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. 2014 Dec 16;89(1):384–405. doi: 10.1128/JVI.02361-14

TABLE 3.

Analysis of control WHVNY DNA samples during the detection of RI-DNA and cccDNA using WHVNY-specific PCR assaysa

Sample and amountb (GE) RI-DNA (total DNA prepn)
 cccDNA (Hirt)
 cccDNA (Hirt + PSD)
GE Recovery (%) GE Recovery (%) GE Recovery (%)
Plasmidc
    1.09 × 107 1.07 × 107 97.8 8.71 × 106 80.0 9.82 × 106 90.1
    1.05 × 104 1.29 × 104 122.3g 1.76 × 104 167.5g 1.23 × 104 116.5g
    9.02 × 103 7.44 × 103 82.5 1.09 × 104 120.8g 4.75 × 103 52.6
    7.43 × 102 7.95 × 102 107.0g 1.28 × 103 172.2g 3.46 × 102 46.6
    1.42 × 102 1.11 × 102 78.0 2.28 × 102 160.5g 1.96 × 102 137.9g
DSLd
    3.30 × 104 1.90 × 104 57.7 0.0 0.0 0.0 0.0
    1.04 × 104 1.69 × 104 162.5g 0.0 0.0 0.0 0.0
    1.97 × 103 1.97 × 103 99.6 0.0 0.0 0.0 0.0
    3.85 × 102 5.55 × 102 144.0g 0.0 0.0 0.0 0.0
    2.03 × 102 2.34 × 102 115.1g 0.0 0.0 0.0 0.0
    4.62 × 101 5.64 × 101 122.2g 0.0 0.0 0.0 0.0
pf-rcDNAe
    2.00 × 106 1.74 × 106 87.0 3.19 × 103 0.16 2.00 × 101 0.001
    2.00 × 106 6.84 × 105 34.2 3.47 × 103 0.17 0.0 0.0
    2.00 × 104 2.38 × 104 118.8g 9.53 × 100 0.05 0.0 0.0
    2.00 × 104 6.05 × 103 30.3 4.52 × 101 0.23 0.0 0.0
    2.00 × 103 1.62 × 102 8.1 0.0 0.0 0.0 0.0
    2.00 × 103 3.76 × 102 18.8 0.0 0.0 0.0 0.0
Serum rcDNAf
    2.00 × 106 1.88 × 106 93.0 1.27 × 102 0.01 0.0 0.0
    2.00 × 106 3.11 × 106 155.7g 3.38 × 102 0.02 0.0 0.0
    2.00 × 104 1.72 × 104 86.0 0.0 0.0 0.0 0.0
    2.00 × 104 2.43 × 104 121.6g 0.0 0.0 0.0 0.0
a

The WHVNY strain-specific qPCR assays that amplified either RI-DNA or cccDNA, the isolation of total DNA, Hirt extract preparation, and subsequent treatment with PSD are described in detail in Materials and Methods.

b

WHVNY DNA control samples were used to evaluate the isolation/treatment procedures, the specificity of the qPCR assays, and the degree of recovery of certain DNA intermediates of WHVNY replication. Each control sample (the number of WHVNY GE is indicated) was added to a piece of liver tissue (approximately 50 to 100 mg) harvested from a WHV-negative (naive) adult woodchuck, and then the preparation of DNA for analysis was conducted as indicated. The results are shown in WHVNY DNA GE and as the percentage of recovery relative to the starting amounts of the input control sample. In addition, all WHVNY-specific control DNA samples listed were tested using analogous WHV7-specific qPCR assays (for the detection of the RI-DNA in total DNA preparation and cccDNA in Hirt and Hirt + PSD preparations), which, as expected, did not detect any of the WHVNY DNA samples.

c

Uncut plasmid pLR-WHVNY-001 harboring WHVNY sequences (described in Materials and Methods) was used as the surrogate to evaluate the parameters of analysis of cccDNA.

d

A surrogate of DSL of WHVNY was prepared by PCR, using the plasmid pJSWHVNY-4C4 as the template, followed by gel purification of the obtained PCR product. The WHVNY DSL mimic is a linear, double-stranded, greater-than-genome-length DNA that, like its natural counterpart, has the left-hand position at nucleotide 1935 and the right-hand position at nucleotide 1945 (38). The details of the preparation of the WHVNY DSL surrogate are described in Materials and Methods. For the surrogate of cccDNA and surrogate of DSL, the initial concentration measurements were performed using a NanoDrop 2000. The dilutions were made based on these measurements, and then the concentrations of diluted samples were verified by qPCR that amplified the RI-DNA (the same qPCR as that used to quantify the rcDNA in sera), and the actual qPCR measurements are shown as the starting amounts of input samples.

e

To evaluate the parameters of analysis of protein-free rcDNA (pf-rcDNA), the rcDNA of WHVNY was isolated from the serum of woodchuck F6541, which was monoinfected with WHVNY.

f

To examine the parameters of analysis of serum-associated rcDNA (i.e., protein-bound rcDNA), the serum that contained rcDNA of WHVNY from woodchuck F6541, which was monoinfected with the strain WHVNY, was used. The concentrations of isolated rcDNA were measured by qPCR specific for RI-DNA, and the dilutions for input material (both kinds of rcDNA control samples) were made based on the actual qPCR measurements.

g

On several occasions the calculated recovery exceeded 100%, which was less than 2-fold different from the actual 100% value, reflected a variation in qPCR measurements, and was considered a complete recovery of the input sample.