New insights into hepatitis B and C virus co-infection
Authors: Volker Brass, Darius Moradpour
Journal: Journal of Hepatology (2009)

Hepatitis B virus and hepatitis C virus interaction in Huh-7 cellsJournal of HepatologyVol. 51Issue 3PreviewCo-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increases the risk of development and the severity of chronic liver disease. Although dominant and suppressive effects of each virus over the other have been reported in vivo, in vitro studies of HBV/HCV co-infection have been limited to analysis of the effects of over-expression of HCV proteins on HBV replication. Full-Text PDF More than 500 million people worldwide are chronically infected with the hepatitis B (HBV) or hepatitis C virus (HCV). Infections with these viruses are the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). As a consequence, major research efforts have been devoted to HBV and HCV. However, relatively little is known about interactions between HBV and HCV in patients co-infected with both viruses. Co-infection with HBV and HCV is common due to shared modes of transmission (reviewed in [1Alberti A. Pontisso P. Chemello L. Fattovich G. Benvegnu L. Belussi F. et al.The interaction between hepatitis B virus and hepatitis C virus in acute and chronic liver disease.J Hepatol. 1995; 22: S38-S41PubMed Google Scholar, 2Raimondo G. Saitta C. Treatment of the hepatitis B virus and hepatitis C virus co-infection: still a challenge for the hepatologist.J Hepatol. 2008; 49: 677-679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar]). Available evidence indicates more frequent development of cirrhosis and an increased incidence of HCC in HBV/HCV co-infection as compared to monoinfection by either virus. As a result, studies have reported an excess liver-related mortality in HBV/HCV co-infection as compared to HBV or HCV infection alone [[3]Amin J. Law M.G. Bartlett M. Kaldor J.M. Dore G.J. Causes of death after diagnosis of hepatitis B or hepatitis C infection: a large community-based linkage study.Lancet. 2006; 368: 938-945Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar]. While liver disease activity and fibrosis progression are generally more severe in the presence of double infection, an inverse relationship in the replicative levels of the two viruses has been noted, suggesting direct or indirect viral interference [1Alberti A. Pontisso P. Chemello L. Fattovich G. Benvegnu L. Belussi F. et al.The interaction between hepatitis B virus and hepatitis C virus in acute and chronic liver disease.J Hepatol. 1995; 22: S38-S41PubMed Google Scholar, 2Raimondo G. Saitta C. Treatment of the hepatitis B virus and hepatitis C virus co-infection: still a challenge for the hepatologist.J Hepatol. 2008; 49: 677-679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar]. Challenging this notion, longitudinal studies revealed that the two viruses may replicate independently from each other in some patients, with fluctuations in the serum level of one virus that appear unrelated to the viremia of the other [[4]Raimondo G. Brunetto M.R. Pontisso P. Smedile A. Maina A.M. Saitta C. et al.Longitudinal evaluation reveals a complex spectrum of virological profiles in hepatitis B virus/hepatitis C virus-co-infected patients.Hepatology. 2006; 43: 100-107Crossref PubMed Scopus (188) Google Scholar]. However, suppression of HBV replication was found in patients with chronic hepatitis B when they developed acute hepatitis C and, conversely, inhibition of HCV replication has been observed in patients with chronic hepatitis C superinfected with HBV [5Liaw Y.F. Chen Y.C. Sheen I.S. Chien R.N. Yeh C.T. Chu C.M. Impact of acute hepatitis C virus superinfection in patients with chronic hepatitis B virus infection.Gastroenterology. 2004; 126: 1024-1029Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 6Sagnelli E. Coppola N. Pisaturo M. Masiello A. Tonziello G. Sagnelli C. et al.HBV superinfection in HCV chronic carriers: a disease that is frequently severe but associated with the eradication of HCV.Hepatology. 2009; 49: 1090-1097Crossref PubMed Scopus (88) Google Scholar]. Finally, HBV reactivation was observed in co-infected patients after successful treatment of HCV with pegylated interferon-α (PEG-IFN-α) and ribavirin [7Potthoff A. Wedemeyer H. Boecher W.O. Berg T. Zeuzem S. Arnold J. et al.The HEP-NET B/C co-infection trial: a prospective multicenter study to investigate the efficacy of pegylated interferon-alpha2b and ribavirin in patients with HBV/HCV co-infection.J Hepatol. 2008; 49: 688-694Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 8Liu C.J. Chuang W.L. Lee C.M. Yu M.L. Lu S.N. Wu S.S. et al.Peginterferon alfa-2a plus ribavirin for the treatment of dual chronic infection with hepatitis B and C viruses.Gastroenterology. 2008; 136: 496-504Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar]. Therefore, while liver disease appears to be enhanced, the two viruses seem to inhibit each other at the replicative level. From the basic perspective, HBV/HCV co-infection implies the exciting scenario of two pathogens with completely divergent life cycles meeting in the same host. HBV belongs to a family of hepatotropic DNA viruses (Hepadnaviridae) (reviewed in [[9]Beck J. Nassal M. Hepatitis B virus replication.World J Gastroenterol. 