The evidence does not support a viral etiology for primary biliary cirrhosis
Authors: Carlo Selmi
Journal: Journal of Hepatology (2010)

The hypothesis that a human endogenous retrovirus (HERV) has a causative role in primary biliary cirrhosis (PBC) has been a recurring theme in the scientific literature on this complex autoimmune disease for over 12 years. With his group, Dr. Andrew Mason has repeatedly reported findings in support of this fascinating proposal – which is presented elsewhere in this issue of the Journal. In a nutshell, epidemiological and experimental evidence incriminate environmental factors in the development of PBC, also supported by the proposed geoepidemiology gradient and the numerous risk factors including recurrent urinary tract infections [[1]Selmi C. Gershwin M.E. The role of environmental factors in primary biliary cirrhosis.Trends Immunol. 2009; 30: 415-420Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar].First, the Mason group reported a high prevalence of anti-retroviral antibodies in the serum of PBC patients [[2]Mason A.L. Xu L. Guo L. Munoz S. Jaspan J.B. Bryer-Ash M. et al.Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders.Lancet. 1998; 351: 1620-1624Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Second, electron microscopy images were presented in support of the presence of viral particles in the lymph nodes of patients with PBC [[3]Xu L. Shen Z. Guo L. Fodera B. Keogh A. Joplin R. et al.Does a betaretrovirus infection trigger primary biliary cirrhosis?.Proc Natl Acad Sci USA. 2003; 100: 8454-8459Crossref PubMed Scopus (201) Google Scholar]. Third, the co-culture of healthy cholangiocytes with periportal lymph nodes from patients with PBC led to the expression of AMA-antigens on the cholangiocyte membrane [[4]Sadamoto T. Joplin R. Keogh A. Mason A. Carman W. Neuberger J. Expression of pyruvate–dehydrogenase complex PDC-E2 on biliary epithelial cells induced by lymph nodes from primary biliary cirrhosis.Lancet. 1998; 352: 1595-1596Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], thus mirroring what had been reported previously in the liver of patients with PBC liver [[5]Tsuneyama K. Van De Water J. Van Thiel D. Coppel R. Ruebner B. Nakanuma Y. et al.Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis.Hepatology. 1995; 22: 1440-1446PubMed Google Scholar] and subsequently defined as the result of immune complex deposition [[6]Fukushima N. Nalbandian G. Van De Water J. White K. Ansari A.A. Leung P. et al.Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC.Hepatology. 2002; 36: 1383-1392PubMed Google Scholar]. A virus was then cloned in the DNA obtained from lymph nodes of two patients with PBC, the sequencing of which revealed a human endogenous retrovirus sharing significant homology with the mouse mammary tumor virus (MMTV) [[7]Xu L. Sakalian M. Shen Z. Loss G. Neuberger J. Mason A. Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis.Hepatology. 2004; 39: 151-156Crossref PubMed Scopus (92) Google Scholar]. Finally, the authors identified retroviral sequences in one of the recently described murine models of PBC as well as the integration of the virus in PBC cholangiocytes, although these latter data were not published in extenso.Based on these observations, the Mason group initiated a multicenter clinical trial of antiretroviral treatment in patients with PBC, but this failed to reach the non histological endpoints commonly utilized in clinical trials in PBC, but instead utilized variables such as upper abdominal pain, that is rarely observed in clinical practice and has not been well validated as an end point in studies in PBC [[8]Mason A.L. Lindor K.D. Bacon B.R. Vincent C. Neuberger J.M. Wasilenko S.T. Clinical Trial: Randomized controlled trial of zidovudine and lamivudine for patients with primary biliary cirrhosis stabilized on ursodiol.Aliment Pharmacol Ther. 2008; 28: 886-894Crossref PubMed Scopus (50) Google Scholar]. In the same trial, the inclusion of upper abdominal pain is a concern, since it tends to be associated with other biliary disorders such as primary sclerosing cholangitis.Two independent groups have investigated a role for retroviruses in PBC with significantly contradictory results. First in 2004, our own group was unable to detect the presence of MMTV using multiple molecular and tissue markers in fresh liver or peripheral blood cells from a large number patients with PBC [[9]Selmi C. Ross S.R. Ansari A.A. Invernizzi P. Podda M. Coppel R.L. et al.Lack of immunological or molecular evidence for a role of mouse mammary tumor retrovirus in primary biliary cirrhosis.Gastroenterology. 