28 Three different constructs were selected, each carrying the mutant (mt) viral isolate representative of the dominant HBV population infecting patients 14, 4, and 8 (pHBV-mtpreS1, pHBV-mtpreS2, and pHBV-mtS, respectively) (Fig. 1A). Linear HBV monomers were released from pHBV-mtpreS1, pHBV-mtpreS2, and pHBV-mtS constructs and from plasmid pUC-HBV (genotype D), used as a WT control, by way of cleavage with the restriction enzyme
SapI (New England Biolabs, Ipswich, MA). After digestion, linear HBV genomes were gel-purified and MK-1775 mw transiently transfected into HepG2 cells using the FuGENE transfection reagent (Roche Applied Science). Briefly, HepG2 cells were seeded at a density of 1 × 106 cells in 100-mm-diameter Petri dishes and transfected 24 hours later with 2 μg of SapI-digested HBV DNA. Culture medium was changed 1 day after transfection, and cells harvested 1 day later. All transfections included 1 μg of reporter plasmid expressing enhanced green fluorescence protein to assess transfection efficiency. All transfection experiments were done at least three times, each time using independently prepared HBV DNA (Qiagen Maxi Preparation Kit). Statistical analysis was performed buy Staurosporine by SPSS version
11.0 software package (SPSS Inc, Chicago, IL). A nonparametric approach was used to examine variables showing absence of a normal distribution, as verified by the Kolmogorov-Smirnov test. The interdependence between numerical variables was performed by the use of the Spearman
rank correlation test, whereas the Mann-Whitney test was applied to perform comparisons of continuously distributed variables between two independent groups. To evaluate the association between categorical variables, the log-likelihood ratio test was applied. P < 0.05 were considered as statistically significant. Quantification analyses showed a significant positive correlation selleck chemicals between HBsAg (median, 2.3 × 103 IU/mL; range, 56-9.4 × 104 IU/mL) and HBV DNA (median, 2.5 × 105 IU/mL; range, 482-2.4 × 108 IU/mL) serum levels (r = 0.416; P = 0.008) in the study population (Fig. 2A). However, when HBeAg-positive and HBeAg-negative subgroups of patients were separately examined, no correlation was found between HBsAg and HBV DNA levels in either subgroup (Fig. 2B,C), likely because of the limited number of individuals included in each of them. HBeAg-positive patients had significantly higher serum HBV DNA levels (median, 1 × 108 IU/mL; range, 2.1 × 104-1.1 × 108; P = 0.017) compared to HBeAg-negative cases (median, 2.3 × 106 IU/mL; range, 482-2.3 × 108), whereas the median HBsAg titer did not differ significantly between the two subgroups (4.9 × 103 IU/mL versus 2 × 103 IU/mL, P = 0.27).