Virus-infected Woodchucks Research Articles

Antiviral efficacy and pharmacokinetics of oral adefovir dipivoxil in chronically woodchuck hepatitis virus-infected woodchucks.

The antiviral efficacy of orally administered adefovir dipivoxil was evaluated in an 18-week study (12 weeks of treatment and 6 weeks of recovery) conducted with woodchucks chronically infected with woodchuck hepatitis virus (WHV). Adefovir dipivoxil is a prodrug of adefovir designed to enhance its oral bioavailability. Following administration of 15 mg of adefovir dipivoxil per kg of body weight in four WHV-infected animals, the mean maximum concentration of adefovir in serum was 0.462 microg/ml, with an elimination half-life of 10.2 h, and the oral bioavailability of adefovir was estimated to be 22.9% (+/-11.2%). To study antiviral efficacy, the animals were divided into three groups. There were six animals each in a high-dose group (15 mg/kg/day) and a low-dose group (5 mg/kg/day). A vehicle control group consisted of five animals because WHV DNA was detectable only by PCR at the time of the study in one of the original six animals. Efficacy was evaluated by determining the levels of WHV DNA in serum. The geometric mean WHV DNA level for the high-dose group diminished by >40-fold (>1.6 log(10)) after 2 weeks of treatment and >300-fold (>2.5 log(10)) at 12 weeks. There was a >10-fold reduction in five of six low-dose animals by 2 weeks, but levels were unchanged in one animal. By 12 weeks of treatment there was a >45-fold (>1.6 log(10)) reduction of WHV DNA levels, and serum WHV DNA levels were below the limit of quantification in three of six animals. Viral DNA levels returned to pretreatment levels during the 6-week recovery period. There were no clinically significant changes in body weight, hematology, or serum chemistry values, including bicarbonate or lactate, in any of the treated animals. No histologic evidence of liver injury was apparent in the biopsies. Under the conditions of this study, adefovir dipivoxil was an effective antihepadnaviral agent.

Pharmacodynamics of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine in Chronically Virus-Infected Woodchucks Compared to Its Pharmacodynamics in Humans

Pharmacodynamics of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine in Chronically Virus-Infected Woodchucks Compared to Its Pharmacodynamics in Humans

Pharmacodynamics of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine in Chronically Virus-Infected Woodchucks Compared to Its Pharmacodynamics in Humans

The pharmacodynamics of (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) was studied in chronically woodchuck hepatitis virus-infected woodchucks and compared to that in previous studies in hepatitis B virus (HBV)-infected humans. Net depletion rates of serum virus DNA in woodchucks receiving 3TC were modeled as a sum of an exponentially declining virus input and a first-order elimination. Preceding shoulders and pseudo-first-order virus half-lives in serum ranged from 1 to 7 days and were dose dependent. Higher plasma 3TC concentrations were needed in woodchucks for virus depletion similar to that attained in humans. Human HBV depletion curves from a previous clinical study with 3TC (>/=100 mg per day) were described by a biexponential relationship. The average half-life value in humans, normalized to fraction of area under the serum virus load-time curve, was similar to the average half-life value observed in woodchucks given the highest 3TC dose (2.4 and 2.0 days, respectively). On cessation of therapy, virus load rebounds in woodchucks were dose dependent and resembled posttherapy virus "flares" reported to occur in humans. The estimates of drug exposures that could lead to optimal antiviral effects presented indicate that 3TC should not be underdosed and compliance should be monitored. The study of chronically infected woodchucks may prove useful for optimizing drug regimens for hepadnavirus infections.

In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks.

The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.

Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly- l-lysine : Synthesis of the carrier and pharmacological properties of the conjugate

Background/Aims: The hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly- l-lysine ( l-Poly(Lys)) must have a high solubility in order to be injected in a small volume compatible with the intramuscular route. In this paper the molecular weights of Poly(Lys) which allowed the synthesis of conjugates with the properties of high solubility and limited loss by the kidney were determined and a procedure for obtaining Poly(Lys) preparations with the required range of polymerization has been described. Methods: Conjugates were prepared using Poly(Lys) of different molecular weights obtained by the procedure described here or purchased from a commercial source. Their solubility and renal loss in mice was determined. Results: Poly(Lys) with molecular weights ranging from 45000 and 65000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuch hepatitis virus-infected woodchucks for 37 days at a daily dose of 5.8 mg/kg exerted a strong antiviral activity. These preparations were devoid of acute toxicity in rat and caused no toxic effects when injected intramuscularly daily for 28 days at a dose ten times higher than that active in woodchucks. Conclusions: The results support the possibility of a clinical use of l-Poly(Lys) to obtain liver targeting of adenine arabinoside monophosphate for the treatment of chronic hepatitis B virus infection.