2004; 127: 493-501Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar]. Subsequently, as published in this journal in 2009, the presence of MMTV sequences was demonstrated in healthy liver tissues and in a variable proportion of samples from patients with chronic liver disease with a variety of aetiologies, including primary liver cancer and PBC [[10]Johal H. Scott G.M. Jones R. Camaris C. Riordan S. Rawlinson W.D. Mouse mammary tumour virus-like virus (MMTV-LV) is present within the liver in a wide range of hepatic disorders and unrelated to nuclear p53 expression or hepatocarcinogenesis.J Hepatol. 2009; 50: 548-554Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar]. Albeit in contrast, these studies did include a large number of clinical samples thus increasing the power of the comparisons.HERV result from ancestral infections and there are sequences within the human genome resembling infectious retroviruses. Following retrovirus integration into the genome, evolutionary processes disabled their potential damaging effects through mutations that prevent viral protein expression and virulence [[5]Tsuneyama K. Van De Water J. Van Thiel D. Coppel R. Ruebner B. Nakanuma Y. et al.Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis.Hepatology. 1995; 22: 1440-1446PubMed Google Scholar]. The genetic sequences of infectious retroviruses encode several proteins that, in order to facilitate viral replication, stimulate the cells in which they are expressed and enable the virus to evade an immune response. Similar proteins can be encoded by HERV, a property that confers the potential to cause disease [[11]Moyes D. Griffiths D.J. Venables P.J. Insertional polymorphisms: a new lease of life for endogenous retroviruses in human disease.Trends Genet. 2007; 23: 326-333Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. Briefly, HERV may induce pathology by: (i) disrupting genes at their integration site; (ii) suppressing or stimulating the immune response; or (iii) expressing accessory proteins that have direct effects on the cells in which they are expressed. The main hurdle to this scenario is that the integration sites of the vast majority of HERV are identical in different individuals and it becomes increasingly difficult to propose that a given HERV can cause a disease in one individual and not another, as we would expect a strikingly high concordance for the clinical phenotype among individuals. Similarly, one would also expect that HERV might increase the neoplastic potential of chronic inflammation, as exemplified by occult hepatitis B infection or MMTV itself. However, the incidence rates of hepatocellular carcinoma and breast cancer in PBC are similar to other etiologies of liver cirrhosis [[12]Cavazza A. Caballeria L. Floreani A. Farinati F. Bruguera M. Caroli D. et al.Incidence, risk factors, and survival of hepatocellular carcinoma in primary biliary cirrhosis: comparative analysis from two centers.Hepatology. 2009; 50: 1162-1168Crossref PubMed Scopus (82) Google Scholar] and to control women [[13]Gershwin M.E. Selmi C. Worman H.J. Gold E.B. Watnik M. Utts J. et al.Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview-based study of 1032 patients.Hepatology. 2005; 42: 1194-1202Crossref PubMed Scopus (454) Google Scholar], respectively.These observations raise major concerns for the proposed evidence. Firstly, the virus sequence should be detected in all somatic cells and not only (or mostly) in lymph nodes [[7]Xu L. Sakalian M. Shen Z. Loss G. Neuberger J. Mason A. Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis.Hepatology. 2004; 39: 151-156Crossref PubMed Scopus (92) Google Scholar]. Second, if we consider monozygotic twins, we should expect a full concordance of viral integration and consequently, disease prevalence, which is not the case for PBC [[14]Selmi C. Mayo M.J. Bach N. Ishibashi H. Invernizzi P. Gish R.G. et al.Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment.Gastroenterology. 2004; 127: 485-492Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar]. One possible explanation could be a mechanism of gene silencing present in the healthy twin, but we recently completed a genome-wide DNA methylation analysis and found no evidence to support this hypothesis as we could not identify consistent differences in DNA methylation at viral insertion sites (unpublished data). Third, HERV are expected to cause human diseases via the development of genetic polymorphisms, essentially single-nucleotide (SNP) or sequence insertions [[15]Balada E. Ordi-Ros J. Vilardell-Tarres M. Molecular mechanisms mediated by human endogenous retroviruses (HERVs) in autoimmunity.Rev Med Virol. 2009; 19: 273-286Crossref PubMed Scopus (83) Google Scholar] but no HERV gene was reported by the recent GWAS performed in large series of PBC cases and controls [[16]Liu X. Invernizzi P. Lu Y. Kosoy R. Bianchi I. Podda M. et al.Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis.Nat Genet. 2010; 42: 658-660Crossref PubMed Scopus (326) Google Scholar]. Last and possibly most importantly, the proposed evidence was obtained in a very small number of cases; the need for specimens that are not readily available (i.e. lymph nodes) and the complexity of the experimental approach (as in the case of viral integration) do not appear sufficient to overcome this major limitation.In conclusion, the available evidence that has been proposed over the past decade falls well short of supporting a viral etiology for PBC. The small numbers of cases included in all published studies, as well as the conflicting results generated by different groups, raise major concerns for this hypothesis. This hypothesis may be another in the long series of retroviral associations proposed for autoimmune diseases [[17]Nelson P.N. Carnegie P.R. Martin J. Davari Ejtehadi H. Hooley P. Roden D. et al.Demystified. Human endogenous retroviruses.Mol Pathol. 2003; 56: 11-18Crossref PubMed Scopus (135) Google Scholar] that have failed to be confirmed independently or that have any practical implication.Conflict of interestThe author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The hypothesis that a human endogenous retrovirus (HERV) has a causative role in primary biliary cirrhosis (PBC) has been a recurring theme in the scientific literature on this complex autoimmune disease for over 12 years. With his group, Dr. Andrew Mason has repeatedly reported findings in support of this fascinating proposal – which is presented elsewhere in this issue of the Journal. In a nutshell, epidemiological and experimental evidence incriminate environmental factors in the development of PBC, also supported by the proposed geoepidemiology gradient and the numerous risk factors including recurrent urinary tract infections [[1]Selmi C. Gershwin M.E. The role of environmental factors in primary biliary cirrhosis.Trends Immunol. 2009; 30: 415-420Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar]. First, the Mason group reported a high prevalence of anti-retroviral antibodies in the serum of PBC patients [[2]Mason A.L. Xu L. Guo L. Munoz S. Jaspan J.B. Bryer-Ash M. et al.Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders.Lancet. 1998; 351: 1620-1624Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Second, electron microscopy images were presented in support of the presence of viral particles in the lymph nodes of patients with PBC [[3]Xu L. Shen Z. Guo L. Fodera B. Keogh A. Joplin R. et al.Does a betaretrovirus infection trigger primary biliary cirrhosis?.Proc Natl Acad Sci USA. 2003; 100: 8454-8459Crossref PubMed Scopus (201) Google Scholar]. Third, the co-culture of healthy cholangiocytes with periportal lymph nodes from patients with PBC led to the expression of AMA-antigens on the cholangiocyte membrane [[4]Sadamoto T. Joplin R. Keogh A. Mason A. Carman W. Neuberger J. Expression of pyruvate–dehydrogenase complex PDC-E2 on biliary epithelial cells induced by lymph nodes from primary biliary cirrhosis.Lancet. 1998; 352: 1595-1596Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], thus mirroring what had been reported previously in the liver of patients with PBC liver [[5]Tsuneyama K. Van De Water J. Van Thiel D. Coppel R. Ruebner B. Nakanuma Y. et al.Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis.Hepatology. 1995; 22: 1440-1446PubMed Google Scholar] and subsequently defined as the result of immune complex deposition [[6]Fukushima N. Nalbandian G. Van De Water J. White K. Ansari A.A. Leung P. et al.Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC.Hepatology. 2002; 36: 1383-1392PubMed Google Scholar]. A virus was then cloned in the DNA obtained from lymph nodes of two patients with PBC, the sequencing of which revealed a human endogenous retrovirus sharing significant homology with the mouse mammary tumor virus (MMTV) [[7]Xu L. Sakalian M. Shen Z. Loss G. Neuberger J. Mason A. Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis.Hepatology. 2004; 39: 151-156Crossref PubMed Scopus (92) Google Scholar]. Finally, the authors identified retroviral sequences in one of the recently described murine models of PBC as well as the integration of the virus in PBC cholangiocytes, although these latter data were not published in extenso. Based on these observations, the Mason group initiated a multicenter clinical trial of antiretroviral treatment in patients with PBC, but this failed to reach the non histological endpoints commonly utilized in clinical trials in PBC, but instead utilized variables such as upper abdominal pain, that is rarely observed in clinical practice and has not been well validated as an end point in studies in PBC [[8]Mason A.L. Lindor K.D. Bacon B.R. Vincent C. Neuberger J.M. Wasilenko S.T. Clinical Trial: Randomized controlled trial of zidovudine and lamivudine for patients with primary biliary cirrhosis stabilized on ursodiol.Aliment Pharmacol Ther. 2008; 28: 886-894Crossref PubMed Scopus (50) Google Scholar]. In the same trial, the inclusion of upper abdominal pain is a concern, since it tends to be associated with other biliary disorders such as primary sclerosing cholangitis. Two independent groups have investigated a role for retroviruses in PBC with significantly contradictory results. First in 2004, our own group was unable to detect the presence of MMTV using multiple molecular and tissue markers in fresh liver or peripheral blood cells from a large number patients with PBC [[9]Selmi C. Ross S.R. Ansari A.A. Invernizzi P. Podda M. Coppel R.L. et al.Lack of immunological or molecular evidence for a role of mouse mammary tumor retrovirus in primary biliary cirrhosis.Gastroenterology. 2004; 127: 493-501Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar]. Subsequently, as published in this journal in 2009, the presence of MMTV sequences was demonstrated in healthy liver tissues and in a variable proportion of samples from patients with chronic liver disease with a variety of aetiologies, including primary liver cancer and PBC [[10]Johal H. Scott G.M. Jones R. Camaris C. Riordan S. Rawlinson W.D. Mouse mammary tumour virus-like virus (MMTV-LV) is present within the liver in a wide range of hepatic disorders and unrelated to nuclear p53 expression or hepatocarcinogenesis.J Hepatol. 2009; 50: 548-554Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar]. Albeit in contrast, these studies did include a large number of clinical samples thus increasing the power of the comparisons. HERV result from ancestral infections and there are sequences within the human genome resembling infectious retroviruses. Following retrovirus integration into the genome, evolutionary processes disabled their potential damaging effects through mutations that prevent viral protein expression and virulence [[5]Tsuneyama K. Van De Water J. Van Thiel D. Coppel R. Ruebner B. Nakanuma Y. et al.Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis.Hepatology. 1995; 22: 1440-1446PubMed Google Scholar]. The genetic sequences of infectious retroviruses encode several proteins that, in order to facilitate viral replication, stimulate the cells in which they are expressed and enable the virus to evade an immune response. Similar proteins can be encoded by HERV, a property that confers the potential to cause disease [[11]Moyes D. Griffiths D.J. Venables P.J. Insertional polymorphisms: a new lease of life for endogenous retroviruses in human disease.Trends Genet. 