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. 1998 Aug;42(8):2132–2137. doi: 10.1128/aac.42.8.2132

Long-Term Therapy with the Guanine Nucleoside Analog Penciclovir Controls Chronic Duck Hepatitis B Virus Infection In Vivo

Enjarn Lin 1, Carolyn Luscombe 1,†, Danni Colledge 1, Yan Yan Wang 1, Stephen Locarnini 1,*
PMCID: PMC105885  PMID: 9687423

Abstract

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the antiviral guanine nucleoside analog penciclovir for 12 or 24 weeks at a dosage of 10 mg/kg of body weight per day. By the completion of both 12 and 24 weeks of therapy, molecular hybridization studies of the liver tissue revealed that the viral DNA, RNA, and protein levels were significantly reduced compared to those in the placebo-treated controls. Penciclovir treatment for 12 or 24 weeks was not associated with any toxicity, establishing the efficacy and safety of long-term penciclovir therapy in chronic DHBV infection.

Hepatitis B virus (HBV) infects more than 350 million people throughout the world and is a major cause of hepatic disease resulting in significant morbidity and mortality (10). The current licensed treatment for chronic hepatitis B in most countries is alpha interferon, which is only partially effective in a small group of highly selected carriers (7, 17). One of the possible reasons cited for failure of interferon is the persistence of viral supercoiled (SC) or covalently closed circular (CCC) DNA in the liver (45).

The duck HBV (DHBV) model (13, 14, 34, 35, 39) has been used extensively in vitro and in vivo to screen potential inhibitors of the hepadnaviral family and provide mechanistic insights into the actions of these agents (6, 12, 20, 22, 33, 44). In vivo studies with nucleoside analogs have revealed that despite effective inhibition with therapy, the persistence of the replicative intermediate known as the CCC or SC DNA form typically occurs (5, 6, 18, 22, 25, 43). Penciclovir, a purine nucleoside analog originally developed against herpes simplex virus and varicella-zoster virus (3, 40, 41), has been shown to be able to inhibit hepadnaviral replication (37, 38) as well as to suppress CCC DNA expression by 50% within 4 weeks of treatment (15). Such inhibition of the CCC DNA might be maintained and/or enhanced with longer-term therapy. In this study, we report that 3 and 6 months of therapy with penciclovir significantly inhibited all viral replicative intermediates including the CCC DNA form and substantially reduced the levels of RNA and protein expression without demonstrating any toxicity in vivo. This is important because famciclovir, the oral form of penciclovir, significantly reduced the level of HBV DNA replication in patients with chronic hepatitis B infection (2, 21, 36).

Pekin-Aylesbury crossbred ducks congenitally infected with an Australian strain of DHBV (1) were used in the study. Five-week-old ducks with stable virus titers were used for the treatments (1). The titer of DHBV DNA was determined by dot blot hybridization of alkaline-denatured duck sera as described previously (1). Ducks were randomly assigned to the following treatment groups, as outlined in Fig. 1: (i) Nine ducks were treated with penciclovir (SmithKline Beecham, Surrey, United Kingdom) at a dosage of 10 mg/kg of body weight/day given by intraperitoneal injection (32) in two equal (morning and evening) doses dissolved in 2 ml of 1% (vol/vol) dimethyl sulfoxide (DMSO). After 12 weeks (3 months) of treatment, three ducks were killed and after 24 weeks (6 months) of treatment, three further ducks were killed, with the remaining three ducks continuing with 4 weeks of treatment-free follow-up. (ii) Nine ducks were treated with isotonic saline (placebo) containing 1% (vol/vol) DMSO rather than penciclovir. Similar to the procedure for the penciclovir-treated ducks, three ducks were killed after 12 weeks of treatment, three ducks were killed at 24 weeks of treatment, and two ducks continued with 4 weeks of treatment-free follow-up. Unfortunately, one placebo-control bird died during the trial due to reasons unrelated to the study (Fig. 1). Placebo- and penciclovir-treated ducks were housed separately. Blood samples were taken from all ducks before treatment began and weekly thereafter for the duration of the study. Serum or blood samples were obtained weekly and were analyzed for markers of liver, renal, and hematological function as described previously (18, 43). Similarly, all ducks were weighed prior to treatment and monthly thereafter.

FIG. 1.

FIG. 1

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Schematic representation of the treatment protocol for this study. Eighteen birds, separated randomly into treatment and control groups, were entered into the study. One placebo-control bird died due to reasons not associated with the study. Treatment ducks received a daily dose of penciclovir (PCV) at 10 mg/kg via the intraperitoneal (i.p.) route, while control birds were given saline as placebo. b.d., twice daily.

