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Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients
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Hepatology, Volume 40, Issue 3, September 2004
Danny Ka-Ho Wong 1, Man-Fung Yuen 1, HeJun Yuan 1 2, Simon Siu-Man Sum 1, Chee-Kin Hui 1, Jeff Hall 3, Ching-Lung Lai 1 *
1Division of Gastroenterology and Hepatology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong 2Department of Medicine, Fudan University, Zhongshan Hospital, Shanghai, China 3Third Wave Technologies, Inc., 502 South Rosa Road, Madison, WI
email: Ching-Lung Lai (hrmelcl@hkucc.hku.hk)
*Correspondence to Ching-Lung Lai, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China D. K.-H. W. and M.-F. Y. contributed equally to this study. fax: +852-28162863
Funded by: Bristol-Myers Squibb Unrestricted Biomedical Research Grant for distinction in Infectious Diseases
Abstract
This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P = .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r = 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r = -0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P = .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r = 0.778, P < .001) and intrahepatic cccDNA (r = 0.481, P = .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. Serum and intrahepatic total HBV DNA and cccDNA levels become lower as the disease progresses from HBeAg-positive to anti-HBe-positive phase, with cccDNA becoming the predominant form of intrahepatic HBV DNA. (HEPATOLOGY 2004;40:727-737.)
Received: 8 November 2003; Accepted: 19 May 2004
Article Text
Chronic hepatitis B virus (HBV) infection affects approximately 400 million people in the world. One crucial step in the HBV life cycle is the formation of a covalently closed circular form of the viral genome through DNA repair of the relaxed circular replicative HBV DNA inside the nuclei of hepatocytes. Covalently closed circular DNA (cccDNA) provides the template for viral and pregenomic messenger RNA. In vitro studies have shown that lamivudine has a profound effect on relaxed circular DNA (rcDNA) while having little or no effect on cccDNA. This is one possible reason for the rebound of HBV DNA to pretreatment levels often seen after lamivudine withdrawal.
Another nonreplicative form of HBV DNA is the double-stranded linear (DL) form produced by in situ priming during HBV replication. DL DNA is a possible precursor to HBV DNA integration. It can also form cccDNA through nonhomologous recombination at its ends via a process called illegitimate replication.
cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of the natural history and management of chronic hepatitis B (CHB). Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods, amplification of residual rcDNA can still occur after self-annealing of the partial elongation products. The problem of nonspecific amplification of rcDNA is a major obstacle in the development of an accurate cccDNA quantitation assay. A non-PCR assay, the Invader assay, has been developed for the quantitative detection of HBV cccDNA. This study aimed to verify the validity of the Invader assay and to employ this assay to quantify cccDNA in liver biopsies and sera from CHB patients in different stages of disease.
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