Table 1. cccDNA copy number in HBV-infected HepG2-NTCP-K7 cells.
| Multiplicity of infection | Southern blota | qPCRb | ||
|---|---|---|---|---|
| per cell | per infected cell | per cell | per infected cell | |
| 100 vp/cell | 2.5 ± 1.0 | 9.2 ±3.8 | 1.4±0.6 | 5.0±2.3 |
| 300 vp/cell | 5.3 ± 0.9 | 9.9 ± 1.8 | 3.0 ± 1.2 | 5.7±2.2 |
| 1000 vp/cell | 9.6 ± 0.9 | 12.5 ±1.2 | 8.2 ± 2.1 | 10.7±2.7 |
HBV-infected HepG2-NTCP-K7 cells were harvested at 3 dpi for DNA extraction and subjected to cccDNA copy number determination. Prior to DNA extraction, cells were trypsinized and counted. The number of infected cells was determined by counting core protein-positive cells (Fig. 1D). Mean ± SD is given.
Protein-free DNAs extracted by modified Hirt method were subjected to Southern blot analysis. Standard curves were generated by preparing serial dilution of a 3.2 kb double-stranded linear HBV genome. The experiment was performed twice.
Total cellular DNA was subjected to cccDNA-selective qPCR without prior T5 exonuclease treatment. Standard curves were generated by preparing serial dilution of a plasmid DNA-containing HBV monomer for qPCR. The experiment was repeated three times. cccDNA, covalently closed circular DNA; dpi, days post infection; HBV, hepatitis B virus; qPCR, real-time PCR; vp, virus particle.