Overexpression of a p-glycoprotein in hepatocellular carcinomas from woodchuck hepatitis virus-infected woodchucks (Marmota monax)

The leading cause of human hepatocellular carcinomas (HCCs) is hepatitis B virus (HBV) infection. Woodchucks infected with a closely related hepadnavirus, woodchuck hepatitis virus (WHV), serve as a model for HBV because woodchucks chronically infected with WHV also develop hepatocellular carcinomas. Increased expression of p-glycoprotein (pgp) in human HCCs is a common obstacle in successful cancer chemotherapy. Pgps are encoded by a family of multidrug-resistance (MDR) genes. Livers from uninfected and WHV-infected woodchucks were examined to determine if pgp was expressed in HCCs and if there was a difference in expression between HCCs and nonneoplastic liver. A 170-kd protein was identified by Western blot in HCCs, whereas, constitutive pgp was not detected in normal liver taken from the same animals in 3 of 3 cases. Immunolocalization of the pgp with a panel of monoclonal antibodies revealed intensification of staining in 7 of 20 foci and 12 of 22 HCCs from six animals. Using primers for the human MDR1 gene, a single product was detected by reverse-transcribed polymerase chain reaction (RT-PCR) from HCCs. We have shown an increase in pgp in HCCs compared with normal liver from WHV-infected woodchucks. This is the first example of the induction of a pgp in a naturally hepadnavirus infected rodent system. It suggests the woodchuck can be a useful model for the study of the acquisition of resistance to chemotherapeutic agents in virally induced HCCs. (Hepatology 1996 Apr;23(4):662-8)

Overexpression of a p-glycoprotein in hepatocellular carcinomas from woodchuck hepatitis virus-infected woodchucks (Marmota monax).

The leading cause of human hepatocellular carcinomas (HCCs) is hepatitis B virus (HBV) infection. Woodchucks infected with a closely related hepadnavirus, woodchuck hepatitis virus (WHV), serve as a model for HBV because woodchucks chronically infected with WHV also develop hepatocellular carcinomas. Increased expression of p-glycoprotein (pgp) in human HCCs is a common obstacle in successful cancer chemotherapy. Pgps are encoded by a family of multidrug-resistance (MDR) genes. Livers from uninfected and WHV-infected woodchucks were examined to determine if pgp was expressed in HCCs and if there was a difference in expression between HCCs and nonneoplastic liver. A 170-kd protein was identified by Western blot in HCCs, whereas, constitutive pgp was not detected in normal liver taken from the same animals in 3 of 3 cases. Immunolocalization of the pgp with a panel of monoclonal antibodies revealed intensification of staining in 7 of 20 foci and 12 of 22 HCCs from six animals. Using primers for the human MDR1 gene, a single product was detected by reverse-transcribed polymerase chain reaction (RT-PCR) from HCCs. We have shown an increase in pgp in HCCs compared with normal liver from WHV-infected woodchucks. This is the first example of the induction of a pgp in a naturally hepadnavirus infected rodent system. It suggests the woodchuck can be a useful model for the study of the acquisition of resistance to chemotherapeutic agents in virally induced HCCs.

Assessment of Peripheral Blood Mononuclear Cell Proliferation by [2-3H]Adenine Uptake in the Woodchuck Model

The levels of incorporated exogenous [3H]thymidine of peripheral blood mononuclear cells (PBMC) of the woodchuck were low after stimulation with mitogens concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) when compared with other cell systems. The use of EDTA as an anticoagulant for blood sampling and AIM-V medium for culturing of PBMC improved the [3H]thymidine uptake of PBMC. A pronounced uptake is observed after use of [3H]adenine instead of [3H]thymidine for PBMC proliferation measurement. One likely explanation for the difference in [3H]adenine versus [3H]thymidine uptake is that the alternative pathway for thymidine monophosphate synthesis is important: the conversion of uridine to uridine monophosphate and, thereafter, to thymidine monophosphate. The optimal conditions for mitogen-induced proliferation of PBMC of the woodchuck were 2 μg/ml ConA and PHA at day 4 and 0.14 μg of PWM/ml at day 5. No consistent differences of [3H]adenine uptake were observed between PBMC from four woodchuck hepatitis virus-infected woodchucks and five uninfected animals.

Detection of an RNase H activity associated with hepadnaviruses

Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex.

