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The evolution and clinical impact of hepatitis B virus genome diversity

Abstract

The global burden of hepatitis B virus (HBV) is enormous, with 257 million persons chronically infected, resulting in more than 880,000 deaths per year worldwide. HBV exists as nine different genotypes, which differ in disease progression, natural history and response to therapy. HBV is an ancient virus, with the latest reports greatly expanding the host range of the Hepadnaviridae (to include fish and reptiles) and casting new light on the origins and evolution of this viral family. Although there is an effective preventive vaccine, there is no cure for chronic hepatitis B, largely owing to the persistence of a viral minichromosome that is not targeted by current therapies. HBV persistence is also facilitated through aberrant host immune responses, possibly due to the diverse intra-host viral populations that can respond to host-mounted and therapeutic selection pressures. This Review summarizes current knowledge on the influence of HBV diversity on disease progression and treatment response and the potential effect on new HBV therapies in the pipeline. The mechanisms by which HBV diversity can occur both within the individual host and at a population level are also discussed.

Key points

  • Hepatitis B virus (HBV) is an ancient virus with deep ancestry in the animal kingdom.

  • HBV seems to undergo very little long-term mutational variation despite multiple host–virus factors driving short-term viral variations.

  • Viral diversity is generated by features of the unique replication cycle of HBV as well as by cellular host factors.

  • A possible bottleneck in the establishment of new viral variants could be the limited number of HBV-susceptible hepatocytes in the chronically infected liver.

  • HBV viral diversity contributes to variations in natural history, disease progression and treatment response in those with chronic infection.

  • Viral diversity must be considered in the development of new therapeutic regimens.

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Fig. 1: Phylogenetic trees of Hepadnaviridae.
Fig. 2: A proposed model for evolution of the Hepadnaviridae genome.
Fig. 3: Mechanisms for the generation and constraint of HBV diversity.
Fig. 4: Hepatitis delta replication cycle.

References

  1. 1.

    Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol. Hepatol. 3, 383–403 (2018).

    Google Scholar 

  2. 2.

    World Health Organization. Global Hepatitis Report, 2019 https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/ (WHO, 2017).

  3. 3.

    Parkin, D. M. Global cancer statistics in the year 2000. Lancet Oncol. 2, 533–543 (2001).

    CAS  PubMed  Google Scholar 

  4. 4.

    Revill, P. A. et al. A global scientific strategy to cure hepatitis B. Lancet Gastroenterol. Hepatol. 4, 545–558 (2019).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Alter, H. et al. A research agenda for curing chronic hepatitis B virus infection. Hepatology 67, 1127–1131 (2018).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Revill, P., Testoni, B., Locarnini, S. & Zoulim, F. Global strategies are required to cure and eliminate HBV infection. Nat. Rev. Gastroenterol. Hepatol. 13, 239–248 (2016). First ‘call to arms’ for hepatitis B cure, that led to the formation of the International Coalition to Eliminate Hepatitis B (ICE-HBV).

    CAS  PubMed  Google Scholar 

  7. 7.

    Wei, L. & Lok, A. S. Impact of new hepatitis C treatments in different regions of the world. Gastroenterology 146, 1145–1150 (2014).

    PubMed  Google Scholar 

  8. 8.

    Salpini, R. et al. A snapshot of virological presentation and outcome of immunosuppression-driven HBV reactivation from real clinical practice: evidence of a relevant risk of death and evolution from silent to chronic infection. J. Viral Hepat. 26, 846–855 (2019).

    CAS  PubMed  Google Scholar 

  9. 9.

    Hoofnagle, J. H. Reactivation of hepatitis B. Hepatology 49, S156–S165 (2009).

    CAS  PubMed  Google Scholar 

  10. 10.

    Comas, I. et al. Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45, 1176–1182 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Linz, B. et al. An African origin for the intimate association between humans and Helicobacter pylori. Nature 445, 915–918 (2007).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wolfe, N. D., Dunavan, C. P. & Diamond, J. Origins of major human infectious diseases. Nature 447, 279–283 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Blaser, M. J. & Kirschner, D. The equilibria that allow bacterial persistence in human hosts. Nature 449, 843–84 (2007).

    CAS  PubMed  Google Scholar 

  14. 14.

    Kahila Bar-Gal, G. et al. Tracing hepatitis B virus to the 16th century in a Korean mummy. Hepatology 56, 1671–1680 (2012). First report of ancient HBV genome isolated from ancient human remains, a 400-year-old Korean mummy.

    PubMed  Google Scholar 

  15. 15.

    Patterson Ross, Z. et al. The paradox of HBV evolution as revealed from a 16th century mummy. PLoS Pathog. 14, e1006750 (2018).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Muhlemann, B. et al. Ancient hepatitis B viruses from the Bronze age to the Medieval period. Nature 557, 418–423 (2018). First report of Bronze age HBV genomes extracted from a number of archaeological sites across Europe, including a now extinct human genotype.

    CAS  PubMed  Google Scholar 

  17. 17.

    Krause-Kyora, B. et al. Neolithic and medieval virus genomes reveal complex evolution of hepatitis B. Elife 7, e36666 (2018).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Yuen, L. K. W. et al. Tracing ancient human migrations into Sahul using hepatitis B virus genomes. Mol. Biol. Evol. 36, 942–954 (2019). First example of HBV evolution and diversity being used to trace human migration.

    CAS  PubMed  Google Scholar 

  19. 19.

    MacLachlan, J. H. & Cowie, B. C. Hepatitis B virus epidemiology. Cold Spring Harb. Perspect. Med. 5, a021410 (2015).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Graham, S. et al. Chronic hepatitis B prevalence among Aboriginal and Torres Strait Islander Australians since universal vaccination: a systematic review and meta-analysis. BMC Infect. Dis. 13, 403 (2013).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Parker, C. et al. Hepatocellular carcinoma in Australia’s Northern Territory: high incidence and poor outcome. Med. J. Aust. 201, 470–474 (2014).

    PubMed  Google Scholar 

  22. 22.

    Ormaeche, M., Whittembury, A., Pun, M. & Suarez-Ognio, L. Hepatitis B virus, syphilis, and HIV seroprevalence in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin, 2007-2008. Int. J. Infect. Dis. 16, e724–e730 (2012).

    PubMed  Google Scholar 

  23. 23.

    Viana, S., Parana, R., Moreira, R. C., Compri, A. P. & Macedo, V. High prevalence of hepatitis B virus and hepatitis D virus in the western Brazilian Amazon. Am. J. Trop. Med. Hyg. 73, 808–814 (2005).

    PubMed  Google Scholar 

  24. 24.

    Schweitzer, A., Mmath, J. H., Mikolaiczyk, 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 386, 1546–1555 (2015).

    Google Scholar 

  25. 25.

    Bouckaert, R., Simons, B. C., Krarup, H., Friesen, T. M. & Osiowy, C. Tracing hepatitis B virus (HBV) genotype B5 (formerly B6) evolutionary history in the circumpolar Arctic through phylogeographic modelling. PeerJ. 5, e3757 (2017).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    McMahon, B. J. et al. Elimination of hepatocellular carcinoma and acute hepatitis B in children 25 years after a hepatitis B newborn and catch-up immunization program. Hepatology 54, 801–807 (2011).

    PubMed  Google Scholar 

  27. 27.

