36.    Analysis of fitness differences of HBV viral variants by co-transfection assay

Hepatitis B Virus (HBV) due to its quasispecies population structure circulates as a collection of genetically related variants that evolve throughout the chronic infection. As a result of the evolutionary process, those viral variants that have the greatest fitness for each particular situation are fixed. We recently showed different fitness for variants involved in two epidemiological situations. To further understand these fitness differences, we evaluated the extracellular HBV DNA levels, the synthesis of HBV DNA intermediates, and HBeAg and HBsAg expression in transfection and cotransfection assays. Our results show that for the sgtD1del (deletion 1763-1770), which presents lower fitness, extracellular DNA levels and intracellular replicative intermediaries were highly diminished comparing with both sgtD1wt and sgtD1mut (G1896A), which had higher fitness. In addition, in the co-transfection assay sgtD1del inhibited sgtD1mut but not sgtD1wt replication. We also showed that sgtF1b, which presents higher fitness, produces significantly higher levels of both extracellular DNA and intracellular replicative intermediaries comparing to the lower fitness sgtF4. In summary, these results evidence the relationship between fitness and replicative ability of HBV genome in transfection assay. In addition, the cotransfection with sgtD1del and sgtD1mut constitutes the first evidence of the impact on HBV replication when two viral variants replicate simultaneously in the same cellular system.