Over-gap PCR amplification to identify presence of replication-competent HBV DNA from integrated HBV DNA: An updated occult HBV infection definition

Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infectionJournal of HepatologyVol. 69Issue 2PreviewOccult hepatitis B virus (HBV) infection (OBI) refers to the presence of intrahepatic HBV DNA in the absence of detectable hepatitis B surface antigen (HBsAg).1 OBI is secondary to overt HBV infections; it guarantees the persistence of the virus in a cryptic form protected from the immune response of the host. The virologic key is the covalently closed circular DNA (cccDNA), an HBV DNA form generated as a plasmid-like episome from the protein-linked relaxed circular DNA genome; it resides in the nucleus of infected cells and gives rise to viral sequences, which act as a transcription template for all viral RNAs. Full-Text PDF Reply to: “Over-gap PCR amplification to identify presence of replication-competent HBV DNA from integrated HBV DNA: An updated occult HBV infection definition”Journal of HepatologyVol. 70Issue 3PreviewRecently, we developed a novel digital droplet PCR (ddPCR) assay for the selective quantitation of intrahepatic HBV covalently-closed-circular DNA (cccDNA), the plasmid-like episome form of HBV DNA that serves as template for all viral RNAs.1 We used this method to determine the prevalence and quantity of HBV cccDNA in individuals with occult HBV infection (OBI) recruited among a cohort of 100 hepatitis B surface (HBsAg)-negative/antibody to hepatitis B core antigen (anti-HBc)-positive liver donors. Full-Text PDF With great interest, we read the manuscript “Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection” by Caviglia et al. published in Journal of Hepatology.[1]Caviglia G.P. Abate M.L. Tandoi F. Ciancio A. Amoroso A. Salizzoni M. et al.Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection.J Hepatol. 2018; 69: 301-307Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Using a highly sensitive in-house droplet digital PCR assay (ddPCR) method, the authors indicated that intrahepatic HBV covalently closed circular (cccDNA) was detectable in about half (52%, 27/52) of the defined cases of occult HBV infection (OBI). We wonder whether the pretreatment with plasmid-safe ATP dependent DNase (PSAD) plus double-over-gap cccDNA ‘specific’ primers spanning the HBV relaxed circular DNA (rcDNA) gap region used in this paper could totally eliminate the interference of rcDNA, though this method had been widely used in the detection of cccDNA.[2]Guo F. Zhao Q. Sheraz M. Cheng J. Qi Y. SU Q. et al.HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.PLoS Pathog. 2017; 13e1006658Crossref PubMed Scopus (80) Google Scholar Here we evaluated the capacity of the above approach to discriminate between the cccDNA, the rcDNA and the integrated double strand linear HBV DNA (dslDNA). In addition, several sets of mono-over-gap rcDNA primers (Table S1) were also tested, which theoretically can amplify both rcDNA and cccDNA.[3]Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.Gut. 2015; 64: 1972-1984Crossref PubMed Scopus (550) Google Scholar First, to exclude the likely cccDNA contaminant leaked from cells, the supernatant of HepAD38[2]Guo F. Zhao Q. Sheraz M. Cheng J. Qi Y. SU Q. et al.HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.PLoS Pathog. 2017; 13e1006658Crossref PubMed Scopus (80) Google Scholar and serum specimens from patients with HBV infection[4]Li X. Zhang J. Yang Z. Kang J. Jiang S. Zhang T. et al.The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma.J Hepatol. 2014; 60: 975-984Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar were treated with DNase I prior to viral DNA extraction and PCR amplification. The elimination efficiency was confirmed by the failed amplification of plasmid DNA containing 1.2xHBV genome (Fig. 1A). In contrast, the HBV rcDNA in Dane particles could still be detected by using the supposed cccDNA ‘specific’ primers, which provided a similar result compared to rcDNA primers. Moreover, the gradual increase of HBV DNA level was observed in parallel with the increased amount of rcDNA, when either the supposed cccDNA primers or the rcDNA primers were used (Fig. 1B). As previously reported,[5]Kock J. Rosler C. Zhang J.J. Blum H.E. Nassal M. Thoma C. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner.PLoS Pathog. 