    Robinson, T., Bullen, C., Humphries, W., Hornell, J. & Moyes, C. The New Zealand hepatitis B screening programme: screening coverage and prevalence of chronic hepatitis B infection. NZ Med. J. 118, U1345 (2005).

    Google Scholar 

  28. 28.

    Huang, C. F. et al. HBV infection in indigenous children, 20 years after immunization in Taiwan: a community-based study. Prev. Med. 48, 397–400 (2009).

    PubMed  Google Scholar 

  29. 29.

    Wilson, N., Ruff, T. A., Rana, B. J., Leydon, J. & Locarnini, S. The effectiveness of the infant hepatitis B immunisation program in Fiji, Kiribati, Tonga and Vanuatu. Vaccine 18, 3059–3066 (2000).

    CAS  PubMed  Google Scholar 

  30. 30.

    McMahon, B. J. Viral hepatitis in the Arctic. Int. J. Circumpolar Health 63 (Suppl. 2), 41–48 (2004).

    PubMed  Google Scholar 

  31. 31.

    Kramvis, A. Molecular characteristics and clinical relevance of African genotypes and subgenotypes of hepatitis B virus. S. Afr. Med. J. 108, S17–S21 (2018).

    CAS  Google Scholar 

  32. 32.

    European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J. Hepatol. 67, 370–398 (2017). The current European Clinical Practice Guidelines for the management of hepatitis B virus infection.

    Google Scholar 

  33. 33.

    Bayliss, J. et al. Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B. Gut 66, 2013–2023 (2017).

    CAS  PubMed  Google Scholar 

  34. 34.

    Sonneveld, M. J. et al. Presence of precore and core promoter mutants limits the probability of response to peginterferon in hepatitis B e antigen-positive chronic hepatitis B. Hepatology 56, 67–75 (2012).

    CAS  PubMed  Google Scholar 

  35. 35.

    Seeger, C., Zoulim, F. & Mason, W. S. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2185–2221 (Lippincott Williams & Wilkins, 2013).

  36. 36.

    International Committee on Taxonomy of Viruses. ICTV https://talk.ictvonline.org (2019).

  37. 37.

    Hahn, C. M. et al. Characterization of a novel hepadnavirus in the white sucker (Catostomus commersonii) from the Great Lakes region of the United States. J. Virol. 89, 11801–11811 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Dill, J. A. et al. Distinct viral lineages from fish and amphibians reveal the complex evolutionary history of hepadnaviruses. J. Virol. 90, 7920–7933 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Littlejohn, M., Locarnini, S. & Yuen, L. Origins and evolution of hepatitis B virus and hepatitis D virus. Cold Spring Harb. Perspect. Med. 6, a021360 (2016). Detailed review summarizing and discussing current theories on the origin and evolution of HBV and HDV.

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Lauber, C. et al. Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses. Cell Host Microbe 22, 387–399 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    de Carvalho Dominguez Souza, B. F. et al. A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses. J. Hepatology 68, 1114–1122 (2018).

    Google Scholar 

  42. 42.

    Aghazadeh, M. et al. A novel hepadnavirus identified in an immunocompromised domestic cat in Australia. Viruses 10, E269 (2018).

    PubMed  Google Scholar 

  43. 43.

    Geoghegan, J. L. et al. Hidden diversity and evolution of viruses in market fish. Virus Evol. 4, vey031 (2018).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Gong, Z. & Han, G. Z. Insect retroelements provide novel insights into the origin of hepatitis B viruses. Mol. Biol. Evol. 35, 2254–2259 (2018).

    CAS  PubMed  Google Scholar 

  45. 45.

    Gilbert, C. & Feschotte, C. Genomic fossils calibrate the long-term evolution of hepadnaviruses. PLoS Biol. 8, e1000495 (2010). First report of endogenous hepadnaviruses, discovered in the genome of the zebra finch.

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Gilbert, C. et al. Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes. Proc. Biol. Sci. 281, 20141122 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Suh, A. et al. Early mesozoic coexistence of amniotes and hepadnaviridae. PLoS Genet. 10, e1004559 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Geoghegan, J. L., Duchene, S. & Holmes, E. C. Comparative analysis estimates the relative frequencies of co-divergence and cross-species transmission within viral families. PLoS Pathog. 13, e1006215 (2017).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Drexler, J. F. et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc. Natl Acad. Sci. USA 110, 16151–16156 (2013). Report of the discovery of a number of HBV-like viruses in bats, including experiments showing that bat HBV can infect human hepatocytes.

    CAS  PubMed  Google Scholar 

  50. 50.

    Wieland, S. F. The chimpanzee model for hepatitis B virus infection. Cold Spring Harb. Perspect. Med. 5, a021469 (2015).

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Dupinay, T. et al. Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island. Hepatology 58, 1610–1620 (2013).

    CAS  PubMed  Google Scholar 

  52. 52.

    Dickens, C., Kew, M. C., Purcell, R. H. & Kramvis, A. Occult hepatitis B virus infection in chacma baboons, South Africa. Emerg. Infect. Dis. 19, 598–605 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Tatematsu, K. et al. A genetic variant of hepatitis B virus divergent from known human and ape genotypes isolated from a Japanese patient and provisionally assigned to new genotype J. J. Virol. 83, 10538–10547 (2009). Description of the single isolate of HBV ‘putative genotype’ J.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Begun, D. R. Planet of the apes. Sci. Am. 289, 74–83 (2003).

    PubMed  Google Scholar 

  55. 55.

    Begun, D. R., Nargolwalla, M. C. & Kordos, L. European Miocene hominids and the origin of the African ape and human clade. Evol. Anthropol. 21, 10–23 (2012).

    PubMed  Google Scholar 

  56. 56.

    Zhou, Y. & Holmes, E. C. Bayesian estimates of the evolutionary rate and age of hepatitis B virus. J. Mol. Evol. 65, 197–205 (2007).

    CAS  PubMed  Google Scholar 

  57. 57.

    Orito, E. et al. Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences. Proc. Natl Acad. Sci. USA 86, 7059–7062 (1989).

    CAS  PubMed  Google Scholar 

  58. 58.

    Paraskevis, D. et al. Dating the origin of hepatitis B virus reveals higher substitution rate and adaptation on the branch leading to F/H genotypes. Mol. Phylogenet. Evol. 93, 44–54 (2015).

    PubMed  Google Scholar 

  59. 59.

    Paraskevis, D. et al. Dating the origin and dispersal of hepatitis B virus infection in humans and primates. Hepatology 57, 908–916 (2013).

    PubMed  Google Scholar 

  60. 60.

    Tedder, R. S., Bissett, S. L., Myers, R. & Ijaz, S. The ‘Red Queen’ dilemma – running to stay in the same place: reflections on the evolutionary vector of HBV in humans. Antivir. Ther. 18, 489–496 (2013). An interesting paper outlining experiments confirming a theory explaining the slow evolutionary rate of human HBV.

    CAS  PubMed  Google Scholar 

  61. 61.

    Simmonds, P. & Smith, D. B. in Viral Hepatitis 4th edn (eds Thomas, H. C., Lok, A. S. F., Locarnini, S. A. & Zuckerman, A. J.) 575–586 (Wiley, 2014).

  62. 62.

    Simmonds, P., Aiewsakun, P. & Katzourakis, A. Prisoners of war – host adaptation and its constraints on virus evolution. Nat. Rev. Microbiol. 17, 321–328 (2018).

