Establishment of DHBV infection.
Three groups of five 39- to 40-day-old DHBV-negative ducks were inoculated intravenously with either 107 (group A), 108 (group B), or 1010 (group C) virions. Sections of liver biopsy tissue from all 15 inoculated ducks collected at day 3 to 4 p.i. were stained using anti-pre-S monoclonal antibodies (1H1) (23, 27) and showed a proportional relationship between the inoculum size and frequency of DHBsAg-positive cells. Ducks from group A showed a mean of 0.00085% DHBsAg-positive total liver cells (0.00038 to 0.0015%), group B showed 0.011% (0.0066 to 0.013%), and group C showed 2.64% (1.5 to 3.8%) (Fig. , Table ). In each case, the single and occasional pairs of DHBsAg-positive cells were evenly and widely distributed throughout the lobules of the liver (Fig. ).
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 (more ...)
Detection of DHBV-infected cells and DHBV DNA in liver biopsy tissue from the group C ducks at day 3 p.i.
Non-protein-bound and total cellular DNA was extracted from frozen liver samples collected at day 3 or 4 p.i. and day 31 p.i. and examined for cccDNA and for total DHBV DNA, respectively, by Southern blot hybridization. cccDNA and total DHBV DNA were undetectable at days 3 to 4 p.i. in the livers of the group A and B ducks (data not shown), but were detected in liver tissue from five out of five group C ducks (Fig. , respectively) and quantitated by phosphorimager analysis (Table ). Levels of cccDNA ranged from 0.035 to 0.096 copies per liver cell or 1.6 to 2.6 copies per DHBsAg-positive cell, while levels of total DHBV DNA ranged from 2.7 to 5.2 copies per liver cell or 78 to 347 copies per DHBsAg-positive cell (Table ). The average ratio of cccDNA to total DHBV DNA in the samples collected at day 3 p.i. from the group C ducks was 1.2%, in contrast to the much higher percentage (40 to 80%) found in liver samples collected from the same ducks with residual DHBV infection at 9 months p.i. as described below.
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 (more ...)
As can be seen in Fig. , two of the group C ducks, 3435 and 4243, had high levels of cccDNA (Fig. , lanes 6 and 10) and total DHBV DNA (Fig. , lanes 6 and 10) at day 31 p.i. These ducks went on to develop persistent DHBV infection (see below) while the three remaining ducks in group C had low levels of cccDNA (Fig. , lanes 7, 8, and 9) and undetectable levels of total DHBV DNA at day 31 (Fig. , lanes 7, 8, and 9) and resolved their transient DHBV infection.
Outcome of infection.
Eleven out of 15 inoculated ducks from groups A, B, and C had transient DHBV infection as shown by the clearance of DHBsAg-positive cells from the liver by 31 days p.i. (data not shown) which was maintained until autopsy at 9 months p.i. Autopsy liver tissues were examined by immunoperoxidase staining for DHBsAg in an attempt to identify the cell type harboring residual DHBV DNA. However, only one DHBsAg-positive hepatocyte was detected in the liver of one duck with residual infection (duck 158; Fig. , panel D). The duck had residual DHBV DNA detected 6.5 months after inoculation with 108
DHBV genomes at 41 days of age. The specificity of DHBsAg detection was confirmed by staining the same cell of a consecutive liver section and by the use of negative control monoclonal antibodies (21
). This result suggested that residual DHBV DNA is present in hepatocytes. However, the biological significance of such a low rate of positive cells is not clear, and we are not confident to conclude that hepatocytes are the only cell type containing residual DHBV DNA.
Of the remaining 4 out of 15 ducks, two of the group C ducks, 3435 and 4243, developed persistent DHBV infection with high levels of virus cccDNA and total DHBV DNA in liver, widespread DHBsAg staining in liver, and detectable DHBsAg in serum from days 15 and 21 p.i. Another two ducks (one each from groups A and B) died prematurely and were unavailable for autopsy. A single series of serum samples from duck 3637 in group C with transient infection were assayed up to day 45 p.i. for viral DNA by quantitative PCR. Viral DNA extracts corresponding to 40 μl of serum were tested by PCR with primers P3 and P4. Samples from days 3, 8, 15, and 30 p.i. were DHBV DNA positive, while those from days 11, 21, and 45 p.i. and samples collected at autopsy at 9 months p.i. were DHBV DNA negative. At day 3 p.i. the estimated copy number of DHBV DNA in serum was 1,525 copies/ml, while copy numbers in the other positive samples were lower. Thus, during transient DHBV infection (days 3 to 30 p.i.) this duck showed very low levels of serum DHBV DNA that fluctuated around the limit of detection by quantitative PCR before becoming undetectable (<25 copies per ml) at day 45 and 9 months p.i.
