Differences in sequences between HBV-relaxed circular DNA and covalently closed circular DNA

Abstract

The hepatitis B virus (HBV) genome exists in two forms: circular covalently closed DNA (cccDNA) and relaxed circular DNA (RCDNA). Here, we investigated the presence of differences in the sequences of both forms in paired samples of serum and liver tissue. The serum and liver biopsy samples were collected at the same time from 67 chronically infected patients. The genotyping of the RCDNA and cccDNA was performed using mass spectrometry analysis. The HBV mutations located in the HBV pol (P) and the HBV basal core promoter/pre-core (BCP/PC) regions were included. The BCP/PC and P sequences of the RCDNA extracted from liver and blood samples were different in 39% and 16% of patients, respectively. Differences were also found between RCDNA and cccDNA extracted from the same liver specimen. Moreover, the cccDNA BCP/PC region sequence had an impact on various virological and clinical parameters. We demonstrated that there are differences between the RCDNA and cccDNA sequences that were extracted from the same liver tissue. However, further investigations are needed to analyze whether the mutations in the cccDNA are conserved and whether cccDNA serves as a ‘mutation storage’ pool for HBV. This result could have profound implications for the subsequent therapy choices for treatment-experienced patients.

Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses

“…For comparison, the HBeAg-positive capuchin monkey HBV (CMHBV) showed significantly more SNPs in serum (1.8 SNPs per 100 nucleotides; χ 2 , P < 0.0001) (24). This difference is noteworthy because liver contains the covalently closed circular DNA reservoir showing relatively higher virus diversity than the relaxed circular DNA found in circulating virions (31). Of note, significant differences in SNP occurrence rates were observed neither between coding nor noncoding shrew HBV genomic regions, nor between single or overlapping ORFs, nor between the different shrew genera (SI Appendix, Table S5).…”

Insights From Deep Sequencing of the HBV Genome—Unique, Tiny, and Misunderstood

“…Simultaneous, competing evolutionary pressures can create different subpopulations of HBV within patients (quasispecies) and at the population level. These can produce a more diverse RC-DNA population and a less diverse and stable cccDNA population, with different sequence polymorphisms potentially archived in the cccDNA pool 128 (Figure 2 Bv ).…”

A Highly Sensitive and Robust Method for Hepatitis B Virus Covalently Closed Circular DNA Detection in Single Cells and Serum

“…15 Rolling circle amplification is a potential alternative to increase the sensitivity of cccDNA detection, 16,17 but this has not been widely accepted. 6,18 To increase the specificity, a cccDNA PCR, using specific primers and probes to selectively amplify cccDNA and exclude rcDNA, was developed. This is achieved as a result of using primers spanning the nick of rcDNA and hybridizing the probe to its gap region.…”

“…19 Reducing rcDNA copy number is another way to increase the accuracy of cccDNA detection. Pretreatment of HBV-DNA with Plasmid-Safe ATP-Dependent DNase (PSAD) (Epicentre, Madison, WI) 15 or T5 exonuclease (NEB, Beverly, MA) 18 to degrade DNA with free ends can improve discriminatory power up to 1000-fold. 15 Although cccDNA-selective PCR provides a relative discrimination between cccDNA and rcDNA, additional means are still required to reduce the false-positive amplification of rcDNA.…”