Pregenomic RNA (pgRNA) is generated from covalently closed circular DNA (cccDNA) and plays important roles in viral genome amplification and replication. Hepatic pgRNA and cccDNA expression levels indicate viral persistence and replication activity. This study was aimed to measure hepatic pgRNA and cccDNA expression levels in various states of hepatitis B virus (HBV) infection. Thirty-eight hepatocellular carcinoma (HCC) patients, including 14 positive for hepatitis B surface antigen (HBsAg) and 24 negative for HBsAg but positive for anti-hepatitis B core (anti-HBc) antibody, were enrolled in this study. In HBsAg-negative but anti-HBc-positive group, HBV-DNA was detected in 20 of 24 (83%) noncancerous liver tissues for at least two genomic regions based on polymerase chain reaction (PCR) analysis. pgRNA and cccDNA expression levels in occult HBV-infected patients were significantly lower than those in HBsAg-positive patients ( ). pgRNA and cccDNA in cancerous tissues were also detected without significant difference from those in noncancerous tissues. In conclusion, cccDNA and pgRNA are detected and represented HBV replication not only in noncancerous but also in cancerous liver tissues. In addition, the replication is shown in not only patients with HBsAg-positive but also occult HBV-infected patients, suggesting the contribution to HCC development. 1. Introduction Hepatitis B virus (HBV) infection remains a major health problem, even though methods for preventing vertical transmission and treatment guidelines have been introduced. Approximately, 2 billion people worldwide are infected with HBV. Three hundred fifty million of them have chronic infection. HBV is particularly endemic in sub-Saharan Africa, the Pacific, and Asia [1]. It frequently causes chronic hepatitis, which, subsequently, progresses to liver cirrhosis and hepatocellular carcinoma (HCC). HBV is responsible for most of the worldwide attributable risk of liver cirrhosis and HCC. The risk is even higher in endemic regions [2]. HBV is an incomplete double-stranded circular DNA virus, with a DNA length of approximately 3,200？bp. After HBV infects the liver cells, the relaxed-circular DNA is transferred into the nucleus and is repaired to covalently closed circular DNA (cccDNA) by DNA polymerase. The cccDNA plays a key role in the life cycle of the virus. It acts as a template for the generation of mRNAs, including pregenomic RNA (pgRNA) [3–5]. The transcription of cccDNA generates four kinds of mRNA: PreC/C (3.5？kb), PreS1 (2.4？kb), S (2.1？kb), and X (0.7？kb). The transcription of cccDNA
References
[1]
D. Lavanchy, “Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures,” Journal of Viral Hepatitis, vol. 11, no. 2, pp. 97–107, 2004.
[2]
E. Tabor, “Hepatocellular carcinoma: global epidemiology,” Digestive and Liver Disease, vol. 33, no. 2, pp. 115–117, 2001.
[3]
J. Beck and M. Nassal, “Hepatitis B virus replication,” World Journal of Gastroenterology, vol. 13, no. 1, pp. 48–64, 2007.
[4]
G. Raimondo, T. Pollicino, I. Cacciola, and G. Squadrito, “Occult hepatitis B virus infection,” Journal of Hepatology, vol. 46, no. 1, pp. 160–170, 2007.
[5]
D. K. H. Wong, F. Y. Huang, C. L. Lai et al., “Occult hepatitis B infection and HBV replicative activity in patients with cryptogenic cause of hepatocellular carcinoma,” Hepatology, vol. 54, no. 3, pp. 829–836, 2011.
[6]
T. Pollicino, G. Squadrito, G. Cerenzia et al., “Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection,” Gastroenterology, vol. 126, no. 1, pp. 102–110, 2004.
[7]
F. Sugauchi, M. Mizokami, E. Orito et al., “A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness,” Journal of General Virology, vol. 82, no. 4, pp. 883–892, 2001.
[8]
A. Laras, J. Koskinas, E. Dimou, A. Kostamena, and S. J. Hadziyannis, “Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients,” Hepatology, vol. 44, no. 3, pp. 694–702, 2006.
[9]
N. Saitou and M. Nei, “The neighbor-joining method: a new method for reconstructing phylogenetic trees,” Molecular Biology and Evolution, vol. 4, no. 4, pp. 406–425, 1987.
[10]
K. Tamura, J. Dudley, M. Nei, and S. Kumar, “MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1596–1599, 2007.
[11]
H. Nordenstedt, D. L. White, and H. B. El-Serag, “The changing pattern of epidemiology in hepatocellular carcinoma,” Digestive and Liver Disease, vol. 42, no. 3, pp. 206–214, 2010.
