Woodchuck Research Articles

INTRASEXUAL TERRITORIALITY IN WOODCHUCKS (MARMOTA MONAX)

Species demonstrating intraspecific variation in social systems can be powerful models for understanding evolution of those systems. As a group, marmots exhibit several types of spacing systems, usually involving some degree of territoriality. Researchers have described populations of 1 species, the woodchuck, Marmota monax, as territorial and as nonterritorial, and such variation has been linked to ecological conditions. I used direct observations of individually recognized animals to describe the spacing system of a high-density population of Marmota monax in southern Maine. This population exhibited intrasexual territoriality. Home range overlap generally was higher between males and females than between same-sex individuals, and woodchucks tended to approach more members of the same sex more quickly compared with members of the opposite sex. Time spent scent marking varied across the active season but did not vary by sex. Males had larger home ranges than females, and home range sizes varied over time, perhaps in response to resources. Amount of overlap also was greater in some years than others, and such changes may be related to kinship. Philopatry and timing of dispersal also vary in this species and have implications for the evolution of sociality.

Hepatocellular carcinoma in the woodchuck model of hepatitis B virus infection

The Eastern woodchuck ( Marmota monax ) harbors a DNA virus (Woodchuck hepatitis virus [WHV]) that is similar in structure and replicative life cycle to the human hepatitis B virus (HBV). Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of hepatocellular carcinoma (HCC) within the first 2-4 years of life. The woodchuck model has been important in the preclinical evaluation of safety and efficacy of the antiviral drugs now in use for treatment of HBV infection and continues to serve as an important, predictive model for innovative forms of therapy of hepatitis B using antiviral nucleosides and immune response modifiers alone or in combination. Almost all woodchucks that become chronic WHV carriers after experimental neonatal inoculation develop HCC with a median HCC-free survival of 24 months and a median life expectancy of 30-32 months. The woodchuck model of viral-induced HCC has been used effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and magnetic resonance imaging. The chemoprevention of HCC using long-term antiviral nucleoside therapy has been shown in the woodchuck, and "proof of principal" has been established for some of the innovative, molecular methods for treatment of HCC. The model is available for fundamental investigations of the viral and molecular mechanisms responsible for hepatocarcinogenesis and should have substantial value for future development of innovative methods for chemoprevention and gene therapy of human HCC.

The win locus involved in activation of the distal N- myc2 gene upon WHV integration in woodchuck liver tumors harbors S/MAR elements

Woodchuck hepatitis virus (WHV) and the woodchuck ( Marmota monax) are models for hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV). In woodchuck liver tumors, the N- myc2 proto-oncogene is frequently activated by WHV integration either close to the gene or in the b3n and win downstream loci, located 10 and 150 kb from N- myc2, respectively. A scaffold/matrix attachment region (S/MAR) regulative element was shown to be in b3n, possibly mediating activation of the upstream N- myc2 gene upon WHV integration. To investigate if S/MAR elements are in win too, a 17-kb DNA fragment corresponding to the major region of WHV insertion in this locus was cloned and sequenced. Overlapping subcloned fragments spanning candidate S/MARs predicted by sequence analysis were tested by standard in vitro binding assays. Results showed the presence of two S/MAR elements in win. The distribution of previously described WHV insertions relative to the S/MARs reinforces the hypothesis that S/MARs nearby distal WHV insertions might be involved in long-range activation of N- myc2.

Suppression of lamivudine-resistant B-domain mutants by adefovir dipivoxil in the woodchuck hepatitis virus model