2010; 6e1001082Crossref PubMed Scopus (111) Google Scholar the rcDNA could not be eliminated completely pretreatment by PSAD and this was further confirmed by T5 Exonuclease and Exonuclease III, respectively (Fig. 1C, D). Hence, PSAD digestion plus double-over-gap PCR may not guarantee the discrimination of cccDNA from rcDNA. The term ‘occult hepatitis B virus infection’ has been introduced to describe a status characterized as an absence of serum HBV surface antigen and presence of replication-competent HBV DNA in the liver.6Pollicino T. Raimondo G. Occult hepatitis B infection.J Hepatol. 2014; 61: 688-689Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 7Raimondo G. Balsano C. Craxi A. Farinati F. Levrero M. Mondelli M. et al.Occult hepatitis B virus infection.Dig Liver Dis. 2000; 32: 822-826Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 8Raimondo G. Allain J.P. Brunetto M.R. Buendia M.A. Chen D.S. Colombo M. et al.Statements from the Taormina expert meeting on occult hepatitis B virus infection.J Hepatol. 2008; 49: 652-657Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar Since cccDNA is the resource for viral replication and the reason for HBV infection persistence, the presence of cccDNA for OBI is indispensable. The integrated HBV DNA fragments, on the other hand, have an incomplete viral genome which lost the capacity to serve as the template for HBV replication. Therefore, it is reasonable to postulate that the detection of cccDNA, but not the presence of integrated HBV DNA fragment, is essential for true OBI. Moreover, it may not be necessary to distinguish cccDNA from rcDNA for the definition of OBI because the rcDNA originates solely from the transcriptionally active cccDNA.[9]Wang G.H. Seeger C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis.Cell. 1992; 71: 663-670Abstract Full Text PDF PubMed Scopus (320) Google Scholar Integration of HBV DNA fragments is a common event during HBV infection. Our previous study revealed that the breakpoints of the integrated HBV DNA fragments were mainly found within the DR1 and DR2 regions (Fig. 1E).[4]Li X. Zhang J. Yang Z. Kang J. Jiang S. Zhang T. et al.The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma.J Hepatol. 2014; 60: 975-984Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar This is in accordance with the suggestion that HBV dslDNA is the preferred form for viral DNA integration into the host genome.[10]Yang W. Summers J. Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor.J Virol. 1999; 73: 9710-9717PubMed Google Scholar To test if the integrated HBV DNA could interfere with the amplification specificity of cccDNA or rcDNA primers, a 1.0 × HBV plasmid carrying the full-length HBV genome ranging from 1,821 to 1,825 was constructed to mimic the HBV integration (Fig. 1F). Since the orientation of cccDNA and/or rcDNA paired primers were outward to each other, PCR amplification would be invalid when using 1.0xHBV plasmid as template. As expected, neither cccDNA nor rcDNA primers had valid PCR amplification. In contrast, successful PCR reactions were observed with the 1.2 × HBV plasmid, which could mimic cccDNA from the liver tissues of patients with chronic HBV.[4]Li X. Zhang J. Yang Z. Kang J. Jiang S. Zhang T. et al.The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma.J Hepatol. 2014; 60: 975-984Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Nevertheless, the other three primer sets (Table S2) specific to PreC/C, S and X regions could amplify all three kinds of templates (Fig. 1G). Taken together, these results indicated mono- or double-over-gap PCR primers could distinguish cccDNA and rcDNA from integrated HBV DNA. In summary, we proposed the absence of serum HBV surface antigen and the presence of HBV rcDNA and/or cccDNA as the true OBI. The mono- or double-over-gap PCR primers could specifically amplify the rcDNA and/or cccDNA but not the integrated DNA, which could be used in laboratory-based identification for OBI in the future. This work was supported by grants from the National S & T Major Project for Infectious Diseases of China (Nos. 2017ZX10302201, 2017ZX10202203 and 2017ZX10202202) and the Natural Science Foundation of China (No. 81572366). The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details. Lu F, Liu Y, Liao H designed the research. Liu Y, Zeng W, Xi J, Liu H, Liao H and Yu G performed the research. All authors analyzed the data. Liu Y wrote the paper. Lu F and Chen X revised the paper. 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