Archives of Virology

, Volume 164, Issue 2, pp 447–455 | Cite as

Analysis of fitness differences of hepatitis B virus genotypes D and F using a cotransfection assay

  • Ina SevicEmail author
  • María Mercedes Elizalde
  • María Mora González López Ledesma
  • Diego Martin Flichman
  • Rodolfo Héctor Campos
Original Article


Hepatitis B virus (HBV) circulates as a collection of genetically related variants that evolve throughout the chronic infection. Those viral variants that have the greatest fitness are fixed. We recently showed different fitness for HBV variants involved in two epidemiological situations. To understand these fitness differences better, we determined the levels of extracellular HBV DNA, the synthesis of HBV DNA intermediates, and the expression of HBeAg and HBsAg in transfection and cotransfection assays. Our results show that for the subgenotype (sgt) D1, which has an 8-nucleotide deletion (sgtD1del) and exhibits lower fitness, the levels of extracellular DNA and intracellular replicative intermediates were much lower than with sgtD1wt or sgtD1mut (G1896A), which had higher fitness. In addition, in the cotransfection assay, sgtD1del inhibited sgtD1mut but not sgtD1wt replication. We also found that sgtF1b, which exhibits higher fitness, produces significantly higher levels of both extracellular DNA and intracellular replicative intermediates than does the lower-fitness sgtF4. These results demonstrate a relationship between fitness and the replicative ability of the HBV genome in the transfection assay. In addition, the data obtained by cotransfecting cells with sgtD1del and sgtD1mut provide new information about the impact of simultaneous replication of two viral variants in the same cell system on HBV replication.



This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) [PIP2015-0595CO], Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [PICT2014-1672] and UBACyT [20020130100505BA 2014-2017].

Supplementary material

705_2018_4090_MOESM1_ESM.tif (1.1 mb)
Supplementary figure S1: Washing efficiency. Huh7 cells were transfected with a linear full-length HBV genome derivative from the pCH-9/3091 POL minus plasmid. Every 24 hours, supernatants were collected, cells were washed six times with PBS, and fresh medium was added. HBV DNA was extracted from collected supernatants and a 279-bp fragment of the X gene (the one that was used for real-time quantification) was amplified in order to detect residual input HBV DNA. Although input DNA was detected at 24 h and 48 h post-transfection, at 72 h and 96 h post-transfection, the latter being the time were supernatants were harvested, no remaining input DNA was observed. (TIFF 1170 kb)


  1. 1.
    WHO (2017) Hepatitis B. Fact sheet number 204.
  2. 2.
    Andino R, Domingo E (2015) Viral quasispecies. Virology 479–480:46–51. CrossRefGoogle Scholar
  3. 3.
    Gao S, Duan Z-P, Coffin CS (2015) Clinical relevance of hepatitis B virus variants. World J Hepatol 7:1086–1096. CrossRefGoogle Scholar
  4. 4.
    Sevic I, González López Ledesma MM, Flichman DM, Campos HR (2017) HBV DNA genome co-transfection procedure for the evaluation of relative fitness. PLoS One 12(5):e01:1–12. doi: .
  5. 5.
    Pezzano SC, Torres C, Fainboim HA et al (2011) Hepatitis B virus in Buenos Aires, Argentina: genotypes, virological characteristics and clinical outcomes. Clin Microbiol Infect 17:223–231. CrossRefGoogle Scholar
  6. 6.
    Rodrigo MB, Mojsiejczuk LN, Torres C et al (2016) Analysis of the molecular evolution of hepatitis B virus genotypes in symptomatic acute infections in Argentina. PLoS One 11:e0159509. CrossRefGoogle Scholar
  7. 7.
    Gonzalez Lopez Ledesma MM, Mojsiejczuk LN, Rodrigo B et al (2015) Hepatitis B virus genotype distribution and genotype-specific BCP/precore substitutions in acute and chronic infections in Argentina. PLoS One 10:1–16. CrossRefGoogle Scholar
  8. 8.
    Sede M, Lopez-Ledesma M, Frider B et al (2014) Hepatitis B virus depicts a high degree of conservation during the immune-tolerant phase in familiarly transmitted chronic hepatitis B infection: Deep-sequencing and phylogenetic analysis. J Viral Hepatol 21:650–661. CrossRefGoogle Scholar
  9. 9.
    Günther S, Li BC, Miska S et al (1995) A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69:5437–5444. Google Scholar
  10. 10.
    Yu X, Mertz JE (1996) Promoters for synthesis of the pre-C and pregenomic mRNAs of human hepatitis B virus are genetically distinct and differentially regulated. J Virol 70:8719–8726. Google Scholar
  11. 11.
    Geng Y, Wang X, Lu X et al (2016) Mutation changes in the preC/Core promoter in HBeAg-positive patients with chronic hepatitis b during interferon therapy. Medicine (Baltimore) 95:e2657. CrossRefGoogle Scholar
  12. 12.
    Milich D, Liang TJ (2003) Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology 38:1075–1086. CrossRefGoogle Scholar
  13. 13.
    Jammeh S, Tavner F, Watson R et al (2008) Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 89:901–909. CrossRefGoogle Scholar
  14. 14.
    Sallam TA, Tong CYW (2002) Two distinct types of hepatitis B virus core promoter variants in Yemeni blood donors. J Med Virol 68:328–334. CrossRefGoogle Scholar
  15. 15.
    Moriyama K (1997) Reduced antigen production by hepatitis B virus harbouring nucleotide deletions in the overlapping X gene and precore-core promoter. J Gen Virol 78:1479–1486CrossRefGoogle Scholar
  16. 16.
    Fischer SF, Schmidt K, Fiedler N et al (2006) Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1. World J Gastroenterol 12:6054–6058CrossRefGoogle Scholar
  17. 17.
    Liu D, Cui L, Wang Y et al (2016) Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity. Hepatology 64:390–404. CrossRefGoogle Scholar
  18. 18.
    Elizalde MM, Sevic I, González López Ledesma MM et al (2018) Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations. Virology 513:160–167. CrossRefGoogle Scholar
  19. 19.
    Liang TJ (2016) The X-files of hepatitis B. Nature 531:95–121. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Virología, Facultad de Farmacia y BioquímicaUniversidad de Buenos AiresBuenos AiresArgentina
  2. 2.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos AiresArgentina

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