    PubMed Central  Google Scholar 

  63. 63.

    Lim, S. G. et al. Viral quasi-species evolution during hepatitis Be antigen seroconversion. Gastroenterology 133, 951–958 (2007).

    CAS  PubMed  Google Scholar 

  64. 64.

    Homs, M. et al. Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution. PLoS One 9, e112306 (2014).

    PubMed  PubMed Central  Google Scholar 

  65. 65.

    Charuworn, P. et al. Baseline interpatient hepatitis B viral diversity differentiates HBsAg outcomes in patients treated with tenofovir disoproxil fumarate. J. Hepatol. 62, 1033–1039 (2015).

    CAS  PubMed  Google Scholar 

  66. 66.

    Kramvis, A. Genotypes and genetic variability of hepatitis B virus. Intervirology 57, 141–150 (2014). Excellent summary of human HBV genotype and sub-genotype classification, including a set of minimum criteria for future proposals of novel genotypes and sub-genotypes.

    PubMed  Google Scholar 

  67. 67.

    Kim, B. K., Revill, P. A. & Ahn, S. H. HBV genotypes: relevance to natural history, pathogenesis and treatment of chronic hepatitis B. Antivir. Ther. 16, 1169–1186 (2011).

    CAS  PubMed  Google Scholar 

  68. 68.

    Chan, H. L. et al. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 53, 1494–1498 (2004).

    PubMed  PubMed Central  Google Scholar 

  69. 69.

    Yang, H. I. et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma. J. Natl Cancer Inst. 100, 1134–1143 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Wong, G. L. et al. Meta-analysis: the association of hepatitis B virus genotypes and hepatocellular carcinoma. Aliment. Pharmacol. Ther. 37, 517–526 (2013).

    CAS  PubMed  Google Scholar 

  71. 71.

    Kramvis, A. & Kew, M. C. Molecular characterization of subgenotype A1 (subgroup Aa) of hepatitis B virus. Hepatol. Res. 37, S27–S32 (2007). First characterization of the African A1 HBV subgenotype describing specific sequence elements that may contribute to the increased risk of HCC in patients infected with HBV-A1.

    CAS  PubMed  Google Scholar 

  72. 72.

    Gounder, P. P. et al. Hepatocellular carcinoma risk in Alaska native children and young adults with hepatitis B virus: retrospective cohort analysis. J. Pediatr. 178, 206–213 (2016).

    PubMed  PubMed Central  Google Scholar 

  73. 73.

    Orito, E. & Mizokami, M. Differences of HBV genotypes and hepatocellular carcinoma in Asian countries. Hepatol. Res. 37, S33–S35 (2007).

    CAS  PubMed  Google Scholar 

  74. 74.

    Krarup, H. B. et al. Benign course of long-standing hepatitis B virus infection among Greenland Inuit? Scand. J. Gastroenterol. 43, 334–343 (2008).

    PubMed  Google Scholar 

  75. 75.

    Minuk, G. Y. et al. A paucity of liver disease in Canadian Inuit with chronic hepatitis B virus, subgenotype B6 infection. J. Viral Hepat. 20, 890–896 (2013).

    CAS  PubMed  Google Scholar 

  76. 76.

    Roman, S., Fierro, N., Moreno-Luna, L. & Panduro, A. Hepatitis B virus genotype H and environmental factors associated to the low prevalence of hepatocellular carcinoma in Mexico. J. Cancer Ther. 4, 367–376 (2013).

    Google Scholar 

  77. 77.

    Sozzi, V. 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 519, 190–196 (2018).

    CAS  PubMed  Google Scholar 

  78. 78.

    Sozzi, V. 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. 90, 10054–10064 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Sugiyama, M. et al. Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 44, 915–924 (2006).

    CAS  PubMed  Google Scholar 

  80. 80.

    Bhoola, N. H., Reumann, K., Kew, M. C., Will, H. & Kramvis, A. Construction of replication competent plasmids of hepatitis B virus subgenotypes A1, A2 and D3 with authentic endogenous promoters. J. Virol. Methods 203, 54–64 (2014).

    CAS  PubMed  Google Scholar 

  81. 81.

    Flink, H. J. et al. Treatment with Peg-interferon α-2b for HBeAg-positive chronic hepatitis B: HBsAg loss is associated with HBV genotype. Am. J. Gastroenterol. 101, 297–303 (2006).

    CAS  PubMed  Google Scholar 

  82. 82.

    Buster, E. H. et al. Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa. Gastroenterology 137, 2002–2009 (2009).

    CAS  PubMed  Google Scholar 

  83. 83.

    Shen, F. et al. Hepatitis B virus sensitivity to interferon-α in hepatocytes is more associated with cellular interferon response than with viral genotype. Hepatology 67, 1237–1252 (2018).

    CAS  PubMed  Google Scholar 

  84. 84.

    Marcellin, P. et al. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N. Engl. J. Med. 359, 2442–2455 (2008).

    CAS  PubMed  Google Scholar 

  85. 85.

    Bihl, F. et al. HBV genotypes and response to tenofovir disoproxil fumarate in HIV/HBV-coinfected persons. BMC Gastroenterol. 15, 79 (2015).

    PubMed  PubMed Central  Google Scholar 

  86. 86.

    Marcellin, P. et al. Kinetics of hepatitis B surface antigen loss in patients with HBeAg-positive chronic hepatitis B treated with tenofovir disoproxil fumarate. J. Hepatol. 61, 1228–1237 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Yao, C. C. et al. Incidence and predictors of HBV relapse after cessation of nucleoside analogues in HBeAg-negative patients with HBsAg ≤ 200 IU/mL. Sci. Rep. 7, 1839 (2017).

    PubMed  PubMed Central  Google Scholar 

  88. 88.

    Chen, C. H. et al. The role of hepatitis B surface antigen quantification in predicting HBsAg loss and HBV relapse after discontinuation of lamivudine treatment. J. Hepatol. 61, 515–522 (2014).

    CAS  PubMed  Google Scholar 

  89. 89.

    Kramvis, A., Kostaki, E. G., Hatzakis, A. & Paraskevis, D. Immunomodulatory function of HBeAg related to short-sighted evolution, transmissibility, and clinical manifestation of hepatitis B virus. Front. Microbiol. 9, 2521 (2018).

    PubMed  PubMed Central  Google Scholar 

  90. 90.

    Buckwold, V. E., Xu, Z., Chen, M., Yen, T. S. & Ou, J. H. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J. Virol. 70, 5845–5851 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Warner, N. & Locarnini, S. The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound. Hepatology 48, 88–98 (2008).

    CAS  PubMed  Google Scholar 

  92. 92.

    Kay, A. & Zoulim, F. Hepatitis B virus genetic variability and evolution. Virus Res. 127, 164–176 (2007).

    CAS  PubMed  Google Scholar 

  93. 93.

    Zoulim, F. & Locarnini, S. Optimal management of chronic hepatitis B patients with treatment failure and antiviral drug resistance. Liver Int. 33 (Suppl. 1), 116–124 (2013).

    CAS  PubMed  Google Scholar 

  94. 94.

    Terrault, N. A. et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 67, 1560–1599 (2018). Most recent American HBV treatment guidelines from the AASLD.

    PubMed  PubMed Central  Google Scholar 

  95. 95.