The autopsy procedure and the preparation of tissue for histological analysis were essentially the same as described previously (15, 18). Additionally, small sections (1 mm3) were fixed in 2% gluteraldehyde–0.1 M Sorensen phosphate buffer (vol/vol) (pH 7.3) at 4°C for 2 to 4 h for analysis by electron microscopy. The remaining tissue (liver and pancreas) was prepared and was stored at −70°C for the study of intracellular viral markers (15). All animals received humane care in compliance with the guidelines of the Fairfield Hospital Animal Ethics Committee.

Viremia was measured by a serum dot blot assay as described by Wang et al. (43). The liver DHBV DNA was analyzed by two DNA extraction procedures: one for total DNA (quantitation of viral burden and assessment of viral replicative intermediates) and the other for viral CCC DNA (15, 18, 19), the transcriptional template of hepadnaviruses. The intrahepatic viral load was determined by the formula of Jilbert et al. (9).

Total RNA was extracted from 200 mg of liver tissue by using an RNA extraction kit (Pharmacia, Milwaukee, Wis.) according to the manufacturer’s directions and was analyzed by standard procedures for slot blot hybridization (27, 44). For the slot blot hybridization, serial doubling dilutions starting with 20 μg of total RNA were performed. For probing, DHBV DNA was prepared, labelled, and hybridized exactly as described previously (25). For tissue-based probing, a digoxigenin-labelled DHBV DNA probe was prepared as described previously (11). Intrahepatic virus-specific proteins were analyzed by immunoblotting as described elsewhere (18, 29) and immunohistochemistry was performed as described previously (18, 19). Densitometric analysis was performed with X-ray film by using an imaging densitometer (model GS-670; Bio-Rad Laboratories, Hercules, Calif.) and Molecular Analyst computer software. The data were analyzed with Statworks (a computer software package from Cricket Software, Philadelphia, Pa.). Unpaired t tests were used to determine significance. The test of significance (P) was at the level of <0.05.

Long-term treatment with penciclovir (12 and 24 weeks) was not associated with any drug-related toxicity. Three birds, two placebo-control birds and one penciclovir-treated bird, displayed evidence of mild peritonitis, most probably due to the physical trauma of twice-daily intraperitoneal injections over 24 weeks. No differences were found between penciclovir-treated and placebo-control-treated ducks with respect to the laboratory markers of hematological, renal, or liver function (data not shown). Weight loss did not occur, and the mean weights of both groups of ducks were comparable during the study (data not shown). There was no appreciable difference in the histology of the liver or pancreas of the treated or control animals. Mild steatosis was observed in both penciclovir- and placebo-treated birds. There was no evidence of any apoptotic morphological changes in any of the tissues examined (data not shown) (16). Electron microscopic examination of the liver tissue revealed no significant differences between control and treated animals, with the birds in each group displaying normal mitochondria and mitochondrial cristae as well as abundant glycogen granules (data not shown).

Serum DHBV DNA levels in the placebo-treated, congenitally infected ducks remained stable throughout the treatment period (Fig. 2). Penciclovir therapy resulted in a fall in the level of viremia in all treated birds, and the level of viremia remained below the level of detection by DNA hybridization throughout the 24-week treatment period. During the follow-up period, serum DHBV DNA concentrations returned to near pretreatment levels (Fig. 2).

FIG. 2.

FIG. 2

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(A) DHBV DNA dot blot hybridization of serum samples from the placebo control birds and samples obtained at the end of treatment from penciclovir-treated birds treated for 6 months. Pre, pretreatment serum samples; numbers correspond to the number of weeks from the start of treatment. (B) Results at the 4-week drug-free follow-up period. Cloned DHBV DNA standards (STD) are given as picograms of DNA added to each well. Pre and the lane numbers are as described above for panel A.

The effect of antiviral therapy on the hepatic levels of viral DNA at the end of the treatment and follow-up periods was determined by using semiquantitative slot blot analysis (15, 19, 44), and the results are presented in Table 1. All penciclovir-treated ducks responded to treatment, with intrahepatic viral DNA levels being reduced, on average, by 95 and 92% after 12 and 24 weeks of therapy, respectively. During the follow-up period intrahepatic DHBV DNA levels returned to levels near those for age-matched controls. Southern analysis of total DNA (data not shown) showed that both relaxed circular (RC) and double-stranded linear (DSL) forms of viral DNA were reduced to undetectable or almost undetectable levels after 12 or 24 weeks of therapy with penciclovir. Both viral species returned during the treatment-free follow-up period to levels greater than those for age-matched controls. All results were statistically significant except the values obtained during the follow-up period (Table 1).