2007; 13: 48-64PubMed Google Scholar]). The viral genome consists of an incompletely double-stranded circular DNA of about 3.2-kbp length. The viral RNAs are transcribed in the nucleus from the covalently closed circular DNA template. Replication occurs within nucleocapsids via the reverse transcription of packaged pregenomic RNA. HCV is classified in the Hepacivirus genus within the Flaviviridae family. It contains a 9.6-kb positive-strand RNA genome that encodes a polyprotein precursor of about 3000 amino acids (reviewed in [[10]Moradpour D. Penin F. Rice C.M. Replication of hepatitis C virus.Nat Rev Microbiol. 2007; 5: 453-463Crossref PubMed Scopus (1127) Google Scholar]). The polyprotein precursor is co- and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. HCV replication takes place in a cytoplasmic membrane-associated complex (membranous web). Interactions between HBV and HCV have been difficult to study because of the lack of appropriate model systems. Indeed, robust infection of cultured cells by HBV remains a challenge, with only few cell types, including HepaRG and primary human or tupaia hepatocytes at disposition [11Gripon P. Dict C. Guguen-Guillouzo C. Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration.Virology. 1993; 192: 534-540Crossref PubMed Scopus (139) Google Scholar, 12Köck J. Nassal M. MacNelly S. Baumert T.F. Blum H.E. von Weizsäcker F. Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus.J Virol. 2001; 75: 5084-5089Crossref PubMed Scopus (127) Google Scholar, 13Gripon P. Rumin S. Urban S. Le Seyec J. Glaise D. Cannie I. et al.Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci USA. 2002; 99: 15655-15660Crossref PubMed Scopus (990) Google Scholar]. However, HepaRG cells do not appear to be susceptible to efficient HCV infection and replication (George Koutsoudakis and Ralf Bartenschlager, personal communication) and primary human as well as tupaia cells support only low level HCV replication [14Castet V. Fournier C. Soulier A. Brillet R. Coste J. Larrey D. et al.Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro.J Virol. 2002; 76: 8189-8199Crossref PubMed Scopus (111) Google Scholar, 15Zhao X. Tang Z.Y. Klumpp B. Wolff-Vorbeck G. Barth H. Levy S. et al.Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection.J Clin Invest. 2002; 109: 221-232PubMed Google Scholar]. Therefore, the few studies that have addressed interactions between HBV and HCV were based on heterologous overexpression of viral proteins and have yielded conflicting results. For example, some studies demonstrated that HCV core inhibits HBV replication [16Schüttler C.G. Fiedler N. Schmidt K. Repp R. Gerlich W.H. Schaefer S. Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein.J Hepatol. 2002; 37: 855-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 17Chen S.Y. Kao C.F. Chen C.M. Shih C.M. Hsu M.J. Chao C.H. et al.Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein.J Biol Chem. 2003; 278: 591-607Crossref PubMed Scopus (132) Google Scholar] while others did not [[18]Pasquinelli C. Shoenberger J.M. Chung J. Chang K.-M. Guidotti L.C. Selby M. et al.Hepatitis C virus core and E2 protein expression in transgenic mice.Hepatology. 1997; 25: 719-727Crossref PubMed Scopus (123) Google Scholar]. Similarly, HCV NS5A was found to enhance [[19]Pan Y. Wei W. Kang L. Wang Z. Fang J. Zhu Y. et al.NS5A protein of HCV enhances HBV replication and resistance to interferon response.Biochem Biophys Res Commun. 2007; 359: 70-75Crossref PubMed Scopus (29) Google Scholar] or inhibit [[20]Guo H. Zhou T. Jiang D. Cuconati A. Xiao G.H. Block T.M. et al.Regulation of hepatitis B virus replication by the phosphatidylinositol 3-kinase-akt signal transduction pathway.J Virol. 2007; 81: 10072-10080Crossref PubMed Scopus (120) Google Scholar] HBV replication. Thus, it remains unclear whether there is a direct interference between HBV and HCV. Against this background, the study by Eyre et al. published in this issue of the Journal provides important new insights into HBV/HCV co-infection [[21]Eyre N.S. Phillips R.J. Bowden S. Yip E. Dewar B. Locarnini S.A. et al.Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells.J Hepatol. 2009; 51: 446-457Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar]. The authors used Huh-7 human HCC cells to study interactions between HBV and HCV. Huh-7 cells support HBV replication and virion formation as well as the entire HCV life cycle, including viral entry, RNA replication, and infectious particle release [22Chang C.M. Jeng K.-S. Hu C.-P. Lo S. Su T.-S. Ting L.-P. et al.Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.EMBO J. 1987; 6: 675-680PubMed Google Scholar, 23Lindenbach B.D. Evans M.J. Syder A.J. Wölk B. Tellinghuisen T.L. Liu C.C. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1980) Google Scholar, 24Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2450) Google Scholar, 25Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. et al.Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1529) Google Scholar]. As these cells cannot be infected by HBV, the authors used a recombinant adenovirus vector (AdHBV) to intracellularly deliver a replication-competent HBV genome. A recombinant adenovirus expressing the green fluorescent protein (AdGFP) served as control for nonspecific effects. Huh-7 cells were also infected with cell culture-derived HCV (HCVcc) [23Lindenbach B.D. Evans M.J. Syder A.J. Wölk B. Tellinghuisen T.L. Liu C.C. et al.Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1980) Google Scholar, 24Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2450) Google Scholar, 25Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. et al.Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1529) Google Scholar]. Importantly, HCV replication and virion production were not affected when cells were co-infected simultaneously with AdHBV and HCVcc or when HCVcc-infected cells were subsequently exposed to the recombinant adenovirus. For unknown reasons, HCV RNA replication was even increased when the cells were preinfected with AdHBV. On the other hand, HBV DNA levels in cells and supernatants were either unaffected or slightly increased by HCVcc infection. Thus, in this replicating context, there appeared to be no or only a minor influence of HBV on HCV and vice versa. The key findings of this study are in line with a recent study by Bellecave et al. [[26]Bellecave P. Gouttenoire J. Gajer M. Brass V. Koutsoudakis G. Blum H.E. et al.Hepatitis B and C virus co-infection: a novel model system reveals the absence of direct viral interference.Hepatology. 2009; 50: 46-55Crossref PubMed Scopus (133) Google Scholar]. In this study, Huh-7 cells were engineered to inducibly replicate and produce infectious HBV using a tetracycline-regulated gene expression system. HCV was introduced into these stable cell lines as well as into control Huh-7 cells inducibly expressing the GFP either in the form of selectable HCV replicons or HCVcc. In this highly reproducible model system, HBV and HCV were found to replicate in the same cell without overt interference. In addition, specific inhibition of one virus did not affect the replication and gene expression of the other. Finally, cells harboring replicating HBV could be infected by HCVcc, arguing against superinfection exclusion, and they also supported efficient production of infectious HCV. Taken together, the studies by Eyre et al. and Bellecave et al. reveal the absence of direct interference between HBV and HCV in vitro. This remarkable observation indicates that – at least in Huh-7 cells – the two viruses rely on distinct and non-competing sets of host factors for their replication. At the same time, these results suggest that the viral interference observed in co-infected patients is likely due to indirect mechanisms mediated by innate and/or adaptive host immune responses. In this context, both HBV and HCV replication are susceptible to antiviral cytokines, including type I and type II IFNs as well as tumor necrosis factor-α, that are produced by the infected cell or infiltrating T-cells [27Guidotti L.G. Chisari F.V. Noncytolytic control of viral infections by the innate and adaptive immune response.Annu Rev Immunol. 2001; 19: 65-91Crossref PubMed Scopus (869) Google Scholar, 28Jo J. Aichele U. Kersting N. Klein R. Aichele P. Bisse E. et al.Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model.Gastroenterology. 2009; 136: 1391-1401Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. An alternative explanation for the viral interference observed in vivo may relate to host factors that become limiting in human hepatocytes in the liver but not in Huh-7 cells in vitro. Clearly, the development of in vitro and in vivo model systems that will allow to study the effect of innate and adaptive responses in HBV/HCV co-infection as well as the establishment of experimental conditions that more closely resemble human hepatocytes or the naturally infected liver represents the next challenge. In conclusion, the study by Eyre et al. appearing in this issue of the Journal [[21]Eyre N.S. Phillips R.J. Bowden S. Yip E. Dewar B. Locarnini S.A. et al.Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells.J Hepatol. 2009; 51: 446-457Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar] and the study by Bellecave et al. published earlier this year [[26]Bellecave P. Gouttenoire J. Gajer M. Brass V. Koutsoudakis G. Blum H.E. et al.Hepatitis B and C virus co-infection: a novel model system reveals the absence of direct viral interference.Hepatology. 2009; 50: 46-55Crossref PubMed Scopus (133) Google Scholar] demonstrate that HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in co-infected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV/HCV co-infection and may contribute to its clinical management in the future. Research in the authors' laboratories is supported by the Swiss National Science Foundation (3100A0-122447), the Swiss Cancer League/Oncosuisse (OCS-01762-08-2005), the Leenaards Foundation, and the Deutsche Forschungsgemeinschaft (Br 3440/2-1).