    Littlejohn, M. Antiviral drug resistance and hepatitis B: a continuing public health problem. Antivir. Ther. 22, 643–644 (2017).

    PubMed  Google Scholar 

  96. 96.

    Liu, Y. et al. No detectable resistance to tenofovir disoproxil fumarate in HBeAg+ and HBeAg− patients with chronic hepatitis B after 8 years of treatment. J. Viral Hepat. 24, 68–74 (2017).

    CAS  PubMed  Google Scholar 

  97. 97.

    Park, E. S. et al. Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. J. Hepatol. 70, 1093–1102 (2019).

    CAS  PubMed  Google Scholar 

  98. 98.

    Cathcart, A. L. et al. No resistance to tenofovir alafenamide detected through 96 weeks of treatment in patients with chronic hepatitis B infection. Antimicrob. Agents Chemother. 62, e01064-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  99. 99.

    Lok, A. S., Zoulim, F., Dusheiko, G. & Ghany, M. G. Hepatitis B cure: from discovery to regulatory approval. Hepatology 66, 1296–1313 (2017).

    PubMed  PubMed Central  Google Scholar 

  100. 100.

    Zeisel, M. B. et al. Towards an HBV cure: state-of-the-art and unresolved questions—report of the ANRS workshop on HBV cure. Gut 64, 1314–1326 (2015).

    CAS  PubMed  Google Scholar 

  101. 101.

    Revill, P. A., Penicaud, C., Brechot, C. & Zoulim, F. Meeting the challenge of eliminating chronic hepatitis B infection. Genes 10, 260 (2019).

    CAS  PubMed Central  Google Scholar 

  102. 102.

    Petersen, J. et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat. Biotechnol. 26, 335–341 (2008).

    CAS  PubMed  Google Scholar 

  103. 103.

    Thi, E. P. et al. ARB-1740, a RNA interference therapeutic for chronic hepatitis B infection. ACS Infect. Dis. 5, 725–737 (2018).

    PubMed  Google Scholar 

  104. 104.

    Wooddell, C. I. et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci. Transl. Med. 9, eaan0241 (2017). Groundbreaking study demonstrating that in HBeAg-negative individuals, most of the secreted HBsAg is produced by RNA transcribed from integrated HBV DNA.

    PubMed  PubMed Central  Google Scholar 

  105. 105.

    Bazinet, M. et al. Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial. Lancet Gastroenterol. Hepatol. 2, 877–889 (2017).

    PubMed  Google Scholar 

  106. 106.

    Roehl, I. et al. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy. Mol. Ther. Nucleic Acids 8, 1–12 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Long, K. R. et al. Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice. Antivir. Res. 149, 41–47 (2018).

    CAS  PubMed  Google Scholar 

  108. 108.

    Klumpp, K. et al. Efficacy of NVR 3-778, alone and in combination with pegylated interferon, vs entecavir In uPA/SCID mice with humanized livers and HBV infection. Gastroenterology 154, 652–662 (2018).

    CAS  PubMed  Google Scholar 

  109. 109.

    Lam, A. M. et al. Preclinical characterization of NVR 3-778, a first-in-class capsid assembly modulator against hepatitis B virus. Antimicrob. Agents Chemother. 63, e01734-18 (2019).

    PubMed  Google Scholar 

  110. 110.

    Berke, J. M. et al. Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro. Antivir. Res. 144, 205–215 (2017).

    CAS  PubMed  Google Scholar 

  111. 111.

    Ruan, L., Hadden, J. A. & Zlotnick, A. Assembly properties of hepatitis B virus core protein mutants correlate with their resistance to assembly-directed antivirals. J. Virol. 92, e01082-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  112. 112.

    Wang, J. et al. Influences on viral replication and sensitivity to GLS4, a HAP compound, of naturally occurring T109/V124 mutations in hepatitis B virus core protein. J. Med. Virol. 89, 1804–1810 (2017).

    CAS  PubMed  Google Scholar 

  113. 113.

    Zhou, Z. et al. Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) inhibit hepatitis B virus replication by different molecular mechanisms. Sci. Rep. 7, 42374 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. 114.

    Seeger, C. & Sohn, J. A. Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA. Mol. Ther. 24, 1258–1266 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. 115.

    Carman, W. F. et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet 336, 325–329 (1990).

    CAS  PubMed  Google Scholar 

  116. 116.

    Carman, W. F. The clinical significance of surface antigen variants of hepatitis B virus. J. Viral Hepat. 4 (Suppl. 1), 11–20 (1997).

    PubMed  Google Scholar 

  117. 117.

    Hsu, H. Y., Chang, M. H., Ni, Y. H. & Chen, H. L. Survey of hepatitis B surface variant infection in children 15 years after a nationwide vaccination programme in Taiwan. Gut 53, 1499–1503 (2004).

    PubMed  PubMed Central  Google Scholar 

  118. 118.

    Wang, J. et al. Molecular evolution of hepatitis B vaccine escape variants in China, during 2000–2016. Vaccine 35, 5808–5813 (2017).

    PubMed  Google Scholar 

  119. 119.

    Mokaya, J. et al. A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: a call for urgent action. PLoS Negl. Trop. Dis. 12, e0006629 (2018).

    PubMed  PubMed Central  Google Scholar 

  120. 120.

    Locarnini, S. & Shouval, D. Commonly found variations/mutations in the HBsAg of hepatitis B virus in the context of effective immunization programs: questionable clinical and public health significance. J. Virol. 88, 6532 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 121.

    Jilg, W. et al. Reduced prevalence of HBsAg variants following a successful immunization program in China. J. Virol. 88, 4605–4606 (2014).

    PubMed  PubMed Central  Google Scholar 

  122. 122.

    Tu, T., Buhler, S. & Bartenschlager, R. Chronic viral hepatitis and its association with liver cancer. Biol. Chem. 398, 817–837 (2017).

    CAS  PubMed  Google Scholar 

  123. 123.

    Orito, E. et al. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 34, 590–594 (2001).

    CAS  PubMed  Google Scholar 

  124. 124.

    Orito, E. & Mizokami, M. Hepatitis B virus genotypes and hepatocellular carcinoma in Japan. Intervirology 46, 408–412 (2003).

    PubMed  Google Scholar 

  125. 125.

    Ding, X. et al. Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China. Intervirology 44, 43–47 (2001).

    CAS  PubMed  Google Scholar 

  126. 126.

    Pan, M. H. et al. Hepatitis B splice variants are strongly associated with and are indeed predictive of hepatocellular carcinoma. J. Hepatol. 68, s474–s475 (2018).

    Google Scholar 

  127. 127.

    Bayliss, J. et al. Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma. J. Hepatol. 59, 1022–1028 (2013). First study to identify an association between the presence of HBV splice variants in patient serum and liver cancer.

    CAS  PubMed  Google Scholar 

  128. 128.

    Baptista, M., Kramvis, A. & Kew, M. C. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 29, 946–953 (1999).

    CAS  PubMed  Google Scholar 

  129. 129.

    Kao, J. H. Hepatitis B virus genotypes and hepatocellular carcinoma in Taiwan. Intervirology 46, 400–407 (2003).

    PubMed  Google Scholar 

  130. 130.

    Fang, Z. L. et al. HBV core promoter mutations prevail in patients with hepatocellular carcinoma from Guangxi, China. J. Med. Virol. 56, 18–24 (1998).