Anti-DHBs became detectable by day 11 p.i. in four out of five ducks in each of groups A and B (data not shown), while in group C, anti-DHBs antibodies were consistently detected in the three ducks with transient infection (3637, 3839, and 4041), from day 7 p.i. until at least day 90 p.i. (data not shown). In contrast, ducks 3435 and 4243 (the two group C ducks that developed persistent infection) had serum anti-surface antibodies only transiently, before, in one duck, and coinciding, in the second duck, with the onset of detectable DHBsAg (data not shown).
Studies of residual DHBV DNA.
Tissue samples were collected at autopsy from four group A, four group B, and three group C ducks at 9 months p.i., with the exception of duck 910, which died and was autopsied early, at 5 months p.i. Residual DHBV DNA was assayed by nested PCR followed by Southern blot hybridization (sensitivity of 0.00003 copies per cell) on DNA extracted from liver, spleen, kidney, pancreas, adrenal, heart, PBMC, skeletal muscle, and serum (sensitivity, 100 copies per ml), and was found in at least one site in all ducks. Most liver (10/11) and spleen (7/11) samples were positive for DHBV DNA, whereas DHBV DNA was infrequently detected in other sites, two kidney samples, and one adrenal and one heart sample only. DHBV DNA was not detected in pancreas, skeletal muscle, PBMC, or serum in any residually infected ducks at 9 months p.i.
Quantitative PCR showed that as the DHBV inoculum was increased, the amount of residual DHBV DNA in the liver at 9 months p.i. also increased. One out of four liver samples from the ducks in group A (1516), three out of four samples from the group B ducks (525, 910, and 1718), and all three samples from the ducks in group C (3637, 3839, and 4041) showed detectable levels of DHBV DNA, using an assay which had a minimum sensitivity of 10 copies of DHBV DNA in 200 ng total DNA or 80,000 cells (0.00012 copies per cell). Although residual DHBV DNA was detected in the group A and B ducks, the levels were too low to be accurately quantified but were estimated to be between 0.00012 and 0.00025 copies per cell. The three group C ducks that cleared their transient DHBV infection had 190, 200, and 1,260 copies of DHBV DNA in 8 × 104 cell equivalents at 9 months p.i., corresponding to 0.0024 to 0.016 copies/cell or a mean of 0.007 copies/cell (Table ). Liver samples collected at 9 months p.i. from the group C ducks with residual (3637, 3839, and 4041), and persistent (3435 and 4243), DHBV infection were examined under code by a histopathologist. No evidence was found for ongoing liver inflammation in the three group C ducks with residual DHBV infection compared to uninfected, age-matched control ducks. In contrast, the two group C ducks with persistent DHBV infection had mild to moderate liver inflammation.
Quantitation of residual DHBV DNA in autopsy liver and spleen in samples collected at 9 months p.i.
Autopsy spleen samples collected at 9 months p.i. were similarly examined for residual DHBV DNA, and three out of four, two out of four, and three out of three samples from groups A, B, and C, respectively, had detectable DHBV DNA (Table ). Overall, the levels of virus DNA were lower in spleen than in liver, and a clear effect of inoculum dose on the amount of residual virus DNA was not seen in the spleen samples. The samples of kidney, heart, and adrenal which were positive for DHBV DNA by nested PCR were also positive in the quantitative assay, but at levels insufficient for accurate quantification, i.e., between 0.00012 and 0.00025 copies/cell (data not shown).