TABLE 1.

Changes to intrahepatic viral markers during long-term penciclovir therapy

Viral replicative intermediate and time of examination Percenta
% Changeb Signifi- cance (P)c
Control birds Penciclovir-treated birds
vged/cell
 3 mo 495 ± 134 27 ± 13 −95 0.004
 6 mo 433 ± 34 37 ± 17 −92 <0.001
 Follow-up 429 ± 443 391 ± 76 −9 0.883
CCC DNA
 3 mo 100 ± 16.1 51.7 ± 2.6 −48 0.007
 6 mo 100 ± 17.3 49.3 ± 4.7 −51 0.008
 Follow-up 100 ± 37.5 118.3 ± 10.2 +18 0.451
RNA
 3 mo 100 ± 23.0 42.6 ± 6.1 −57 0.014
 6 mo 100 ± 60.2 30.0 ± 12.8 −70 0.129
 Follow-up 100 ± 48.4 223.5 ± 94.6 +124 0.198
Pre-S protein
 3 mo 100 ± 10.9 23.4 ± 4.1 −77 <0.001
 6 mo 100 ± 22.1 49.9 ± 20.3 −50 0.045
 Follow-up 100 ± 21.5 110.7 ± 28.6 +11 0.687
Core protein
 3 mo 100 ± 40.0 7.4 ± 2.3 −93 0.016
 6 mo 100 ± 10.2 17.9 ± 24.5 −82 0.006
 Follow-up 100 ± 11.0 170.5 ± 46 +71 0.136
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a

All the control values except vge/cell were expressed as 100%, and the values for the penciclovir-treated birds were adjusted and expressed accordingly. 

b

Percent change as an increase or decrease compared with the levels for the age-matched controls. 

c

Statistical significance is a P value of <0.05. 

d

vge, viral genome equivalents. vge/cell was calculated by the method of Jilbert et al. (9). 

Southern blot analysis and subsequent densitometric analysis of intrahepatic viral CCC DNA revealed that penciclovir therapy for 12 and 24 weeks was able to reduce CCC DNA levels by 48 and 51%, respectively, compared to the levels for age-matched controls (Fig. 3, lanes 4 to 6 and 10 to 12). Both values were significant (P < 0.05) (Table 1). At the end of the follow-up period, viral CCC DNA levels in treated ducks had risen to a level 18% more than those for the age-matched controls (Fig. 3, lanes 15 to 17).

FIG. 3.

FIG. 3

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Southern blot hybridization analysis of DHBV DNA extracted from liver tissue by the CCC DNA technique as described in the text. Three bands specific for DHBV were noted and correspond to RC DNA, DSL DNA, and CCC DNA species, respectively. Lane M, molecular size markers; (A) Control birds at 3 months; (B) birds treated with penciclovir for 3 months; (C) placebo-treated controls at 6 months; (D) birds treated with penciclovir for 6 months; (E) placebo-treated controls at follow-up; (F) birds at 4 weeks of drug-free follow-up after 6 months of treatment with PCV.

In situ hybridization for DHBV DNA revealed that by the end of either 12 or 24 weeks of penciclovir treatment, virtually all hepatocytes had undetectable levels of DHBV DNA, while the number of bile duct epithelial cells (BDECs) staining positive for DHBV DNA was unaffected (data not shown). During the follow-up period, all parameters returned to the pretreatment levels and/or to the levels for age-matched controls (data not shown). Penciclovir treatment had no effect on the level of viral replication in the pancreatic tissue (data not shown).

After 12 and 24 weeks of therapy with penciclovir, mean reductions of 57 and 70% of DHBV RNA in the liver, respectively, were achieved compared to the levels for the age-matched placebo-treated controls (Table 1). Because of the extent of the variation in the raw data, only the effect at 12 weeks was statistically significant. Relapse occurred during the treatment-free follow-up period, with the treated ducks showing intrahepatic RNA concentrations twice as high as those for the age-matched controls, but this did not reach statistical significance (Table 1).

The results of immunoblot analysis for DHBV pre-S and core proteins are summarized in Table 1, and the blots are shown in Fig. 4A and B. Immunoblots underwent densitometric analysis, which revealed that after 12 weeks of therapy with penciclovir, treated birds displayed 77 and 93% reductions in pre-S and core antigen levels, respectively, compared to those for the placebo-treated controls. After 24 weeks of therapy, penciclovir-treated birds displayed 50 and 82% reductions in pre-S and core antigen levels, respectively. During the follow-up period, viral antigen levels returned to levels comparable to those in placebo-treated controls, with core antigen levels increasing to 171% of those for the controls (Table 1). As before, this relapse did not reach statistical significance.