[12]
A. Zidan, H. Scheuerlein, S. Schule, U. Settmacher, and F. Rauchfuss, “Epidemiological pattern of hepatitis B and hepatitis C as etiological agents for hepatocellular carcinoma in Iran and worldwide,” Hepatitis Monthly, vol. 12, no. 10, Article ID e6894, 2012.
[13]
S. G. Lim, R. Mohammed, M.-F. Yuen, and J.-H. Kao, “Prevention of hepatocellular carcinoma in hepatitis B virus infection,” Journal of Gastroenterology and Hepatology, vol. 24, no. 8, pp. 1352–1357, 2009.
[14]
G. Raimondo, J.-P. Allain, M. R. Brunetto et al., “Statements from the Taormina expert meeting on occult hepatitis B virus infection,” Journal of Hepatology, vol. 49, no. 4, pp. 652–657, 2008.
[15]
C. Brechot, M. Hadchouel, and J. Scotto, “State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigen-positive and -negative liver diseases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 78, no. 6, pp. 3906–3910, 1981.
[16]
J.-H. Kao, P.-J. Chen, M.-Y. Lai, and D.-S. Chen, “Occult hepatitis B virus infection and clinical outcomes of patients with chronic hepatitis C,” Journal of Clinical Microbiology, vol. 40, no. 11, pp. 4068–4071, 2002.
[17]
A. Tamori, “Possible contribution to hepatocarcinogenesis of X transcript of hepatitis b virus in Japanese patients with hepatitis C virus,” Hepatology, vol. 29, no. 5, pp. 1429–1434, 1999.
[18]
B. Rehermann, C. Ferrari, C. Pasquinelli, and F. V. Chisari, “The hepatitis B virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response,” Nature Medicine, vol. 2, no. 10, pp. 1104–1108, 1996.
[19]
N. Yuki, T. Nagaoka, M. Yamashiro et al., “Long-term histologic and virologic outcomes of acute self-limited hepatitis B,” Hepatology, vol. 37, no. 5, pp. 1172–1179, 2003.
[20]
G. Raimondo, T. Pollicino, and G. Squadrito, “What is the clinical impact of occult hepatitis B virus infection?” The Lancet, vol. 365, no. 9460, pp. 638–640, 2005.
[21]
M. Torbenson and D. L. Thomas, “Occult hepatitis B,” Lancet Infectious Diseases, vol. 2, no. 8, pp. 479–486, 2002.
[22]
B. E. Korba, F. V. Wells, B. Baldwin et al., “Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections,” Hepatology, vol. 9, no. 3, pp. 461–470, 1989.
[23]
L. C. Faria, M. Gigou, A. M. Roque-Afonso et al., “Hepatocellular carcinoma is associated with an increased risk of hepatitis B virus recurrence after liver transplantation,” Gastroenterology, vol. 134, no. 7, pp. 1890–1899, 2008.
[24]
Q. Wang, M. I. Fiel, W. Luan, et al., “Impact of intrahepatic hepatitis B DNA and covalently closed circular DNA on survival after hepatectomy in HBV-associated hepatocellular carcinoma patients,” Annals Surgical Oncology, vol. 20, no. 12, pp. 3761–3770, 2013.
[25]
M. Abu-Amara and J. J. Feld, “Does antiviral therapy for chronic hepatitis B reduce the risk of hepatocellular carcinoma?” Seminars in Liver Disease, vol. 33, no. 2, pp. 157–166, 2013.
[26]
M. Levrero, T. Pollicino, J. Petersen, L. Belloni, G. Raimondo, and M. Dandri, “Control of cccDNA function in hepatitis B virus infection,” Journal of Hepatology, vol. 51, no. 3, pp. 581–592, 2009.
[27]
R. J. Lenhoff and J. Summers, “Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus,” Journal of Virology, vol. 68, no. 7, pp. 4565–4571, 1994.
[28]
K. Yamamoto, M. Horikita, F. Tsuda et al., “Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen,” Journal of Virology, vol. 68, no. 4, pp. 2671–2676, 1994.
[29]
M. Cabrerizo, J. Bartolomé, C. Caramelo, G. Barril, and V. Carre？o, “Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen-negative cases,” Hepatology, vol. 32, no. 1, pp. 116–123, 2000.