    CAS  PubMed  Google Scholar 

  131. 131.

    Kim, D. W., Lee, S. A., Hwang, E. S., Kook, Y. H. & Kim, B. J. Naturally occurring precore/core region mutations of hepatitis B virus genotype C related to hepatocellular carcinoma. PLoS One 7, e47372 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132.

    Ni, Y. H., Chang, M. H., Hsu, H. Y. & Tsuei, D. J. Different hepatitis B virus core gene mutations in children with chronic infection and hepatocellular carcinoma. Gut 52, 122–125 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. 133.

    Sung, F. Y. et al. Hepatitis B virus core variants modify natural course of viral infection and hepatocellular carcinoma progression. Gastroenterology 137, 1687–1697 (2009).

    CAS  PubMed  Google Scholar 

  134. 134.

    Liu, W. C. et al. Hepatocellular carcinoma-associated single-nucleotide variants and deletions identified by the use of genome-wide high-throughput analysis of hepatitis B virus. J. Pathol. 243, 176–192 (2017).

    CAS  PubMed  Google Scholar 

  135. 135.

    Yen, C. J. et al. Hepatitis B virus surface gene pre-S2 mutant as a high-risk serum marker for hepatoma recurrence after curative hepatic resection. Hepatology 68, 815–826 (2018).

    CAS  PubMed  Google Scholar 

  136. 136.

    Tsai, H. W. et al. Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma. Oncotarget 7, 27724–27734 (2016).

    PubMed  PubMed Central  Google Scholar 

  137. 137.

    Yen, T. T. et al. Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability. Oncotarget 7, 23346–23360 (2016).

    PubMed  PubMed Central  Google Scholar 

  138. 138.

    Hsieh, Y. H. et al. Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis. J. Pathol. 236, 337–347 (2015).

    CAS  PubMed  Google Scholar 

  139. 139.

    Lai, M. W. et al. Hepatocarcinogenesis in transgenic mice carrying hepatitis B virus pre-S/S gene with the sW172* mutation. Oncogenesis 5, e273 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. 140.

    Su, I. J., Wang, H. C., Wu, H. C. & Huang, W. Y. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J. Gastroenterol. Hepatol. 23, 1169–1174 (2008).

    CAS  PubMed  Google Scholar 

  141. 141.

    Fan, Y. F. et al. Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. Hepatology 33, 277–286 (2001).

    CAS  PubMed  Google Scholar 

  142. 142.

    Lamontagne, R. J., Bagga, S. & Bouchard, M. J. Hepatitis B virus molecular biology and pathogenesis. Hepatoma Res. 2, 163–186 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. 143.

    Salpini, R. et al. The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro. Clin. Microbiol. Infect. 25, 906.e1–906.e7 (2019).

    CAS  Google Scholar 

  144. 144.

    Raimondo, G. et al. Occult hepatitis B virus infection. Dig. Liver Dis. 32, 822–826 (2000).

    CAS  PubMed  Google Scholar 

  145. 145.

    Raimondo, G. et al. Update of the statements on biology and clinical impact of occult hepatitis B virus infection. J. Hepatol. 71, 397–408 (2019). Comprehensive summary on the importance of occult HBV infection.

    PubMed  Google Scholar 

  146. 146.

    Ramachandran, S. et al. Recent and occult hepatitis B virus infections among blood donors in the United States. Transfusion 59, 601–611 (2019).

    CAS  PubMed  Google Scholar 

  147. 147.

    Deguchi, M. et al. Evaluation of the highly sensitive chemiluminescent enzyme immunoassay ‘‘Lumipulse HBsAg-HQ’’ for hepatitis B virus screening. J. Clin. Lab. Anal. 32, e22334 (2018).

    PubMed  Google Scholar 

  148. 148.

    Zhang, K. et al. Antigenicity reduction contributes mostly to poor detectability of HBsAg by hepatitis B virus (HBV) S-gene mutants isolated from individuals with occult HBV infection. J. Med. Virol. 90, 263–270 (2018).

    CAS  PubMed  Google Scholar 

  149. 149.

    Hass, M. et al. Functional analysis of hepatitis B virus reactivating in hepatitis B surface antigen-negative individuals. Hepatology 42, 93–103 (2005).

    CAS  PubMed  Google Scholar 

  150. 150.

    Huang, F. Y. et al. Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection. PLoS One 9, e99028 (2014).

    PubMed  PubMed Central  Google Scholar 

  151. 151.

    Ponde, R. A. Molecular mechanisms underlying HBsAg negativity in occult HBV infection. Eur. J. Clin. Microbiol. Infect. Dis. 34, 1709–1731 (2015).

    CAS  PubMed  Google Scholar 

  152. 152.

    McNaughton, A. L. et al. Insights from deep sequencing of the HBV genome — unique, tiny, and misunderstood. Gastroenterology 156, 384–399 (2019).

    PubMed  Google Scholar 

  153. 153.

    Bill, C. A. & Summers, J. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc. Natl Acad. Sci. USA 101, 11135–11140 (2004).

    CAS  PubMed  Google Scholar 

  154. 154.

    Lucifora, J. et al. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343, 1221–1228 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. 155.

    Nair, S. & Zlotnick, A. Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription. J. Virol. 92, e02190–17 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. 156.

    Suspene, R. et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc. Natl Acad. Sci. USA 102, 8321–8326 (2005).

    CAS  PubMed  Google Scholar 

  157. 157.

    Vartanian, J. P. et al. Massive APOBEC3 editing of hepatitis B viral DNA in cirrhosis. PLoS Pathog. 6, e1000928 (2010).

    PubMed  PubMed Central  Google Scholar 

  158. 158.

    Gout, J. F., Thomas, W. K., Smith, Z., Okamoto, K. & Lynch, M. Large-scale detection of in vivo transcription errors. Proc. Natl Acad. Sci. USA 110, 18584–18589 (2013).

    CAS  PubMed  Google Scholar 

  159. 159.

    Imashimizu, M., Oshima, T., Lubkowska, L. & Kashlev, M. Direct assessment of transcription fidelity by high-resolution RNA sequencing. Nucleic Acids Res. 41, 9090–9104 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  160. 160.

    Carey, L. B. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits. Elife 4, e09945 (2015).

    PubMed  PubMed Central  Google Scholar 

  161. 161.

    Park, S. G., Kim, Y., Park, E., Ryu, H. M. & Jung, G. Fidelity of hepatitis B virus polymerase. Eur. J. Biochem. 270, 2929–2936 (2003).

    CAS  PubMed  Google Scholar 

  162. 162.

    Preikschat, P. et al. Complex HBV populations with mutations in core promoter, C gene, and pre-S region are associated with development of cirrhosis in long-term renal transplant recipients. Hepatology 35, 466–477 (2002).

    CAS  PubMed  Google Scholar 

  163. 163.

    Yeung, P. et al. Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in chronic hepatitis B. J. Infect. Dis. 203, 646–654 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  164. 164.

    Zhao, X. L. et al. Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV. Gut 65, 502–511 (2016).

    CAS  PubMed  Google Scholar 

  165. 165.

    Lewellyn, E. B. & Loeb, D. D. Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus. J. Virol. 81, 6207–6215 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  166. 166.

    Liu, N., Ji, L., Maguire, M. L. & Loeb, D. D. cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus. J. Virol. 78, 642–649 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  167. 167.

