Abstract
Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.
Similar content being viewed by others
References
van Zonneveld M, Honkoop P, Hansen BE et al (2004) Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B. Hepatology 39:804–810
Nguyen VT, Law MG, Dore GJ (2009) Hepatitis B-related hepatocellular carcinoma: epidemiological characteristics and disease burden. J Viral Hepat 16:453–463
Yang SS, Hsu CT, Hu JT, Lai YC, Wu CH (2002) Lamivudine does not increase the efficacy of interferon in the treatment of mutant type chronic viral hepatitis B. World J Gastroenterol 8:868–871
Lewin SR, Ribeiro RM, Walters T et al (2001) Analysis of hepatitis B viral load decline under potent therapy: complex decay profiles observed. Hepatology 34:1012–1020
Yokosuka O, Omata M, Imazeki F, Okuda K, Summers J (1985) Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA. Hepatology 5:728–734
Addison WR, Walters KA, Wong WW et al (2002) Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication. J Virol 76:6356–6363
Lu M, Hilken G, Yang D, Kemper T, Roggendorf M (2001) Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks. J Virol 75:3811–3818
Zhu Y, Yamamoto T, Cullen J et al (2001) Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 75:311–322
Werle-Lapostolle B, Bowden S, Locarnini S et al (2004) Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126:1750–1758
Wursthorn K, Lutgehetmann M, Dandri M et al (2006) Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B. Hepatology 44:675–684
Thompson AJ, Nguyen T, Iser D et al (2010) Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers. Hepatology 51:1933–1944
Takkenberg RB, Zaaijer HL, Molenkamp R et al (2009) Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients. J Med Virol 81:988–995
Takkenberg RB, Zaaijer HL, Menting S et al (2010) Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients. Eur J Gastroenterol Hepatol 22:952–960
Wong DK, Yuen MF, Yuan H et al (2004) Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 40:727–737
Laras A, Koskinas J, Dimou E, Kostamena A, Hadziyannis SJ (2006) Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients. Hepatology 44:694–702
Zhong YW, Liang ZL, Ren XQ et al (2008) Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay. Chin J Exp Clin Virol 22:225–227
Yuen MF, Wong DK, Sum SS et al (2005) Effect of lamivudine therapy on the serum covalently closed-circular (ccc) DNA of chronic hepatitis B infection. Am J Gastroenterol 100:1099–1103
Chen Y, Sze J, He ML (2004) HBV cccDNA in patients’ sera as an indicator for HBV reactivation and an early signal of liver damage. World J Gastroenterol 10:82–85
Lok AS, McMahon BJ (2007) Chronic hepatitis B. Hepatology 45:507–539
Ishak K, Baptista A, Bianchi L et al (1995) Histological grading and staging of chronic hepatitis. J Hepatol 22:696–699
Singla B, Chakraborti A, Sharma BK et al (2013) Hepatitis B virus reverse transcriptase mutations in treatment naïve chronic hepatitis B patients. J Med Virol 85:1155–1162
Yin JL, Shackel NA, Zekry A et al (2001) Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol 79:213–221
Tuttleman JS, Pourcel C, Summers J (1986) Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47:451–460
Newbold JE, Xin H, Tencza M et al (1995) The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 69:3350–3357
Shao J, Wei L, Wang H et al (2007) Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B. World J Gastroenterol 13:2104–2107
Sakugawa H, Nakasone H, Nakayoshi T et al (2001) Correlation between serum transaminase activity and virus load among patients with chronic liver disease type B. Hepatol Res 21:159–168
Wong DK, Seto WK, Fung J et al (2013) Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency. Clin Gastroenterol Hepatol 11:1004–1010
Wang M, Qiu N, Lu S et al (2013) Serum hepatitis B surface antigen is correlated with intrahepatic total HBV DNA and cccDNA in treatment-naïve patients with chronic hepatitis B but not in patients with HBV related hepatocellular carcinoma. J Med Virol 85:219–227
Cabrerizo M, Bartolomé J, Caramelo C, Barril G, Carreno V (2000) Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen-negative cases. Hepatology 32:116–123
Moraleda G, Saputelli J, Aldrich CE, Averett D, Condreay L, Mason WS (1997) Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol 71:9392–9399
Lampertico P, Viganò M, Manenti E, Iavarone M, Lunghi G, Colombo M (2005) Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology 42:1414–1419
Chen CJ, Yang HI, Su J et al (2006) Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 295:65–73
Acknowledgments
The authors have no competing interests. This work was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. (ICMR No: VIR/28/2010-ECD-I). Authors are grateful to ICMR for giving Junior Research Fellowship to Bhupesh Singla (3/1/3JRF-2008/MPD, dated 1/9/2008).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Singla, B., Chakraborti, A., Sharma, B.K. et al. Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients. Mol Biol Rep 41, 4689–4696 (2014). https://doi.org/10.1007/s11033-014-3339-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-014-3339-7