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. 2005 Oct;79(19):12242-52.
doi: 10.1128/JVI.79.19.12242-12252.2005.

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection

Marc F Le Mire  1 , Darren S Miller, Wendy K Foster, Christopher J Burrell, Allison R Jilbert
Affiliations

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection

Marc F Le Mire et al. J Virol. 2005 Oct.

Abstract

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.

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Figures

FIG. 1.
FIG. 1.
Expression of DHBsAg in liver at day 3 to 4 p.i. (A, B, and C) and 6.5 months p.i. (D). Immunohistochemical detection of DHBsAg was performed using monoclonal anti-DHBV pre-S antibodies (1H1) (31). DHBsAg-positive hepatocytes (indicated with arrows in panels A, B, and D) in liver tissue from (A) duck 20 from group A (with a group average of 0.0085% DHBsAg-positive cells); (B) duck 910 from group B (average of 0.11%), and (C) duck 3435 from group C (average of 2.64%). A single DHBsAg-hepatocyte was also detected at 6.5 months p.i. in duck 158 with residual DHBV infection following inoculation with 108 DHBV genomes at 41 days of age. DHBsAg was detected in the cytoplasm of hepatocytes as shown (A, B, C, and D). Sections were counterstained with hematoxylin. Bar, 50 μm.
FIG. 2.
FIG. 2.
Detection of DHBV DNA in non-protein-bound (A) and total (B) DNA extracted from livers of group C ducks on days 3 and 31 p.i. and examined by Southern blot hybridization. Hybridization was performed using a 32P-labeled genome-length DHBV DNA probe with exposure for 16 h. The identifiable DNA forms include relaxed circular (RC), double-stranded linear (DSL), cccDNA (ccc), and a smear of replicative intermediates (RI) of various lengths. Extracted liver collected at day 3 (lanes 1 to 5) and day 31 p.i. (lanes 6 to 10) from ducks 3435, 3637, 3839, 4041, and 4243, respectively. Lanes M1 and M2, 10 and 100 pg of linear 3.0-kb DHBV plasmid DNA. Ducks 3637, 3839, and 4041 had transient DHBV infection with reduction in the level of both cccDNA (panel A, lanes 7, 8, and 9) and total DNA (panel B, lanes 7, 8, and 9) by day 31 p.i. In contrast ducks 3435 and 4243 developed persistent DHBV infection with high levels of virus replication on day 31 p.i. (panels A and B, lanes 6 and 10, respectively).
FIG. 3.
FIG. 3.
Detection of full-length DHBV DNA by PCR using primers FL1 and FL2 (Table 1) and liver DNA extracted from group C ducks at 9 months p.i. (A) An ethidium bromide-stained gel of the products of a full-length PCR. (B) Southern blot hybridization of the same samples using a genome-length 32P-labeled DHBV DNA probe, with autoradiographic exposure for 30 min. Lane 1, λ DNA digested with PstI showing bands of 2.8, 1.7, and 1.2 kbp, indicated by arrows; lane 2, PCR product amplified using total DNA from the liver of a congenitally DHBV-infected duck with high-level persistent infection; lane 3, empty; lanes 4 to 6, products of full-length PCR using autopsy liver from ducks with residual DHBV infection (ducks 4041, 3839, and 3637, respectively). The positive control (lane 2) and the three group C samples show a ∼3-kbp product (indicated by an asterisk) and several smaller products from 1 to 1.8 kbp in length.
FIG. 4.
FIG. 4.
Southern blot hybridization to detect DHBV cccDNA in non-protein-bound DNA extracted from autopsy liver of the three group C ducks with residual DHBV infection. Samples from day 3 and 31 p.i. (3D and 31D, respectively) and 3, 6, and 9 months p.i. (3M, 6M, and 9M, respectively) show cccDNA detected in liver from duck 3637 until 3 months p.i., and in ducks 3839 and 4041 until 9 months p.i. Lane M, 80 pg of 3-kbp plasmid DHBV DNA. Non-protein-bound DNA extracted from 20 mg of liver was used for days 3 and 31 p.i. and from 150 mg of liver at the later time points. After Southern transfer the membrane was hybridized to a genome-length 32P-labeled DHBV DNA probe with autoradiography for 64 h. Copy numbers of cccDNA/per cell are shown for the 9-month samples (<0.003, 0.008, and 0.009).
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