The design and application of a real-time PCR assay to assess rcDNA and cccDNA produced by HBV during infection

摘要

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite theudavailability of anti-viral agents, there is currently no cure. This double stranded DNA virus isudhepatotropic, and active viral replication results in two genomic equivalents, the relaxed circularudDNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNAudcontains a partially synthesised positive DNA stand and a gap region within the negative strand.udAfter infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. Anudimportant objective of HBV therapy is the elimination of cccDNA, as its persistence withinudhepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for thisudreplication intermediate is crucial. The objective of this study was to develop a method basedudon real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gapudwere designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBVudDNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBVudtransgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouseudlivers, but was present at low concentrations in serum samples. The intrahepatic HBV DNAudprofile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells,udonly 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These resultsudwere validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNAudpools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNAuddetection as it allows for the rapid simultaneous amplification and quantification of a specificudDNA target through either non-specific or specific DNA detection chemistries. In conclusion, thisudHBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy