Figure 2. Detection of WHV cccDNA and WHV RNA in liver and PBMC samples obtained at 34–40 months post-infection from woodchucks with POI established by inoculation with 10 WHV/tm5 virions.

For detection of WHV cccDNA (top panel), total DNA from PBMC (P) or liver (L) was digested with a single-strand-specific nuclease and amplified by PCR with primers spanning the nick genomic region of WHV. Southern blot hybridization was applied to validate controls and confirm specificity of the 674-bp amplicons. For the detection of WHV RNA (bottom panel), DNase-treated total RNA was reverse transcribed (RT+) or not (RT−) prior to PCR amplification with WHV X gene-specific primers. The specificity of 192-bp amplicons and controls was confirmed by Southern blot hybridization analysis. In both panels, contamination controls consisted of water added to direct (DW) and nested (NW) reactions instead of test DNA or cDNA. Mock samples (M) were extracted and treated as test samples in all steps. DNA and RNA from the liver of a serum WHsAg-positive woodchuck with chronic hepatitis served as positive controls, respectively.