    Brechot, C., Gozuacik, D., Murakami, Y. & Paterlini-Brechot, P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin. Cancer Biol. 10, 211–231 (2000).

    CAS  PubMed  Google Scholar 

  168. 168.

    Tu, T., Budzinska, M. A., Vondran, F. W. R., Shackel, N. A. & Urban, S. Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles. J. Virol. 92, e02007–17 (2018). This study changed the dogma that HBV integration only occurs late in chronic disease by demonstrating that HBV integration occurs very early after infection.

    CAS  PubMed  PubMed Central  Google Scholar 

  169. 169.

    Lan, P., Zhang, C., Han, Q., Zhang, J. & Tian, Z. Therapeutic recovery of hepatitis B virus (HBV)-induced hepatocyte-intrinsic immune defect reverses systemic adaptive immune tolerance. Hepatology 58, 73–85 (2013).

    CAS  PubMed  Google Scholar 

  170. 170.

    Michler, T. et al. Blocking sense-strand activity improves potency, safety and specificity of anti-hepatitis B virus short hairpin RNA. EMBO Mol. Med. 8, 1082–1098 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  171. 171.

    Li, B. et al. Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo. Antivir. Res. 134, 117–129 (2016).

    CAS  PubMed  Google Scholar 

  172. 172.

    Tu, T., Budzinska, M. A., Shackel, N. A. & Urban, S. HBV DNA integration: molecular mechanisms and clinical implications. Viruses 9, 75 (2017).

    PubMed Central  Google Scholar 

  173. 173.

    Sommer, G. & Heise, T. Posttranscriptional control of HBV gene expression. Front. Biosci. 13, 5533–5547 (2008).

    CAS  PubMed  Google Scholar 

  174. 174.

    Su, T. S. et al. Hepatitis B virus transcript produced by RNA splicing. J. Virol. 63, 4011–4018 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  175. 175.

    Gunther, S., Sommer, G., Iwanska, A. & Will, H. Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA. Virology 238, 363–371 (1997).

    CAS  Google Scholar 

  176. 176.

    Chen, J. et al. Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy. Sci. Rep. 5, 16459 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  177. 177.

    Lam, A. M. et al. Hepatitis B virus capsid assembly modulators, but not nucleoside analogs, inhibit the production of extracellular pregenomic RNA and spliced RNA variants. Antimicrob. Agents Chemother. 61, e00680-17 (2017).

  178. 178.

    Ziemer, M., Garcia, P., Shaul, Y. & Rutter, W. J. Sequence of hepatitis B virus DNA incorporated into the genome of a human hepatoma cell line. J. Virol. 53, 885–892 (1985). One of the first studies showing HBV integration into the human genome.

    CAS  PubMed  PubMed Central  Google Scholar 

  179. 179.

    Soussan, P. et al. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA. J. Clin. Invest. 105, 55–60 (2000). First identification and characterization of a novel protein produced by HBV splice variants.

    CAS  PubMed  PubMed Central  Google Scholar 

  180. 180.

    Soussan, P. et al. Expression of defective hepatitis B virus particles derived from singly spliced RNA is related to liver disease. J. Infect. Dis. 198, 218–225 (2008).

    CAS  PubMed  Google Scholar 

  181. 181.

    Soussan, P. et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J. Hepatol. 38, 343–348 (2003).

    CAS  PubMed  Google Scholar 

  182. 182.

    Chen, W. N. et al. Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion. J. Virol. 86, 13533–13541 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  183. 183.

    Shi, W. et al. Identification of novel inter-genotypic recombinants of human hepatitis B viruses by large-scale phylogenetic analysis. Virology 427, 51–59 (2012).

    CAS  PubMed  Google Scholar 

  184. 184.

    Simmonds, P. & Midgley, S. Recombination in the genesis and evolution of hepatitis B virus genotypes. J. Virol. 79, 15467–15476 (2005). This study was the first to demonstrate that significant recombination events occurred in the evolution of HBV.

    CAS  PubMed  PubMed Central  Google Scholar 

  185. 185.

    Yang, J., Xing, K., Deng, R., Wang, J. & Wang, X. Identification of hepatitis B virus putative intergenotype recombinants by using fragment typing. J. Gen. Virol. 87, 2203–2215 (2006).

    CAS  PubMed  Google Scholar 

  186. 186.

    Bowyer, S. M. & Sim, J. G. Relationships within and between genotypes of hepatitis B virus at points across the genome: footprints of recombination in certain isolates. J. Gen. Virol. 81, 379–392 (2000).

    CAS  PubMed  Google Scholar 

  187. 187.

    Araujo, N. M. Hepatitis B virus intergenotypic recombinants worldwide: an overview. Infect. Genet. Evol. 36, 500–510 (2015).

    PubMed  Google Scholar 

  188. 188.

    Sugauchi, F. et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J. Virol. 76, 5985–5992 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  189. 189.

    Littlejohn, M. et al. Molecular virology of hepatitis B virus, sub-genotype C4 in northern Australian indigenous populations. J. Med. Virol. 86, 695–706 (2014). This is the first description of the sequence and characterization of the novel C4 subgenotype in indigenous Australian peoples.

    CAS  PubMed  Google Scholar 

  190. 190.

    Cui, C. et al. The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid. J. Gen. Virol. 83, 2773–2777 (2002).

    CAS  PubMed  Google Scholar 

  191. 191.

    Cheng, Y., Guindon, S., Rodrigo, A. & Lim, S. G. Increased viral quasispecies evolution in HBeAg seroconverter patients treated with oral nucleoside therapy. J. Hepatol. 58, 217–224 (2013).

    CAS  PubMed  Google Scholar 

  192. 192.

    Wu, S. et al. Evolution of hepatitis B genotype C viral quasi-species during hepatitis B e antigen seroconversion. J. Hepatol. 54, 19–25 (2011).

    PubMed  Google Scholar 

  193. 193.

    Nie, H., Evans, A. A., London, W. T., Block, T. M. & Ren, X. D. Quantitative dynamics of hepatitis B basal core promoter and precore mutants before and after HBeAg seroconversion. J. Hepatol. 56, 795–802 (2012).

    CAS  PubMed  Google Scholar 

  194. 194.

    Stevens, C. E., Neurath, R. A., Beasley, R. P. & Szmuness, W. HBeAg and anti-HBe detection by radioimmunoassay: correlation with vertical transmission of hepatitis B virus in Taiwan. J. Med. Virol. 3, 237–241 (1979).

    CAS  PubMed  Google Scholar 

  195. 195.

    Yan, H. et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1, e00049 (2012). This is the first report of the identification and description of the HBV entry receptor — a major discovery for the HBV field.

    PubMed  PubMed Central  Google Scholar 

  196. 196.

    Meier, A., Mehrle, S., Weiss, T. S., Mier, W. & Urban, S. Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes. Hepatology 58, 31–42 (2013).

    CAS  PubMed  Google Scholar 

  197. 197.

    Hasegawa, K., Huang, J., Rogers, S. A., Blum, H. E. & Liang, T. J. Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. J. Virol. 68, 1651–1659 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  198. 198.

    Baumert, T. F., Rogers, S. A., Hasegawa, K. & Liang, T. J. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J. Clin. Invest. 98, 2268–2276 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  199. 199.