Adult woodchucks ( Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) were treated orally with lamivudine (15 mg/kg per day) for 57 weeks. After 20 weeks of treatment a 2–3 log reduction in serum WHV DNA was detected. Serum titers of WHV then increased gradually, in the presence of lamivudine treatment, reaching pre-treatment values by week 40. Viral recrudescence was associated with development of mutations in the B domain of the WHV polymerase gene. Mutations observed in the highly conserved FLLA motif of the B domain were L564V, L565M, and A566T, with A566T being the most frequently observed. Beginning on week 57 of lamivudine treatment, one group ( n=3) was treated orally with adefovir dipivoxil at a dose of 15 mg/kg per day plus lamivudine, and a second group ( n=3) was treated with H 2O placebo plus lamivudine. In woodchucks treated with adefovir dipivoxil, two had the A566T mutation, and one had both A566T and L565V. In the group maintained on lamivudine monotherapy, A566T alone was present in one animal, another carried both A566T and L565V, and in the third, no B-domain mutations were detected. There was a 4.5 log reduction in serum WHV DNA after 12 weeks of treatment with the adefovir/lamivudine combination, while in the lamivudine monotherapy controls, WHV DNA decreased by only 0.83 log ( P>0.001). A slight recurrence in serum titers of WHV DNA was observed one week after withdrawal of adefovir treatment but no further increase in viral load was observed during the remainder of the 12-week post-treatment follow-up period. The results demonstrate that supplemental adefovir dipivoxil treatment is effective in suppressing replication of lamivudine-resistant B-domain mutants in the woodchuck model of hepatitis B virus infection.

Suppression of lamivudine-resistant B-domain mutants by adefovir dipivoxil in the woodchuck hepatitis virus model

Adult woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) were treated orally with lamivudine (15 mg/kg per day) for 57 weeks. After 20 weeks of treatment a 2–3 log reduction in serum WHV DNA was detected. Serum titers of WHV then increased gradually, in the presence of lamivudine treatment, reaching pre-treatment values by week 40. Viral recrudescence was associated with development of mutations in the B domain of the WHV polymerase gene. Mutations observed in the highly conserved FLLA motif of the B domain were L564V, L565M, and A566T, with A566T being the most frequently observed. Beginning on week 57 of lamivudine treatment, one group (n=3) was treated orally with adefovir dipivoxil at a dose of 15 mg/kg per day plus lamivudine, and a second group (n=3) was treated with H2O placebo plus lamivudine. In woodchucks treated with adefovir dipivoxil, two had the A566T mutation, and one had both A566T and L565V. In the group maintained on lamivudine monotherapy, A566T alone was present in one animal, another carried both A566T and L565V, and in the third, no B-domain mutations were detected. There was a 4.5 log reduction in serum WHV DNA after 12 weeks of treatment with the adefovir/lamivudine combination, while in the lamivudine monotherapy controls, WHV DNA decreased by only 0.83 log (P>0.001). A slight recurrence in serum titers of WHV DNA was observed one week after withdrawal of adefovir treatment but no further increase in viral load was observed during the remainder of the 12-week post-treatment follow-up period. The results demonstrate that supplemental adefovir dipivoxil treatment is effective in suppressing replication of lamivudine-resistant B-domain mutants in the woodchuck model of hepatitis B virus infection.

Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors.

Integrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N-myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks (Marmota monax) chronically infected with WHV. Fifty-five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N-myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N-myc rearrangement. Fifty-one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N-myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N-myc rearrangements were present. Twenty-four grade 3 HCCs had integrations of WHV DNA in 79% and N-myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N-myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N-myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV infection.

Insights into cardioprotection obtained from study of cellular Ca2+ handling in myocardium of true hibernating mammals.

Mammalian hibernators exhibit remarkable resistance to low body temperature, whereas non-hibernating (NHB) mammals develop ventricular dysfunction and arrhythmias. To investigate this adaptive change, we compared contractile and electrophysiological properties of left ventricular myocytes isolated from hibernating (HB) woodchucks (Marmota monax) and control NHB woodchucks. The major findings of this study were the following: 1) the action potential duration in HB myocytes was significantly shorter than in NHB myocytes, but the amplitude of peak contraction was unchanged; 2) HB myocytes had a 33% decreased L-type Ca2+ current (I(Ca)) density and twofold faster I(Ca) inactivation but no change in the current-voltage relationship; 3) there were no changes in the density of inward rectifier K+ current, transient outward K+ current, or Na+/Ca2+ exchange current, but HB myocytes had increased sarcoplasmic reticulum Ca2+ content as estimated from caffeine-induced Na+/Ca2+ exchange current values; 4) expression of the L-type Ca2+ channel alpha(1C)-subunit was decreased by 30% in HB hearts; and 5) mRNA and protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), phospholamban, and the Na+/Ca2+ exchanger showed a pattern that is consistent with functional measurements: SERCA2a was increased and phospholamban was decreased in HB relative to NHB hearts with no change in the Na+/Ca2+ exchanger. Thus reduced Ca2+ channel density and faster I(Ca) inactivation coupled to enhanced sarcoplasmic reticulum Ca2+ release may underlie shorter action potentials with sustained contractility in HB hearts. These changes may account for natural resistance to Ca2+ overload-related ventricular dysfunction and point to an important cardioprotective mechanism during true hibernation.