FIG. 4.

FIG. 4

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(A) Representative immunoblot analysis of liver cell lysates probed with pre-S antisera, after which detection of bound antibodies was achieved by enhanced chemiluminescence. Lane M, markers; lane 1, control bird at 3 months; lane 2, bird treated with penciclovir for 3 months; lane 3, control bird at 6 months; lane 4, bird treated with penciclovir for 6 months; lane 5, control bird at follow-up; lane 6, drug-free follow-up after 6 months of penciclovir treatment. (B) Immunoblot similar to that in panel A which was probed with anti-DHBV core antigen. Lane M, markers; lane 1, control bird at 3 months; lane 2, control bird at 6 months; lane 3, bird treated with penciclovir for 3 months; lane 4, bird treated with penciclovir for 6 months; lane 5, control bird at follow-up; lane 6, drug-free follow-up after 6 months of penciclovir treatment.

Immunohistochemical staining of the livers of the control animals for pre-S and core antigen demonstrated that every hepatocyte and virtually all BDECs were positive for both viral proteins, and the BDECs typically stained more intensely than the adjacent hepatocytes (Fig. 5A). No obvious changes in this staining pattern were noted between the control birds at the different time points. After 3 and 6 months of penciclovir therapy, the liver tissue displayed a significant reduction in the intensity of staining for both viral markers, with most hepatocytes staining negative for pre-S (Fig. 5B and C) and core antigens. Small groups of hepatocytes as well as occasional isolated hepatocytes within the lobule retained some staining despite therapy (Fig. 5B and C). These cells might represent reinfection by cell-to-cell contact from resistant reservoirs such as the BDECs. Virus replication in the BDECs was unaffected by therapy and was readily distinguished due to the contrast of the surrounding negative periportal hepatocytes (Fig. 5C). In the follow-up period, staining of the tissue revealed a return to the level of viral protein expression similar to that for the placebo-treated age-matched control. None of the penciclovir-treated livers demonstrated any selective accumulation or increase in the level of expression of either viral marker. Penciclovir treatment for 3 or 6 months did not affect the level of pre-S or core antigen expression in the pancreatic islets (Fig. 5D).

FIG. 5.

FIG. 5

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Immunohistochemical detection of DHBV pre-S proteins in duck tissue before and at the completion of 6 months of PCV treatment. (A) Intrahepatic staining from a placebo-control-treated animal. (B) Intrahepatic staining from a penciclovir-treated animal at the end of 24 weeks of therapy showing general lobular area. (C) Portal tract from a penciclovir-treated animal at the completion of 24 weeks of penciclovir therapy. (D) Pancreatic islet from a penciclovir-treated animal after 24 weeks of therapy. Positive staining of isolated hepatocytes (HEP) and bile duct (BD) cells are marked. Magnifications, ×400.

This study confirms the efficacy of short-term treatment with penciclovir therapy for chronic DHBV infection previously reported from this laboratory (15). Long-term treatment with penciclovir was able to control active DHBV replication, reducing viremia to undetectable levels and intrahepatic DNA and RNA replication by 92 and 70%, respectively, after 24 weeks of therapy. Similarly, the amount of the transcriptional template (CCC DNA) was reduced by half at the end of therapy. Viral antigen expression was also significantly affected by penciclovir treatment, with the levels of pre-S and core antigen expression in the liver being reduced by 77 and 93%, respectively, after 12 weeks of therapy. The profile of immunohistochemical staining for the viral protein supported the in situ hybridization results for viral DNA and again identified the persistence of viral DNA and pre-S antigen in BDECs and extrahepatic sites such as the pancreas (15, 18, 37) during antiviral therapy with guanine nucleoside analogs. Importantly, ducks treated with penciclovir did not display any evidence of cytotoxicity at the clinical, biochemical, histological, or ultrastructural level.

