TABLE 5.
Detection of RI-DNA and cccDNA of WHVNY and WHV7 in collected tissue samplesa
| Animal and tissue type | Tissue sample | WHVNYb |
WHV7b |
||
|---|---|---|---|---|---|
| RI-DNA | cccDNAc | RI-DNA | cccDNA (Hirt + PSD) | ||
| F7808 | |||||
| Liver | LM | 2.08 ± 2.05 | ND | (6.04 ± 1.65) × 108 | (7.17 ± 3.19) × 105 |
| RL | 1.01 ± 0.90 | ND | (8.52 ± 7.05) × 108 | (9.14 ± 4.98) × 105 | |
| LL | 2.50 ± 1.35 | ND | (54.8 ± 5.30) × 107 | (6.43 ± 3.82) × 105 | |
| HCC | T1 | 6.46 ± 1.70 | ND | (1.54 ± 1.60) × 108 | 1.71 × 104 ± NA |
| T2 | 0.32 ± NA | ND | (6.04 ± 4.26) × 106 | (14.0 ± 9.34) × 103 | |
| T3 | 0.79 ± 0.36 | 6.44 × 10−4 ± NA | (753 ± 2.15) × 105 | (19.6 ± 5.81) × 104 | |
| F7806 | |||||
| Liver | LM | 9.49 ± 10.96 | 0.38 ± 0.53 | (23.9 ± 5.99) × 107 | (2.73 ± 1.69) × 105 |
| RL | 4.65 ± 2.16 | ND | (42.6 ± 9.69) × 107 | (4.16 ± 3.08) × 105 | |
| LL | 6.53 ± 9.76 | 0.27 ± 0.38 | (2.27 ± 1.85) × 108 | (7.35 ± 5.86) × 105 | |
| HCC | T1 | 1.26 ± 1.11 | 0.01 ± 0.01 | (2.17 ± 2.60) × 107 | (3.82 ± 2.95) × 104 |
| T2 | 8.29 ± 6.24 | 4.00 × 10−5 ± NA | (26.8 ± 4.77) × 107 | (1.13 ± 1.01) × 105 | |
| T3 | 3.43 ± 0.74 | ND | (9.22 ± 3.39) × 107 | (6.96 ± 4.36) × 104 | |
| M7761 | |||||
| Liver | LM | (5.30 ± 2.23) × 102 | 14.4 ± 5.53 | (13.8 ± 5.43) × 107 | (1.21 ± 1.26) × 106 |
| RL | (4.95 ± 1.15) × 102 | 6.50 ± 1.54 | (15.6 ± 1.47) × 107 | (1.61 ± 1.81) × 106 | |
| LL | (5.35 ± 1.95) × 102 | 9.30 ± 1.25 | (11.6 ± 1.67) × 107 | (1.11 ± 1.08) × 106 | |
| HCC | T1 | 2.00 ± 2.60 | ND | (16.4 ± 3.70) × 105 | (3.24 ± 2.27) × 102 |
| T2 | (47.8 ± 1.81) × 101 | 7.71 ± 1.02 | (10.6 ± 2.43) × 107 | (9.80 ± 7.17) × 105 | |
| T3 | (2.80 ± 1.11) × 101 | 2.53 ± 0.33 | (8.79 ± 2.74) × 106 | (3.94 ± 2.89) × 104 | |
| T4 | (4.90 ± 2.70) × 101 | 0.87 ± 1.10 | (10.7 ± 5.79) × 106 | (20.1 ± 7.55) × 103 | |
| T5 | (5.08 ± 2.58) × 102 | 7.96 ± 7.80 | (15.1 ± 5.63) × 107 | (7.35 ± 7.86) × 105 | |
The description of the animals and collected tissue samples that were used for the analysis is provided in the footnotes to Table 1. For each tissue assayed, the RI-DNA and cccDNA of WHV7 were detected during each independent DNA preparation. The RI-DNA and cccDNA of WHVNY were not always detected in each independent DNA preparation conducted for the same tissue sample. The frequency of detection of RI-DNA and cccDNA for WHVNY is summarized in Table 6. The values shown for RI-DNA and cccDNA of WHV are the averages from the analysis of at least two independent DNA preparations, with exceptions for the cases when WHVNY DNA was found in only one independent isolation per tissue sample. The results are expressed as WHV GE/μg of total DNA. The amounts of total DNA used for the calculations are based on the results of the measurements in the total DNA preparations. Thus, the cccDNA numbers also were calculated per μg of DNA in the corresponding total DNA preparations. For calculating the average values, only data from experiments when WHV DNA was detected have been used. The standard deviations are provided. NA (not applicable) indicates that WHVNY DNA was detected in only one DNA preparation.
The preparation of DNA samples for the subsequent quantification of either RI-DNA or cccDNA and WHV strain-specific qPCR assays used are described in Materials and Methods.
Hirt extracts were used to quantify cccDNA of WHVNY in the samples from woodchucks F7808 and F7806; Hirt extracts treated with PSD were used to measure cccDNA of WHVNY in the samples from animal M7761. ND, not detected.