Inhibition of HCV replication by ODNs assessed by replicon-expressing Huh-7 clone 9b cells. In figures (A) to (E), 6 × 104, 4 × 104 or 2 × 104 cells were seeded for 24 h, 48 h, or 72 h of incubation, respectively, as indicated above the graphs, 24 h prior to transfection as described in the text. ‘-‘denotes untransfected control cells otherwise treated identically. (A) Cells were transfected with FITC-labeled ODN A. After 24 h, cells were detached by trypsinization, paraformaldehyde-fixed and investigated with an inverted Leica SP5 fluorescence microscope. FITC was excited at 488 nm. (B) to (E), cells were transfected with 160 nM of the indicated ODNs, unless noted otherwise. Levels of significance were determined by two-sided Student’s t-Tests, *P≤0.05, **P≤0.01, against ODN A in (C) and (E), and against untransfected control cells in (D). Bars indicate mean values ± S.E.M of at least two independent experiments in duplicate. (F) Sequence comparison of the ODN 320 target region in the HCV 5’ NTR (bold) and flanking sequences, among genotypes 1 to 6. Accession numbers are [GenBank:AF009606], [GenBank:NC_009823], [GenBank:NC_009824], [GenBank:NC_009825], [GenBank:NC_009826], [GenBank:NC_009827] for genotypes 1 through 6. n.d., not determined; n.s., not significant (t-Test).