 9-17
        • Micco L.
        • Peppa D.
        • Loggi E.
        • Schurich A.
        • Jefferson L.
        • Cursaro C.
        • et al.
        Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B.
        J Hepatol. 2013; 58: 225-233
        • ter Borg M.J.
        • Hansen B.E.
        • Herrmann E.
        • Zeuzem S.
        • Cakaloglu Y.
        • Karayalcin S.
        • et al.
        Modelling of early viral kinetics and pegylated interferon-alpha2b pharmacokinetics in patients with HBeag-positive chronic hepatitis B.
        Antivir Ther. 2007; 12: 1285-1294
        • Rehermann B.
        • Thimme R.
        Insights from antiviral therapy into immune responses to hepatitis B and C virus infection.
        Gastroenterology. 2019; 156: 369-383
        • Wiegand J.
        • Hasenclever D.
        • Tillmann H.L.
        Should treatment of hepatitis B depend on hepatitis B virus genotypes? A hypothesis generated from an explorative analysis of published evidence.
        Antivir Ther. 2008; 13: 211-220
        • Bock C.T.
        • Malek N.P.
        • Tillmann H.L.
        • Manns M.P.
        • Trautwein C.
        The enhancer I core region contributes to the replication level of hepatitis B virus in vivo and in vitro.
        J Virol. 2000; 74: 2193-2202
        • Torres C.
        • Pineiro Y.
        • Leone F.G.
        • Pezzano S.C.
        • Mbayed V.A.
        • Campos R.H.
        New perspectives on the evolutionary history of hepatitis B virus genotype F.
        Mol Phylogenet Evol. 2011; 59: 114-122
        • Arauz-Ruiz P.
        • Norder H.
        • Robertson B.H.
        • Magnius L.O.
        Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America.
        J Gen Virol. 2002; 83: 2059-2073