Measurement of cell-mediated immune response in woodchucks.

Infection with hepatitis B virus (HBV) is a major health problem, with 350 million people chronically infected worldwide. Chronic HBV infection is characterized by severe hepatic disease sequelae, including chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma (1). The immunologic mechanisms that predispose to the development of chronic HBV infection are not completely understood. However, the cell-mediated immunity (CMI) to HBV is believed to play a crucial role in protection against viral infection, and development of efficient antiviral CMI is probably needed to avoid viral persistence and progression to chronic hepatitis (1). The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been used extensively as an animal model for HBV infection, disease, and prevention (e.g., 2). This model is being applied further to study the immunopathogenesis, antiviral therapy, and immunotherapy of chronic WHV infection (3–8). Such studies have demonstrated the need for more detailed investigation of the immune response of woodchucks, and to develop new molecular and cellular immunologic assays for measuring this response (Table 1). For example, in vitro proliferation assays for measuring the responses of woodchuck peripheral blood mononuclear cells (PBMCs) to viral antigens are a prerequisite for studying the CMI of woodchucks during WHV infection and therapy (3,4–7). The use of a universal anti-CD3 antibody reagent indicated that woodchuck PBMCs that did not adhere to nylon wool and that proliferated after stimulation with WHV recombinant core antigen (rWHcAg) were CD3+ lymphocytes (4). However, antibodies to woodchuck CD4 and CD8 positive lymphocytes are needed to enable more detailed lymphocyte phenotyping in PBMC cultures, and for applications of separation and depletion of such lymphocytes. Functionally active woodchuck cytokines and anti-cytokine antibodies are also needed to develop enzyme-linked immunosorbent assay (ELISA) methods for determining the responding cell type in PBMC cultures, for example, for differenti-

Malignant Pleural Mesothelioma in a Woodchuck (Marmota monax)

A spontaneous pleural mesothelioma was observed in a 4-year-old female woodchuck (Marmota monax). At necropsy, multifocal, tan to white, firm discrete nodules, 2 to 40 mm in diameter, were scattered over the ventral parts of the lungs and their corresponding parts of the diaphragm and the thoracic wall. Histopathologically, the tumor cells were large, cuboidal-shaped and variable size, and were weakly positive with PAS and Alcian blue. Immunohistochemically, neoplastic cells were positive for both vimentin and cytokeratin, indicating mesothelial origin. This report represents, as far as we know, the first reported case of a spontaneous mesothelioma in woodchucks.

Lead accumulation in woodchucks (Marmota monax) at small arms and skeet ranges

Increasing concern regarding the stewardship of US Army lands requires a proactive program to evaluate sites of potential risk. Small arms and upland skeet ranges are a potentially significant source of lead exposure for burrowing mammals. Woodchucks (Marmota monax) were evaluated for lead exposure in a previously used upland skeet range and a small arms range, respective to animals collected at two nearby reference locations. Soil lead concentrations collected at burrow entrances on the firing ranges were compared with blood, bone, kidney, liver, and fecal concentrations of woodchucks collected from the reference areas. No statistical differences were found in the lead concentrations in tissue between woodchucks in reference and firing ranges; concentrations of lead in liver and kidney were below detection limits. Levels in bone, blood, and feces suggest the bioavailability of lead at these various sites, although other factors (e.g., differences in foraging areas, age structure, habitat preferences, and environmental conditions) were also likely to influence exposure. Blood levels were below that which suggests toxicity. Further analysis of other ranges with higher lead concentrations and of small mammal species with smaller home ranges is recommended to further elucidate trends that could be extrapolated to other sites.