Sequential liver samples taken at day 3 to 4 and 1, 3, 6, and 9 months p.i. were next analyzed by quantitative PCR for total DHBV DNA to assess fluctuation in DHBV DNA levels over time. For two of the time points for the group C ducks, results of Southern blot hybridization were used because insufficient remaining biopsy material was available. Virus DNA in the group C ducks showed a fall to low levels of during the first month p.i., after which the levels of residual DHBV DNA remained relatively constant, ranging from 0.0044 to 0.007 copies/cell (Table ).
Time course of total DHBV DNA levels in the liver measured by quantitative PCR or Southern blot hybridization
Contribution of cccDNA to the total residual DHBV DNA.
The above samples of autopsy liver DNA were next assayed by quantitative PCR for cccDNA using primers spanning the cohesive overlap region. Liver samples from one out of four ducks in group A (duck 20) and one out of four ducks in group B (duck 525) contained detectable cccDNA. However, the levels of residual DHBV cccDNA were insufficient for accurate quantitation (data not shown). Liver samples from all three group C ducks (3637, 3839, and 4041) contained cccDNA at 0.014, 0.0010, and 0.0019 copies/cell, respectively (Table ), compared with 0.016, 0.0024, and 0.0025 copies of total DHBV DNA per cell, respectively (Table ). The levels of cccDNA were 40 to 80% of the levels of total DHBV DNA, making cccDNA the predominant form of residual DHBV DNA in ducks with residual DHBV infection. This is in marked contrast to what was seen in the same ducks during the early phase of their transient infection, when cccDNA in liver tissue at day 3 p.i. represented only 1.2% of total DHBV DNA (Table ).
All 11 spleen samples and the remaining samples from other organs which were positive by quantitative PCR for total DHBV DNA (two kidney, one heart, and one adrenal sample) were also tested by quantitative PCR for cccDNA. All samples tested were negative for cccDNA by this assay (i.e., <10 copies/80,000 cells) (data not shown). Thus, within the limits of this methodology for detecting very small amounts of DNA, cccDNA was not shown to be a significant contributor to the residual DHBV DNA in these other organs.
Further analysis of residual DHBV DNA genomes.
Further experiments were performed (i) to examine whether full-length DHBV genomes were present and (ii) to confirm, using Southern blot hybridization, that the residual DHBV DNA detected using the cccDNA PCR was indeed in the characteristic cccDNA form.
In the first experiment, PCR amplification of full-length DHBV DNA was performed as previously described (9
) using 1 μg of liver DNA extracted from the three group C ducks at autopsy at 9 months p.i. Extracted DNA was amplified using primers FL1 and FL2 (Table ), which were designed to amplify full-length DHBV DNA and should amplify a cccDNA template with a greater efficiency than relaxed circular DNA, since relaxed circular DNA is not continuous between the primers. The PCR products were analyzed by gel electrophoresis on a 1% agarose gel stained with ethidium bromide (Fig. ). A 3-kb product was seen in all three autopsy samples (Fig. , lanes 4, 5, and 6), as well as in a positive control sample that consisted of extracted liver DNA from a duck with high-level DHBV infection (Fig. , lane 2). Southern blot hybridization using a genome-length 32
P-labeled DHBV DNA probe demonstrated that the 3-kbp product contained DHBV sequences (Fig. , lanes 4, 5, and 6). Also seen in all three samples were discrete smaller than genome-length products, which also hybridized to the DHBV DNA probe and may represent PCR products amplified from defective cccDNA molecules with large deletions.
FIG. 3. Detection of full-length DHBV DNA by PCR using primers FL1 and FL2 (Table ) 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 (more ...)
In the second experiment, DNA was extracted as described above to enrich for non-protein-bound DNA (hence cccDNA), from sequential samples of liver from the three ducks in group C (3637, 3839, and 4041) collected up to 9 months p.i. In order to increase the sensitivity of the Southern blot hybridization a larger than usual quantity of cccDNA extract was loaded onto the agarose gel (38 μl containing cccDNA extracted from 5 × 107 cells compared with the usual volume of 18 μl containing cccDNA from 7 × 106 cells). Samples showed cccDNA at all time points in two out of three ducks analyzed (Fig. ) and up to 3 months p.i. in the remaining duck (number 3637). The sensitivity of Southern blot hybridization based on the plasmid DNA marker (M) was estimated as equivalent to 0.003 genomes per cell, allowing approximate quantitation of the DNA signal. The residual DHBV cccDNA in liver at 9 months p.i. in ducks 3637, 3839, and 4041 was estimated as ≤0.003 (not detected), 0.008, and 0.009 copies per cell by Southern blot hybridization, compared with estimates of 0.014, 0.0010, and 0.0019 copies per cell by quantitative PCR for cccDNA (Table ). Thus, Southern blot hybridization provided independent confirmation of the PCR finding of cccDNA in residual infection.
