Molecular characterization of intrahepatic and extrahepatic hepatitis B virus (HBV) reservoirs in patients on suppressive antiviral therapy.
ABSTRACT The hepatitis B virus (HBV) replicates via an error-prone reverse transcriptase generating potential drug-resistant quasispecies. The degree of HBV variability in liver vs peripheral blood mononuclear cells (PBMC) in patients on long-term suppressive antivirals is unclear. We characterized HBV replication, drug resistance and molecular diversity in patients with plasma HBV DNA undetectable by clinical assays. Explant liver (n=9), PBMC (n=6) and plasma (n=7) from nine such patients undergoing liver transplantation were evaluated for HBV genomes by sensitive PCR/nucleic acid hybridization assay. Cases with HBV DNA in liver and PBMC were tested for covalently closed circular DNA (HBV cccDNA). HBV polymerase (P) amplicons were cloned, sequenced and both P and overlapping surface (S) gene sequences were analysed. HBV DNA was detected in 43% (3/7) of plasma, 100% (9/9) of liver and 83% (5/6) of PBMC samples. HBV cccDNA was detected in all liver and one PBMC sample. Four patients had a clinical diagnosis of resistance. HBV P gene sequencing revealed 100% wild type (wt) in plasma (2/2), 83% wt in PBMC (5/6) but livers of 3/9 (33%) contained wt and 6/9 (66%) carried resistance to lamivudine and/or adefovir. The translated S gene revealed no changes affecting HBV antigenicity. Sequences from livers with antiviral resistant mutants revealed greater interpatient quasispecies diversity. Despite apparent HBV suppression, the liver continues to support HBV replication and extrahepatic HBV can be detected. PBMC may be a sanctuary for wt virus during antiviral therapy, while the liver harbours more drug-resistant viruses. Drug resistance correlates with intrahepatic viral diversity.
- SourceAvailable from: Tomasz I MichalakLiver Biopsy in Medicine, Edited by Mizuguchi Y, 10/2011: chapter 21: pages 355-378; Intech, Rijeka, Croatia., ISBN: 978-953-307-883-0
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ABSTRACT: The characterization of hepatitis B virus (HBV) quasispecies in different compartments in liver transplant (LT) recipients may be helpful in optimizing prophylaxis regimens. The aims of this study were to evaluate liver, peripheral blood mononuclear cells (PBMC), and plasma samples for HBV and to compare the quasispecies in hepatic and extrahepatic sites in LT recipients on long-term prophylaxis. For 12 patients followed for up to 15 years post-LT, liver, plasma, and PBMC samples [all HBV DNA-negative according to conventional polymerase chain reaction (PCR) assays] were evaluated for HBV DNA by a sensitive nested PCR method [covalently closed circular DNA (cccDNA) for liver and PBMC samples] and by the sequencing and phylogenetic analysis of polymerase quasispecies. For the 10 patients on prophylaxis with no clinical recurrence (median time post-LT = 15.5 months, range = 12-96 months), liver samples were HBV DNA-reactive in 9 of 10 cases, plasma samples were HBV DNA-reactive in 3 of 10 cases, and PBMC samples were HBV DNA-reactive in 2 of 7 cases (including 1 case with HBV cccDNA in PBMCs). The sequence analysis showed that all HBV clones had a wild-type (WT) sequence in the liver and PBMCs. In 2 patients with early HBV recurrence post-LT who were treated with nucleosides only, HBV DNA was detected in serum, PBMC, and liver samples, and HBV cccDNA was found in liver samples. An HBV lamivudine-resistant variant with an M204I mutation was identified in liver (70% and 18% of the clones) and plasma samples (100% of the clones), but a WT sequence was found in 70% and 100% of the PBMC clones. In conclusion, despite prophylaxis and the absence of HBV DNA in serum according to conventional assays, HBV is detectable in the serum, liver, and PBMCs of almost all patients, and this supports the use of continued anti-HBV therapy in this group. Antiviral drug-resistant variants are more frequent in the liver versus PBMCs, but both compartments are potential sources of reinfection.Liver Transplantation 08/2011; 17(8):955-62. DOI:10.1002/lt.22312 · 3.79 Impact Factor
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ABSTRACT: To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV-) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV-DNA was measured by a quantitative real-time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV- patients, HBV-DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV-DNA was found in 6/7 (86%) PBMC of occult-positive and none of occult-negative LT. Significantly lower HBV-DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult-positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper-mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV-DNA detection in PBMC may offer a useful tool to identify OHB in serum-negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.Journal of Viral Hepatitis 01/2013; 20(1):42-9. DOI:10.1111/j.1365-2893.2012.01623.x · 3.31 Impact Factor