THE DESIGN AND APPLICATION OF A REAL-TIME PCR ASSAY TO ASSESS rcDNA AND cccDNA PRODUCED BY HBV DURING INFECTION
Year: 2010
Type: dissertation
Abstract: Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the \navailability of anti-viral agents, there is currently no cure. This double stranded DNA virus is \nhepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular \nDNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA \ncontains a partially synthesised positive DNA stand and a gap region within the negative strand. \nAfter infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An \nimportant objective of HBV therapy is the elimination of cccDNA, as its persistence within \nhepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this \nreplication intermediate is crucial. The objective of this study was to develop a method based \non real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap \nwere designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV \nDNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV \ntransgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse \nlivers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA \nprofile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, \nonly 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results \nwere validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA \npools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA \ndetection as it allows for the rapid simultaneous amplification and quantification of a specific \nDNA target through either non-specific or specific DNA detection chemistries. In conclusion, this \nHBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy
Author: Kristie Bloom
Language: English
Topic: Hepatitis B Virus Studies
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2276801080