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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
  • 69 Downloads

Abstract

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.

Notes

Funding

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)

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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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