Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the
availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is
hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular
DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA
contains a partially synthesised positive DNA stand and a gap region within the negative strand.
After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An
important objective of HBV therapy is the elimination of cccDNA, as its persistence within
hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this
replication intermediate is crucial. The objective of this study was to develop a method based
on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap
were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV
DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV
transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse
livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA
profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells,
only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results
were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA
pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA
detection as it allows for the rapid simultaneous amplification and quantification of a specific
DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this
HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/8576 |
| Date | 30 August 2010 |
| Creators | Bloom, Kristie Michelle |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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