Background

The Hepatitis B virus (HBV), a DNA virus belonging to the family Hepadnaviridae, is a human pathogen persisting in approximately 240 million people globally. HBV infection leads to higher risks of liver cirrhosis and hepatocellular carcinoma (Liang et al., 2015). At present, chronic HBV infection is not curable as current treatments do not eradicate the replicative reservoir, covalently closed circular (ccc)DNA (Levrero et al., 2016). In in vitro infected hepatocytes, cccDNA is formed by cellular repair of relaxed circular duplex (rc)DNA, the genomic form in virions (Guo et al., 2007; Long et al., 2017; Schreiner and Nassal, 2017). cccDNA serves as the viral template for pregenomic RNA which becomes encapsidated into nucleocapsids and is reverse transcribed to generate rcDNA and double-stranded linear (dsl)DNA by the viral polymerase (Bartenschlager and Schaller, 1992; Tu et al., 2017). Of note, albeit the reconstitution of hepatoma cells with the receptor human sodium taurocholate cotransporting polypeptide (NTCP), high excess of inoculated virions with high multiple genome equivalents (mges) (mges > 100) is required to achieve moderate infection rates (e.g., > 20% in HepG2^NTCP cells) (Qu et al., 2018). This raises the problem that cccDNA-containing samples taken early after inoculation with HBV contain large amounts of rcDNA from input virions.

Therefore, adequate methodologies for absolute or relative quantification of cccDNA are required. They are divided into two categories: (I) hybridization after separation by gel electrophoresis (Southern blot) and (II) PCR amplification (Li et al., 2017). Southern blot is still a gold standard but not very sensitive, and demands multiple experimental processes and a high-copy load of cccDNA (> 2 x 10⁶ copies using ³²P-radioactive isotope/digoxigenin/biotin probe; > 1 x 10⁴ copies using branched DNA technique) (Yu et al., 2015). Therefore, hybridization-based methods are complicated, time-consuming and not practical for a large number of samples (e.g., > 20) to be analyzed in parallel. PCR methods include real-time quantitative (q)PCR, nested qPCR (Xu et al., 2011), digital-droplet PCR (Mu et al., 2015), and rolling circle amplification (Margeridon et al., 2008). Real-time qPCR is the fastest and the most robust method for almost all laboratories. However, unlike Southern blot which gel electrophoretically separates cccDNA, PCR methods are not strictly specific, especially when rcDNA and other HBV replicative intermediates are present in excess, such as in in vitro infection (e.g., MGE > 300), even when cccDNA-specific primer pairs are used (Nassal, 2015; Qu et al., 2018).

To solve this problem, we have developed a qPCR assay using validated cccDNA selective primer pairs and a digestion step by T5 exonuclease, which removes cellular DNA and all HBV intermediates via its exonuclease activity targeting free ends of rcDNA and dslDNA but leaves cccDNA intact (Qu et al., 2018). This assay allowed fast and specific quantification of cccDNA within one working day as shown in Figure 1 (2 h of total DNA extraction; 1.5 h of T5 exonuclease reaction and 2 h of qPCR), accurate calculation of “cccDNA copies/(infected) cell” (Figure 2) and drug efficacy testing on cccDNA levels (Figure 3). The method also provided quantitative judgment on whether cccDNA is formed in new cell models and identified low amount of cccDNA in in vitro infection of non-primate hepatocytes (Lempp et al., 2016 and 2017). This protocol is adapted from Qu et al. (2018) and herein more detailed information on this qPCR quantification after T5 exonuclease digestion is included, and different primer pairs are compared to address the applicability of HBV genotypes. Taken together, this protocol will facilitate studies on cccDNA and help clinicians, technicians and graduate students to analyze cccDNA in samples derived from in vitro infection.