    Nishizawa, T. et al. Enhanced pregenomic RNA levels and lowered precore mRNA transcription efficiency in a genotype A hepatitis B virus genome with C1766T and T1768A mutations obtained from a fulminant hepatitis patient. J. Gen. Virol. 97, 2643–2656 (2016).

    CAS  PubMed  Google Scholar 

  200. 200.

    Seeger, C. & Mason, W. S. Molecular biology of hepatitis B virus infection. Virology 479–480, 672–686 (2015).

    PubMed  Google Scholar 

  201. 201.

    Tu, T. & Urban, S. Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections. Curr. Opin. Virol. 30, 68–79 (2018).

    CAS  PubMed  Google Scholar 

  202. 202.

    Allweiss, L. et al. Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut 67, 542–552 (2018).

    CAS  PubMed  Google Scholar 

  203. 203.

    Farci, P. & Niro, G. A. Current and future management of chronic hepatitis D. Gastroenterol. Hepatol. 14, 342–351 (2018).

    Google Scholar 

  204. 204.

    Wranke, A. et al. Clinical and virological heterogeneity of hepatitis delta in different regions world-wide: the Hepatitis Delta International Network (HDIN). Liver Internatl. 38, 842–850 (2018).

    CAS  Google Scholar 

  205. 205.

    Magnius, L. et al. ICTV virus taxonomy profile: deltavirus. J. Gen. Virol. 99, 1565–1566 (2018).

    CAS  PubMed  Google Scholar 

  206. 206.

    Le Gal, F. et al. Genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains. Hepatology 66, 1826–1841 (2017).

    PubMed  Google Scholar 

  207. 207.

    Ryu, W. S., Bayer, M. & Taylor, M. Assembly of hepatitis delta virus. J. Virol. 66, 2310–2315 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  208. 208.

    Wang, C. J., Chen, P. J., Wu, J. C., Patel, D. & Chen, D. S. Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles. J. Virol. 65, 6630–6636 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  209. 209.

    Polo, J. M. et al. Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle. J. Virol. 69, 4880–4887 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  210. 210.

    Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 20, 675–691 (2019).

    CAS  PubMed  Google Scholar 

  211. 211.

    Barrett, S. P. & Salzman, J. Circular RNAs: analysis, expression and potential functions. Development 143, 1838–1847 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  212. 212.

    Perrotta, A. T. & Been, M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature 350, 434–436 (1991).

    CAS  PubMed  Google Scholar 

  213. 213.

    Ferre-D’Amare, A. R., Zhou, K. & Doudna, J. A. Crystal structure of a hepatitis delta virus ribozyme. Nature 395, 567–574 (1998).

    CAS  PubMed  Google Scholar 

  214. 214.

    Webb, C. H., Nguyen, D., Myszka, M. & Luptak, A. Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes. Sci. Rep. 6, 28179 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  215. 215.

    Webb, C. H. & Luptak, A. HDV-like self-cleaving ribozymes. RNA Biol. 8, 719–727 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  216. 216.

    Salehi-Ashtiani, K., Luptak, A., Litovchick, A. & Szostak, J. W. A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene. Science 313, 1788–1792 (2006).

    CAS  PubMed  Google Scholar 

  217. 217.

    Wille, M. et al. A divergent hepatitis D-like agent in birds. Viruses 10, E720 (2018).

    PubMed  Google Scholar 

  218. 218.

    Hetzel, U. et al. Identification of a novel deltavirus in boa constrictors. MBio 10, e00014–19 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  219. 219.

    Chang, W. S. et al. Novel hepatitis D-like agents in vertebrates and invertebrates. Virus Evol. 5, vez021 (2019).

    PubMed  PubMed Central  Google Scholar 

  220. 220.

    Perez-Vargas, J. et al. Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo. Nat. Commun. 10, 2098 (2019).

    PubMed  PubMed Central  Google Scholar 

  221. 221.

    Weller, M. L. et al. Hepatitis delta virus detected in salivary glands of Sjögren’s syndrome patients and recapitulates a Sjögren’s syndrome-like phenotype in vivo. Pathog. Immun. 1, 12–40 (2016).

    PubMed  PubMed Central  Google Scholar 

  222. 222.

    Szirovicza, L. et al. Snake deltavirus utilizes envelope proteins of different viruses to generate infectious particles. MBio 11, e03250-19 (2019).

    Google Scholar 

  223. 223.

    Rizzetto, M. Hepatitis D virus: introduction and epidemiology. Cold Spring Harb. Perspect. Med. 5, a021576 (2015).

    PubMed  PubMed Central  Google Scholar 

  224. 224.

    Wang, T. C. & Chao, M. RNA recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells. J. Virol. 79, 2221–2229 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  225. 225.

    Lin, C. C. et al. RNA recombination in hepatitis delta virus: identification of a novel naturally occurring recombinant. J. Microbiol. Immunol. Infect. 50, 771–780 (2017).

    CAS  PubMed  Google Scholar 

  226. 226.

    Hsu, S. C. et al. Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications. Hepatology 35, 665–672 (2002).

    CAS  PubMed  Google Scholar 

  227. 227.

    Wu, J. C. et al. Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D. Lancet 346, 939–941 (1995).

    CAS  PubMed  Google Scholar 

  228. 228.

    Ivaniushina, V. et al. Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia. J. Gen. Virol. 82, 2709–2718 (2001).

    CAS  PubMed  Google Scholar 

  229. 229.

    Casey, J. L. et al. Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F. J. Infect. Dis. 174, 920–926 (1996).

    CAS  PubMed  Google Scholar 

  230. 230.

    Watanabe, H. et al. Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease. J. Gen. Virol. 84, 3275–3289 (2003).

    CAS  PubMed  Google Scholar 

  231. 231.

    Madejon, A. et al. Hepatitis B and D viruses replication interference: influence of hepatitis B genotype. World J. Gastroenterol. 22, 3165–3174 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  232. 232.

    Kiesslich, D. et al. Influence of hepatitis B virus (HBV) genotype on the clinical course of disease in patients coinfected with HBV and hepatitis delta virus. J. Infect. Dis. 199, 1608–1611 (2009).

    CAS  PubMed  Google Scholar 

  233. 233.

    Schaper, M. et al. Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D. J. Hepatol. 52, 658–664 (2010).

    CAS  PubMed  Google Scholar 

  234. 234.

    Su, C. W. et al. Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients. Gastroenterology 130, 1625–1635 (2006).

    CAS  PubMed  Google Scholar 

  235. 235.

    Smedile, A. et al. Hepatitis B virus replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D. Hepatology 13, 413–416 (1991).

    CAS  PubMed  Google Scholar 

  236. 236.

    Quintero, A. et al. Hepatitis delta virus genotypes I and III circulate associated with hepatitis B virus genotype F in Venezuela. J. Med. Virol. 64, 356–359 (2001).

    CAS  PubMed  Google Scholar 

  237. 237.

    Shih, H. H. et al. Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus. J. Virol. 82, 2250–2264 (2008).

    CAS  PubMed  Google Scholar 

  238. 238.

    Vietheer, P. T., Netter, H. J., Sozzi, T. & Bartholomeusz, A. Failure of the lamivudine-resistant rtM204I hepatitis B virus mutants to efficiently support hepatitis delta virus secretion. J. Virol. 79, 6570–6573 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  239. 239.