2007; 23: 326-333Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. Briefly, HERV may induce pathology by: (i) disrupting genes at their integration site; (ii) suppressing or stimulating the immune response; or (iii) expressing accessory proteins that have direct effects on the cells in which they are expressed. The main hurdle to this scenario is that the integration sites of the vast majority of HERV are identical in different individuals and it becomes increasingly difficult to propose that a given HERV can cause a disease in one individual and not another, as we would expect a strikingly high concordance for the clinical phenotype among individuals. Similarly, one would also expect that HERV might increase the neoplastic potential of chronic inflammation, as exemplified by occult hepatitis B infection or MMTV itself. However, the incidence rates of hepatocellular carcinoma and breast cancer in PBC are similar to other etiologies of liver cirrhosis [[12]Cavazza A. Caballeria L. Floreani A. Farinati F. Bruguera M. Caroli D. et al.Incidence, risk factors, and survival of hepatocellular carcinoma in primary biliary cirrhosis: comparative analysis from two centers.Hepatology. 2009; 50: 1162-1168Crossref PubMed Scopus (82) Google Scholar] and to control women [[13]Gershwin M.E. Selmi C. Worman H.J. Gold E.B. Watnik M. Utts J. et al.Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview-based study of 1032 patients.Hepatology. 2005; 42: 1194-1202Crossref PubMed Scopus (454) Google Scholar], respectively. These observations raise major concerns for the proposed evidence. Firstly, the virus sequence should be detected in all somatic cells and not only (or mostly) in lymph nodes [[7]Xu L. Sakalian M. Shen Z. Loss G. Neuberger J. Mason A. Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis.Hepatology. 2004; 39: 151-156Crossref PubMed Scopus (92) Google Scholar]. Second, if we consider monozygotic twins, we should expect a full concordance of viral integration and consequently, disease prevalence, which is not the case for PBC [[14]Selmi C. Mayo M.J. Bach N. Ishibashi H. Invernizzi P. Gish R.G. et al.Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment.Gastroenterology. 2004; 127: 485-492Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar]. One possible explanation could be a mechanism of gene silencing present in the healthy twin, but we recently completed a genome-wide DNA methylation analysis and found no evidence to support this hypothesis as we could not identify consistent differences in DNA methylation at viral insertion sites (unpublished data). Third, HERV are expected to cause human diseases via the development of genetic polymorphisms, essentially single-nucleotide (SNP) or sequence insertions [[15]Balada E. Ordi-Ros J. Vilardell-Tarres M. Molecular mechanisms mediated by human endogenous retroviruses (HERVs) in autoimmunity.Rev Med Virol. 2009; 19: 273-286Crossref PubMed Scopus (83) Google Scholar] but no HERV gene was reported by the recent GWAS performed in large series of PBC cases and controls [[16]Liu X. Invernizzi P. Lu Y. Kosoy R. Bianchi I. Podda M. et al.Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis.Nat Genet. 2010; 42: 658-660Crossref PubMed Scopus (326) Google Scholar]. Last and possibly most importantly, the proposed evidence was obtained in a very small number of cases; the need for specimens that are not readily available (i.e. lymph nodes) and the complexity of the experimental approach (as in the case of viral integration) do not appear sufficient to overcome this major limitation. In conclusion, the available evidence that has been proposed over the past decade falls well short of supporting a viral etiology for PBC. The small numbers of cases included in all published studies, as well as the conflicting results generated by different groups, raise major concerns for this hypothesis. This hypothesis may be another in the long series of retroviral associations proposed for autoimmune diseases [[17]Nelson P.N. Carnegie P.R. Martin J. Davari Ejtehadi H. Hooley P. Roden D. et al.Demystified. Human endogenous retroviruses.Mol Pathol. 2003; 56: 11-18Crossref PubMed Scopus (135) Google Scholar] that have failed to be confirmed independently or that have any practical implication. Conflict of interestThe author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.