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, (more ...) Studies of infectivity of liver, spleen, and serum from ducks with residual DHBV infection.
Several strategies were used in an attempt to demonstrate whether infectious virions were present in tissues and serum from ducks with residual hepadnavirus infection. A total of six experiments were performed involving inoculation into 1- to 2-day-old ducklings of material derived from ducks that had recovered from transient DHBV infection; 1- to 2-day-old ducklings can be infected with a single virion when diluted serum from ducks with congenital and experimental DHBV infection is used (13
Fresh and frozen liver cell homogenates, cultured spleen cells and the culture supernatant, and a sucrose cushion-purified fraction of serum from ducks with residual infection were tested for infectivity in 1- to 2-day-old ducklings by either intraperitoneal or intravenous inoculation. DHBV infection (defined by detectable serum DHBsAg) was not transmitted by any inocula prepared from material from ducks with residual DHBV DNA. Infection was, however, transmitted by a liver homogenate prepared from a congenitally DHBV-infected duck, confirming the validity of the assay. The results of a total of six infection experiments are summarized in Table .
Attempts to demonstrate the presence of infectious DHBV in samples collected from ducks with residual DHBV infection
Effect of immunosuppression on levels of residual DHBV DNA.
DHBV infection was established in a group of five 40-day-old ducks (group D) by inoculation with 1010
DHBV virions. All five ducks had transient DHBV infection that resolved by 1 month p.i. Four months after resolution of infection (5 months p.i.) cyclosporine and dexamethasone were administered to three ducks (1001, 1005, and 1006) and water was administered as placebo to two ducks (1003 and 1007) for 4 weeks. The cyclosporine dose was similar to doses used in published experiments with chickens (7
) and the dexamethasone dose was based on a report on its use in ducks (10
). Liver tissue and serum were collected from all ducks pretreatment and after 2 and 4 weeks of treatment and examined for residual DHBV DNA. As well as liver, spleen samples were collected after 4 weeks of treatment at autopsy and were examined for DHBV DNA and also inoculated into 2-day-old ducklings to test for the presence of infectious virus. The ducks tolerated the immunosuppressive treatment with no changes to physical activity, although their body weight fell by a mean of 400 g in the treated ducks versus 100 g in those given placebo over the 4-week study (data not shown).
Levels of cyclosporine taken as trough (i.e., immediately before dosing) were 58, 134, and 244 μg/liter in the three treated ducks (1006, 1001, and 1005; the therapeutic range in humans is 80 to 250 μg/liter). Histological changes were assessed in sections of autopsy spleen and liver samples by a histopathologist who was not aware of the treatment status of each duck. Depletion of spleen cells was noted in two out of three treated ducks (1001 and 1005), but not in duck 1006 (which had the lowest serum level of cyclosporine) or in the control ducks (1003 and 1007) (data not shown). Liver tissue from the treated and control ducks was also examined, but no changes in liver inflammation or necroinflammatory activity were noted.
In vitro lymphocyte proliferation assays (22
) were also performed using PBMC collected pretreatment and after 4 weeks of immunosuppressive therapy. However, levels of proliferation were inconsistent when PBMC were stimulated with the mitogen phytohemagglutinin, liver-derived DHBcAg, and DHBV particles, making the results difficult to interpret (data not shown).
Levels of residual DHBV DNA were measured in liver DNA extracts by quantitative PCR for total DHBV DNA using primers P3 and P4. No significant change in levels of DHBV DNA was found at 2 or 4 weeks after commencing immunosuppressive treatment compared to the pretreatment samples (Table ). Similar results were seen in the ducks given placebo. It was concluded that immunosuppressive treatment had no effect on levels of residual DHBV DNA as determined in our assays.
Levels of residual DHBV DNA under immunosuppressive treatment