Penciclovir, like most other nucleoside analogs, must be phosphorylated to its active triphosphate form for antiviral activity (30, 40). Penciclovir triphosphate has a long intracellular half-life of 12 to 18 h (3, 31, 40, 41) and blocks both first and second DNA strand synthesis, probably by causing premature DNA chain termination (40). Penciclovir triphosphate has also been shown to inhibit priming of hepadnavirus reverse transcription (28, 42, 46). It also has a high selectivity ratio for the hepadnaviral polymerase, with a Ki against HBV polymerase of 0.04 μM, which is about threefold lower than the Km for dGTP (31). This Ki compares very favorably with the Ki against human DNA polymerase α of 450 μM (8). Studies have also shown that the (R) enantiomer is a much more potent inhibitor of HBV polymerase and reverse transcriptase (31) than the (S) enantiomer, which is preferentially formed by the virally encoded deoxynucleoside kinase in HSV-infected cells (40). At present it is unknown which of the two enantiomers is preferentially formed in hepatocytes. Penciclovir’s lack of toxicity was further confirmed in this study.

In mammals, famciclovir is converted to penciclovir by the sequential action of ubiquitous esterases followed by hepatic aldehyde oxidase (in humans) and/or xanthine oxidase (in other species) (26, 30). Species differences in substrate specificities and the activities of both oxidases have been reported (26, 30). The oxidizing enzyme(s) is relatively less active in ducks than in mammals since its substrate, 6-deoxypenciclovir, is detectable in duck but not mammalian plasma after oral famciclovir treatment (26, 30). Despite this, the pharmacokinetics of the active metabolite, penciclovir, in plasma are similar in all species when they are standardized on the basis of basal metabolic rate (26, 30). In vitro, primary duck hepatocytes and human liver-derived cell lines take up and phosphorylate penciclovir at a similar (low) rate, achieving similar intracellular penciclovir triphosphate concentrations (31). These observations suggest that the relatively greater efficacy of penciclovir as an antihepadnaviral agent in ducks is due to physiological or biochemical differences (e.g., reabsorbtion from the lower bowel or renal portal system and lower intracellular concentrations of dGTP, respectively) than to pharmacological differences. Since the duck model has become more extensively used in preclinical evaluations of hepadnaviral agents (6, 12–15, 22, 29, 37, 38), further studies to define the molecular basis for these differences are warranted.

These investigations have demonstrated the characteristic relapse in virological markers that occurs during the drug-free follow-up period typically seen with nucleoside analogs (18, 22, 43, 44). Again, the persistence of the CCC DNA species is most likely responsible for the relapse phenomenon observed (4, 5, 45), although other factors may be involved, including the resistance of virus replication to nucleoside analog therapy in extrahepatocyte and extrahepatic sites such as within the BDECs and the islet cells in the pancreas. Other sources of this reinfection may be from the spleen and kidney as well as the isolated hepatic foci (18). In situ hybridization identified the presence of replication in the BDECs of the liver and in the islets of Langerhans of the pancreas. These sites of active replication were either resistant to penciclovir therapy or did not receive adequate concentrations of penciclovir due to the absence of the enzymatic machinery required to phosphorylate penciclovir. Additionally, in treated birds, a patch work pattern of staining for viral DNA or antigen (Fig. 5) was observed within the hepatic lobule.

In order to achieve clearance of Hepadnaviridae, a sustained host immune response in combination with the antiviral effect of a nucleoside analog such as penciclovir is probably required. Chronic infection with HBV induces a state of immunotolerance in many individuals, and the hepatitis B e antigen (HBeAg) plays a key role in this process (23). Longer-term therapy with an agent such as penciclovir which effectively reduces the total viral burden and which also removes or reduces the level of HBeAg and presumably the immunosuppressive effect on the immune response could allow time for the immune system to recover. This recovery might be manifested by the chronic carrier moving from the immunotolerant into the immunoelimination phase of the carrier state (17). Combination therapy involving penciclovir and another agent which together would target multiple steps in the hepadnavirus life cycle may result in such an outcome, and future in vivo studies should attempt to address these issues. Finally, future studies into the nature of the priming reaction (46) and the regulation of the viral CCC DNA molecule (24) may permit the design of new antiviral agents and the development of new therapeutic regimens.

Acknowledgments

We thank John Marshall and Jenny Doultree of the Victorian Infectious Diseases Reference Laboratory (VIDRL) for assistance with the electron microscopy, Malcolm Boyd from SmithKline Beecham Pharmaceuticals (Surrey, United Kingdom) for the supply of penciclovir, and our colleagues in the VIDRL Biomedical Reference Laboratories for assistance with the care, treatment, and autopsy of the ducks. We are also grateful for the assistance of Tim Shaw in providing useful comments on the manuscript. Assistance with histopathological and immunohistochemical techniques was kindly provided by the staff of the Department of Anatomical Pathology at the Respatriation Hospital, Melbourne, Australia, and hematological and biochemical assays were performed by the staff of the Clinical Pathology Division of VIDRL.

This study was supported by a grant from the National Health and Medical Research Council of Australia.

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