Landscape Influence on <em>Canis</em> Morphological and Ecological Variation in a Coyote-Wolf <em>C. lupus</em> × <em>latrans</em> Hybrid Zone, Southeastern Ontario

The ecology of Coyote-Wolf (Canis latrans × C. lupus) hybrids has never fully been typified. We studied morphological and ecological variation in Canis within a region of Coyote-Wolf hybridization in southeastern Ontario. We assessed Canis morphology from standard body measurements and ten skull measurements of adult specimens and found that Canis in this region are morphologically intermediate between Algonquin Provincial Park Wolves (C. lupus lycaon) and Coyotes, indicating a latrans × lycaon hybrid origin; however, there is a closer morphological affinity to latrans than lycaon. Analysis of 846 scats indicated dietary habits also intermediate between lycaon and Coyotes. We used a geographic information system (GIS) to assess spatial landscape features (road density, land cover and fragmentation) for six study sites representing three landscape types. We found noticeable variation in Canis morphology and diet in different landscape types. In general, canids from landscape type A (lowest road density, more total forest cover, less fragmentation) displayed more Wolf-like body morphology and consumed a greater proportion of larger prey (Beaver [Castor canadensis] and White-tailed Deer [Odocoileus virginianus]). In comparison, canids from landscape types B and C (higher road density and/or less total forest cover, more fragmentation) were generally more Coyote-like in body and skull morphology and made greater use of medium to small-sized prey (Groundhog [Marmota monax], Muskrat [Ondatra zibethicus] and lagomorphs). These landscape trends in Canis types suggest selection against Wolf-like traits in fragmented forests with high road density. The range of lycaon southeast of Algonquin Provincial Park appears to be limited primarily due to human access and consequent exploitation. We suggest that road density is the best landscape indicator of Canis types in this region of sympatric, hybridizing and unprotected Canis populations.

Molecular characterization of CD28 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) of woodchuck (Marmota monax).

Eastern woodchuck (Marmota monax) became an important animal model to study the immunological processes in hepatitis B virus infection. To facilitate further study of T-cell responses in this model, we cloned and sequenced the cDNAs of Woodchuck CD28 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), which play important roles for the regulation of T-cell activation by delivering the costimulation signals. According to the deduced amino-acid sequences, Woodchuck CD28 showed a similarity of 70% to 80% to its mammalian homologues. Woodchuck CTLA-4 has a higher similarity of 74% to 85% to corresponding mammalian CTLA-4 molecules. The strict conservation of critical amino-acid residues like cystein and asparagine residues in Woodchuck CD28 and CTLA-4 suggests that both molecules may structurally resemble their human or mouse homologues. A hexapeptide motif, MYPPPY, which has been supposed to be essential for the interaction with CD80, is present in both Woodchuck CD28 and CTLA-4. The cloned cDNAs of Woodchuck CD28 and CTLA-4 were placed under the control of the cytomegalovirus (CMV) promoter of the mammalian expression vector pcDNA3. Both proteins were expressed and detected by respective crossreactive antibodies in transiently transfected mammalian cells. By immunohistochemical staining with these antibodies, CD28 and CTLA-4 were also detected on cultured woodchuck peripheral blood lymphocytes. The molecular characterization of Woodchuck CD28 and CTLA-4 will facilitate studies on the T-cell response to hepadnavirus in the woodchuck model.

Serologic evidence for a novel Ehrlichia species in woodchucks (Marmota monax) from Pennsylvania, USA.

Serologic evidence for a novel Ehrlichia species in woodchucks (Marmota monax) from Pennsylvania, USA.

Nitric oxide-dependent vasodilation maintains blood flow in true hibernating myocardium

Patients with chronic coronary artery disease may exhibit chronically depressed regional myocardial function, which can be reversed by revascularization. This was called hibernating myocardium, since it was thought that myocardial blood flow was also chronically reduced and since animals during true hibernation are thought to have downregulated function and blood flow. The missing link in this syllogism, whose discovery became our goal, was to measure myocardial blood flow during true hibernation. Five woodchucks ( Marmota monax) were anesthetized and chronically instrumented. Hemodynamics were recorded prior to placement in a hibernaculum and again after the animals displayed a stable period of hibernation. Body temperature had fallen from 35.7 ± 0.5 to 13.4 ± 0.6 °C. Heart rate, mean aortic pressure, and left ventricular d P/d t had decreased, P < 0.05, by 67 ± 12% from 86 ± 4 bpm, 36 ± 11% from 76 ± 4 mmHg, and 61 ± 11% from 1924 ± 222 mmHg/s, respectively. Although renal blood flow, as assessed by radioactive microspheres, had decreased by 95 ± 2% and overall cardiac output fell by approximately 84%, surprisingly, transmural myocardial blood flow did not fall during hibernation (0.70 ± 0.16 ml/min/g) relative to when awake (0.84 ± 0.09 ml/min/g). Interestingly, during hibernation, nitric oxide (NO) synthase inhibition resulted in a significant decrease, P < 0.05, in myocardial blood flow (–41 ± 5%) and increase in coronary vascular resistance (CVR, +90 ± 17% from 77 ± 13 mmHg min g/ml). Thus, true mammalian hibernation results in downregulation of blood flow to the visceral organs, e.g. to the kidneys, but a surprising maintenance of myocardial blood flow, which appears to be due to an NO mechanism.