Nuclear ploidy of normal and neoplastic hepatocytes from woodchuck hepatitis virus-infected and uninfected woodchucks

Flow cytometric analysis of the ploidy of normal and neoplastic hepatocyte nuclei obtained from adult woodchucks, a model of human hepadnavirus-induced hepatocellular carcinoma, was performed. All 36 samples of nuclei from non-neoplastic liver from woodchuck hepatitis virus-infected or uninfected liver were diploid, indicating that age-related nuclear polyploidization does not occur in this species, unlike other rodents. Individual or multiple hepatic neoplasms were obtained from each of 14 woodchuck hepatitis virus-infected woodchucks. Nineteen samples of hepatocellular carcinoma and eight adenomas were examined. Aneuploid nuclei were detected in 10 of the hepatocellular carcinomas and three of the adenoma samples. Similar DNA indexes, ranging from 1.11 to 1.22, were found in 7 of the 10 aneuploid HCCs and all 3 aneuploid adenomas. Nine of the 19 hepatocellular carcinoma samples and 5 of the 8 adenomas were diploid. Four of the diploid hepatocellular carcinomas had increased proportions of tetraploid nuclei. The presence of aneuploid nuclei was not related to histological appearance of the neoplasms or serum gamma-glutamyltranspeptidase levels. Because none of the hepatocellular carcinomas metastasized, the presence of aneuploidy could not be related to biological behavior. We determined the proportion of uninucleate and binucleate hepatocytes in hepatocellular carcinoma and nonneoplastic liver. Approximately 7% of hepatocytes were binucleate in nonneoplastic liver from woodchuck hepatitis virus-infected and uninfected liver. Only 2% of malignant hepatocytes were binucleate. The results of this study indicate that aneuploidy is a common change in hepatic neoplasms from woodchuck hepatitis virus-infected woodchucks.

Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: Presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections

During long-term studies of the natural history of woodchuck hepatitis virus infection, five cases of histologically confirmed, primary hepatocellular carcinoma were observed in a total of 92 woodchucks which had recovered, by analysis of viral serologic markers (WHsAg-, anti-WHc+, anti-WHs+), from experimental acute woodchuck hepatitis virus infections 20 to 30 months prior to the detection of hepatocellular carcinoma. No hepatocellular carcinoma was observed in 167 uninfected controls at least 3 years of age and held in the same laboratory environment. Southern blot hybridization analysis of liver tissue taken from four of these recovered woodchucks revealed the presence of low levels (0.1 to 0.3 copies per cell) of integrated woodchuck hepatitis virus DNA in hepatocellular carcinoma (four of four animals) and nonneoplastic tissue (three of four animals). Similarly, hepatocellular carcinoma tissue obtained from two wild-caught, naturally infected and serologically recovered woodchucks also contained low levels of integrated woodchuck hepatitis virus DNA. Liver tissues from another 27 of these 92 recovered woodchucks (without hepatocellular carcinoma) were examined for woodchuck hepatitis virus nucleic acids 13 to 31 months following experimental woodchuck hepatitis virus infection. Nonreplicating woodchuck hepatitis virus DNA was present in the liver of eight (30%) and in the peripheral blood lymphocytes from eight (30%) of these 27 animals. These results were in marked contrast to the analysis of woodchuck hepatitis virus DNA in the liver tissue of chronic woodchuck hepatitis virus carriers (20 experimentally infected and nine naturally infected). In these animals, high levels of replicating woodchuck hepatitis virus DNA (up to 2,000 copies per cell) were observed in all hepatocellular carcinoma and nonneoplastic liver tissue. Integrated woodchuck hepatitis virus DNA was found in eight of 60 individual hepatocellular carcinomas detected in 29 chronic carriers, 15 to 40 months postinfection. Integrated woodchuck hepatitis virus DNA was present in the nonneoplastic tissue from four of these 29 chronic woodchuck hepatitis virus carriers.

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.

Rearrangement and enhanced expression of c-myc in hepatocellular carcinoma of hepatitis virus infected woodchucks.

Hepatocellularcarcinoma (HCC) that occur in woodchucks chronically infected with woodchuck hepatitis virus (WHV) were screened for activation of cellular oncogenes. Enhanced expression and allelic alterations of the c-myc oncogene were found in three HCC out of nine. Variations in the size of the c-myc transcripts, ranging from 2.0 kilobases (kb) to 5.6 kb, as well as in the level of c-myc gene expression, 5-50-fold higher than in adjacent liver tissues, were observed among the three HCC. Rearrangements of the c-myc locus were either upstream of the gene or within the first intron. Cloning and sequencing of the break-point region from one of the three tumours showed that the c-myc gene was truncated and joined to a unique cellular sequence of unknown function. WHV DNA was not integrated near the c-myc coding exons, excluding a direct role of the virus in c-myc activation. The novel type of rearrangement and activation of the c-myc gene, reported here in liver tumours of hepatitis virus infected animals, appears strikingly similar to those resulting from chromosomal translocations in human Burkitt's lymphomas, acute B- and T-cell leukaemias and mouse plasmacytomas.

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.