    Colombo, P. et al. Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection. J. Hepatol. 12, 64–69 (1991).

    CAS  PubMed  Google Scholar 

  240. 240.

    Colagrossi, L. et al. HDV can constrain HBV genetic evolution in HBsAg: implications for the identification of innovative pharmacological targets. Viruses 10, E363 (2018).

    PubMed  Google Scholar 

  241. 241.

    Jilbert, A. J. & Locarnini, S. A. in Viral Hepatitis 3rd edn (eds Thomas, H. C. Lemon, S. & Zuckerman, A. J.) 193–209 (Wiley, 2005).

  242. 242.

    Erhardt, A. et al. Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D. Gut 54, 1009–1013 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  243. 243.

    Kao, J. H., Wu, N. H., Chen, P. J., Lai, M. Y. & Chen, D. S. Hepatitis B genotype and the response to interferon therapy. J. Hepatol. 33, 998–1002 (2000).

    CAS  PubMed  Google Scholar 

  244. 244.

    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 122, 1756–1762 (2002).

    CAS  PubMed  Google Scholar 

  245. 245.

    Osiowy, C., Gordon, D., Borlang, J., Giles, E. & Villeneuve, J. P. Hepatitis B virus genotype G epidemiology and co-infection with genotype A in Canada. J. Gen. Virol. 89, 3009–3015 (2008).

    CAS  PubMed  Google Scholar 

  246. 246.

    Huy, T. T. T., Ngoc, T. T. & Abe, K. New complex recombinant genotype of hepatitis B virus identified in Vietnam. J. Virol. 82, 5657–5663 (2008).

    CAS  PubMed Central  Google Scholar 

  247. 247.

    Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    CAS  Google Scholar 

  248. 248.

    Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  249. 249.

    Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018).

    CAS  PubMed  Google Scholar 

  250. 250.

    Li, J., Buckwold, V. E., Hon, M. W. & Ou, J. H. Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation. J. Virol. 73, 1239–1244 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  251. 251.

    Carman, W. F. et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2, 588–591 (1989).

    CAS  PubMed  Google Scholar 

  252. 252.

    Okamoto, H. et al. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J. Virol. 64, 1298–1303 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  253. 253.

    Chotiyaputta, W. & Lok, A. S. Hepatitis B virus variants. Nat. Rev. Gastroenterol. Hepatol. 6, 453–462 (2009).

    CAS  PubMed  Google Scholar 

  254. 254.

    Zoulim, F. & Locarnini, S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137, 1593–1608 (2009).

    CAS  PubMed  Google Scholar 

  255. 255.

    Li, X. et al. PreS deletion profiles of hepatitis B virus (HBV) are associated with clinical presentations of chronic HBV infection. J. Clin. Virol. 82, 27–32 (2016).

    PubMed  Google Scholar 

  256. 256.

    Lin, C. L. et al. Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 22, 1098–1103 (2007).

    CAS  PubMed  Google Scholar 

  257. 257.

    Blondot, M. L., Bruss, V. & Kann, M. Intracellular transport and egress of hepatitis B virus. J. Hepatol. 64, S49–S59 (2016).

    CAS  PubMed  Google Scholar 

  258. 258.

    Qi, Y. et al. DNA polymerase kappa is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus. PLoS Pathog. 12, e1005893 (2016).

    PubMed  PubMed Central  Google Scholar 

  259. 259.

    Walters, K. A., Joyce, M. A., Addison, W. R., Fischer, K. P. & Tyrrell, D. L. Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen. J. Virol. 78, 7925–7937 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  260. 260.

    Ni, Y. et al. The effect of integration on cell susceptibility to HBV and HDV. Presented at the 2019 International Meeting for the Molecular Biology of Hepatitis Viruses (IHBV, 2019).

  261. 261.

    Bogomolov, P. et al. Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: first results of a phase Ib/IIa study. J. Hepatol. 65, 490–498 (2016).

    CAS  PubMed  Google Scholar 

  262. 262.

    Allweiss, L. et al. Strong intrahepatic decline of hepatitis D virus RNA and antigen after 24 weeks of treatment with Myrcludex B in combination with tenofovir in chronic HBV/HDV infected patients: interim results from a multicenter, open-label phase 2b clinical trial [abstract PS-162]. J. Hepatol. 68, S90 (2018).

    Google Scholar 

  263. 263.

    Wedemeyer, H. et al. Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with tenofovir in patients with chronic HBV/HDV co-infection [abstract GS-005]. J. Hepatol. 68, S3 (2018). The final results of the clinical trial that demonstrated treatment with the entry inhibitor Myrcludex B showed efficacy against HDV infection.

    Google Scholar 

  264. 264.

    Freitas, N., Cunha, C., Menne, S. & Gudima, S. O. Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles. J. Virol. 88, 5742–5754 (2014).

    PubMed  PubMed Central  Google Scholar 

  265. 265.

    Mason, W. S., Liu, C., Aldrich, C. E., Litwin, S. & Yeh, M. M. Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection. J. Virol. 84, 8308–8315 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  266. 266.

    Tu, T. et al. Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection. J. Viral Hepat. 22, 737–753 (2015).

    CAS  PubMed  Google Scholar 

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Acknowledgements

T.T. is financially supported by the German Centre for Infection Research (DZIF), TTU Hepatitis Projects 5.807 and 5.704, the Deutsche Forschungsgemeinschaft (DFG) TRR179 (TP 15 and the Australian Centre for HIV and Hepatitis Virology Research). The authors thank R. Schinazi for providing helpful information on capsid inhibitors.

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Glossary

Hepatitis B surface antigen

(HBsAg). HBV encodes three surface (envelope) proteins: large (L), middle (M) and small (S) HBsAg, which share the C-terminal domain but differ in their N-terminal extensions.

HBV genotypes

Based on >7.5% nucleotide divergence, HBV has been phylogenetically classified into nine genotypes (A–I) and one putative genotype (J).

HBV sub-genotypes

HBV genotypes have been further classified into >35 sub-genotypes based on approximately 4–8% nucleotide divergence.

Hepatitis B e antigen

(HBeAg). A secreted protein of the nucleocapsid gene of HBV. Seroconversion to HBeAg-negative is an important clinical marker in the natural history of HBV infection.

Heterochronous

Occurring or sampled at different times.

BCP variant

A variant with nucleotide changes in the basal core promoter of HBV that result in reduced expression of HBeAg.

PC variant

A variant with nucleotide changes in the pre-core region of the HBeAg that result in loss of expression of HBeAg.

HBV variants

Changes in the virus sequence that may or may not have consequences.

Splice variants

Variants produced as a result of splicing of HBV pregenomic RNA. These are incapable of autonomous replication but replicate in the presence of wild-type HBV.

Indels

Insertions or deletions of bases in the virus genome.

Hepatitis B core antigen

(HBcAg). The nucleocapsid (core) protein of HBV. A phosphoprotein that associates with the viral polymerase, DNA and RNA.

HBV quasi-species

The population of related viral species occurring within a patient.

Pseudotyped

Generation of viruses (or viral vectors) in combination with foreign viral envelope proteins.

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Revill, P.A., Tu, T., Netter, H.J. et al. The evolution and clinical impact of hepatitis B virus genome diversity. Nat Rev Gastroenterol Hepatol 17, 618–634 (2020). https://doi.org/10.1038/s41575-020-0296-6

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