PALEOPHYLOGEOGRAPHY: THE TEMPO OF GEOGRAPHIC DIFFERENTIATION IN MARMOTS (MARMOTA)

Quantitative analysis of molar shape may provide a metric for paleontological phylogeography or the study of intraspecific relationships in the fossil record. In this study, outlines of the lower 3rd molars (m3) of North American Marmota were analyzed as between-shape distances after ordination with eigenshape analysis. Based on comparisons with cytochrome b, quantitative divergence in molar shape appears to be a reasonable proxy for phylogenetic reconstruction at the level of populations, subspecies, and the most closely related species of marmots. Phylogeographic analysis of living North American marmots and middle Pleistocene fossils from Porcupine Cave, Colorado, revealed that the latter were more closely related to extant Marmota monax than they are to M. flaviventris, which currently inhabits the Porcupine Cave area, suggesting that the origin of living species occurred in regions different from those they inhabit today. Change in molar shape was significant from level to level through the Porcupine Cave section, which spans a glacial-interglacial cycle, but was not correlated with environmental change. The amount of morphological difference among conspecific populations of living M. monax suggested that they differentiated over many glacial cycles, whereas morphological differences between M. caligata and M. flaviventris were smaller and may have evolved over fewer cycles.

MRNA analysis in flattened fauna: obtaining gene-sequence information from road-kill and game-hunting samples

Whether gene-sequence information could be obtained using mRNA from road-kill and hunting samples was investigated. Adipose tissue was used to clone cDNA fragments of the hormone leptin and brain tissue was used for the enzyme glutamic acid decarboxylase (GAD). Tissues collected from road-killed animals were used to clone leptin from RNA samples of raccoon (Procyon lotor) and woodchuck (Marmota monax). We were able to extract RNA and clone GAD67 from samples of masked shrew (Sorex cinereus), although the time of death was unknown. We collaborated with hunters who provided tissues from which we cloned leptin and GAD isoforms from beaver (Castor canadensis), red squirrel (Tamiasciurus hudsonicus), black bear (Ursus americanus), and moose (Alces alces americana). Molecular phylogenetic analyses confirmed that the sequences obtained did not result from contamination. A time-course experiment showed that even 24 h after the death of rats, sufficient mRNA remains to amplify leptin from adipose tissue. These results suggest that road-kill and hunting samples could be used as a valuable source of gene-sequence information.

Molecular genetic and biochemical analysis of woodchuck (Marmota monax) MHC class I polymorphism.

The woodchuck (Marmota monax) is an animal model that is used in the study of human hepatitis B virus ( HBV ) infection. A knowledge of woodchuck MHC class I (Mamo-I) genes and gene products is therefore essential for understanding the antigen-specific T-cell responses in this animal model. A number of Mamo-I genes have been identified by molecular cloning and sequencing. However, the allelic nature of these genes has not been proven by classical genetics like the segregation analysis in families. In this study, we analyzed the allelic diversity of Mamo-I in two three-generation woodchuck families including 15 members by sequencing of Mamo-I genes and immunoblotting of Mamo-I proteins after one-dimensional isoelectric focusing (1D-IEF). In addition to four published Mamo-I alleles, six new alleles that belonged to the same locus as the known Mamo-I alleles (Mamo-A) were found within the two woodchuck families. A typical Mendelian segregation of Mamo-I gene and antigens was observed in the families studied. For simple and rapid detection of allelic variability of Mamo-I gene, a typing method based on the detection of PCR products amplified by sequence specific primers (SSP) has been developed and tested in 41 unrelated animals. The most prevalent allele was Mamo-A*01 with a frequency of 21.9% followed by Mamo-A*07 (12.2%). Our study established Mamo-A as a classical MHC class I locus by the polymorphic and allelic nature of Mamo-I gene in the woodchuck.

