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Real-Time PCR Quantification of Total Intracellular HBV DNA and cccDNA in HBsAg-Positive Patients with and without HDV co-Infection and in Patients with Occult HBV Infection
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Reported by Jules Levin
AASLD, Nov 2-6, 2007, Boston, MA
T. Pollicno1; G. Raffa1; T. Santantonio2; G. Squadrito1; M. Milella2; G. Colucci3; C. Calvi3; I. Cacciola1; M. Levrero4; G. Raimondo1
1. Internal Medicine, University Hospital of Messina, Messina, Italy.
2. Infectious Diseases, University of Bari, Bari, Italy.
3. Roche Diagnostics , Monza, Italy.
4. Internal Medicine, University "La Sapienza" , Rome, Italy.
Few information is available on the relation between HBVcccDNA levels and its transcriptional activity in chronic HBV infected individuals with different clinical/virological profiles. Aims of this study were (1) to quantify total HBV DNA, HBVcccDNA and pregenomic (pg)RNA in liver tissue from patients with different forms of chronic infection; (2) to evaluate serum HBV DNA and HBsAg titers in the same patients; (3) to investigate the acetylation/methylation status of HBVcccDNA-bound histones in occult HBV infected patients. We analysed serum and liver HBV extracts from 48 patients (36 HBsAg-positive and 12 occult HBV carriers). 5/36 HBsAg-positive cases were HBeAg-positive, 13 were anti-HBe positive with active HBV infection (HBV DNA>104 copies/ml), 14 were HDV infected with inactive HBV infection, and 4 were HDV carriers with active HBV infection. HBVcccDNA quantification method (Werle-Lapostolle, Gastroenterology 2004) was adapted to the TaqMan Cobas technology (Roche). HBsAg was quantified by Architect System kit (Abbott). HBVcccDNA chromatin immuno-precipitation (ChIP) (Pollicino, Gastroenterology 2006) was applied to study the acetylation/methylation status of HBVcccDNA from the liver tissue of 4 occult HBV carriers and 4 HBsAg-positive patients.
Table reports the results concerning the median amount (range) of HBV DNA, pgRNA and HBsAg. HBVcccDNA ChIP experiments showed that in occult HBV carriers (1) the cccDNA-bound histones were hypoacetylated, and (2) histone deacetylase HDAc1, methyl CpG binding protein 2 (MeCP2) and histone methyltransferase SUV39H1 were recruited onto the HBVcccDNA. Conclusions (1) HBVcccDNA is quantifiable in all chronic carriers including occult infected individuals (2) In anti-HBe positive patients with active HBV replication, serum and total intrahepatic HBV DNA levels differ according to their HDV status whereas cccDNA levels are comparable between HDV positive and negative individuals (3) HBV DNA levels may be largely different among HDV infected patients indicating different grade of HDV interference on HBV replication (4) Serum HBsAg amount does not correlate with intracellular cccDNA levels (5) Despite the persistence of HBsAg synthesis, HBVcccDNA levels are similar in HDV patients with inactive HBV and occult HBV carriers (6) HBVcccDNA histones are hypo-acetylated and methylated in occult infection.
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