A novel method to precisely quantify Hepatitis B Virus covalently closed circular (ccc)DNA formation and maintenance.

A novel method to precisely quantify Hepatitis B Virus covalently closed circular (ccc)DNA formation and maintenance. Antiviral Res. 2020 Jul 26;:104865 Authors: Tu T, Zehnder B, Qu B, Ni Y, Main N, Allweiss L, Dandri M, Shackel N, George J, Urban S Abstract Hepatitis B virus (HBV) is the major cause of viral-associated liver disease. Persistent HBV infection is maintained by its episomal genome (covalently closed circular DNA, cccDNA), which acts as a template for viral transcripts. The formation of cccDNA is poorly characterised due to limited ability to quantify it accurately in the presence of replicative intermediates. Here, we describe a novel cccDNA quantification assay (cccDNA inversion quantitative PCR, cinqPCR), which uses restriction enzymes to invert a DNA sequence close to the gap region of Genotype D HBV strains, including the isolate widely-used in experimental studies. Importantly, cinqPCR allows simultaneous normalisation to cellular DNA in a single reaction, provides absolute copy numbers without requiring a standard curve, and has high precision, sensitivity, and specificity for cccDNA compared to previous assays. We first established that cinqPCR gives values consistent with classical approaches in both in vitro and in vivo (humanized mice) HBV infections. We then used cinqPCR to find that cccDNA is formed within 12 hours post-inoculation (hpi). cccDNA formation slowed by 28hpi despite de novo synthesis of HBV DNA...
Source: Antiviral Research - Category: Virology Authors: Tags: Antiviral Res Source Type: research