SEASONAL BODY TEMPERATURE FLUCTUATIONS AND ENERGETIC STRATEGIES IN FREE-RANGING EASTERN WOODCHUCKS (MARMOTA MONAX)

During a 2-year period, radiotelemetry was used to continuously monitor body temperature (Tb) of free-ranging woodchucks (Marmota monax) in southeastern Pennsylvania. Hibernation was preceded by daily Tb fluctuations (‘‘test drops’’) of 2‐4 8C. During hibernation, woodchucks exhibited the characteristic pattern of torpor bouts. Time of arousals occurred randomly, but onset of torpor occurred predominantly between 1800 and 0000 h. Males had shorter hibernation periods (mean of 104.8 days) than did females (121.8 days). Males had shorter torpor bouts, but euthermic bouts were the same length as in females. Males also maintained higher T b during torpor. Overall, the cost of hibernation was greater for males than for females: males spent 38% more energy than did females. The primary energetic expense for both sexes was the periodic maintenance of euthermy throughout hibernation, which accounted for 75.2% of the energy budget for males and 66.8% for females. Compared with the 1999‐2000 hibernation seasons, woodchucks during the 1998‐ 1999 season had longer euthermic bouts, fewer torpor bouts (11.8 compared with 13.1), and spent less time in torpor (68% compared with 75%). These differences conserved more energy during the 1999‐2000 hibernation season and may have been the result of severe drought conditions during summer 1999. After emergence from hibernation, woodchucks generally maintained a constant state of euthermy throughout the active season, with T b fluctuating daily by 1‐2 8C. However, during the summer drought of 1999, daily Tb fluctuated 8‐178C in 5 of 8 woodchucks, presumably to conserve energy and water.

Hepatic Cirrhosis Occurring in a Young Woodchuck (Marmota monax) Due to Vertical Transmission of Woodchuck Hepatitis Virus (WHV)

Histologic and clinicopathologic findings of a woodchuck (Marmota monax) vertically infected with woodchuck hepatitis virus (WHV) are presented. The liver exhibits marked cirrhotic changes, which is characteristic of the pre-transformation phase of WHV. At necropsy, the woodchuck exhibited ascites and the liver had a grossly nodular appearance. Microscopically, focal hepatocyte necrosis and inflammatory cells were observed in midzonal and periportal areas in the liver. In Macchiavellos stained sections, cytoplasmic inclusion bodies appeared reddish granular materials. We believe that this may represent a new suitable and cost-effective cirrhotic model for the disease processes associated with hepadnaviruses in a number of other species, most notably Hepatitis B virus infection in man.

Real-time polymerase chain reaction assays for leukocyte CD and cytokine mRNAs of the Eastern woodchuck ( Marmota monax)

Real-time polymerase chain reaction (PCR) assays were developed for woodchuck leukocyte cluster of differentiation (CD) and cytokine mRNA expression. Plasmid DNA standards of each marker (CD3, CD4, CD8, IL-2, IFN-γ, TNF-α, IL-4, IL-10), and RNA standards from mitogen-stimulated woodchuck peripheral blood mononuclear cells (PBMCs) were used to validate and optimize the assays for TaqMan 7700 ® and iCycler ® PCR instruments. The complementary DNAs (cDNAs) produced by reverse transcription (RT) of RNA were quantified by real-time PCR against the plasmid DNA standards (6–8 log range) with detection of as few as 10–50 copies of amplicon cDNA per reaction. Analysis of unstimulated and concanavalin A-stimulated woodchuck PBMC demonstrated increased CD and cytokine mRNA expression following mitogenic activation. A liver sample from a woodchuck hepatitis virus (WHV) infected woodchuck with histologically confirmed acute hepatitis had increased intrahepatic CD and cytokine mRNAs compared to liver from an uninfected control woodchuck. The real-time PCR assays were highly specific for the woodchuck markers in PBMC and liver samples and were equally applicable for use in alternate real-time PCR instrumentation. These assays will enable the high-throughput analyses of mRNA markers during WHV infection, and thereby facilitate continued modelling of the immunopathogenesis and immunotherapy of human hepatitis B virus (HBV) infection.