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Background: Canine hip dysplasia (HD) is a common polygenic trait characterized by hip malformation that results in osteoarthritis (OA). The condition in dogs is very similar to developmental dysplasia of the human hip which also leads to OA., Methodology/principal Findings: A total of 721 dogs, including both an association and linkage population, were genotyped. The association population included 8 pure breeds (Labrador retriever, Greyhounds, German Shepherd, Newfoundland, Golden retriever, Rottweiler, Border Collie and Bernese Mountain Dog). The linkage population included Labrador retrievers, Greyhounds, and their crosses. Of these, 366 dogs were genotyped at ∼22,000 single nucleotide polymorphism (SNP) loci and a targeted screen across 8 chromosomes with ∼3,300 SNPs was performed on 551 dogs (196 dogs were common to both sets). A mixed linear model approach was used to perform an association study on this combined association and linkage population. The study identified 4 susceptibility SNPs associated with HD and 2 SNPs associated with hip OA., Conclusion/significance: The identified SNPs included those near known genes (PTPRD, PARD3B, and COL15A1) reported to be associated with, or expressed in, OA in humans. This suggested that the canine model could provide a unique opportunity to identify genes underlying natural HD and hip OA, which are common and debilitating conditions in both dogs and humans.

Objective: To determine the frequency of viral detection in conjunctival samples from client-owned domestic dogs with naturally acquired idiopathic conjunctivitis and to identify signalment, historical, and clinical findings positively associated with viral detection., Design: Case-control study., Animals: 30 dogs with naturally acquired idiopathic conjunctivitis and a control population of 30 dogs without ocular disease., Procedures: Complete physical and ophthalmic examinations were performed for each dog. Conjunctival swab specimens were analyzed by use of virus isolation and PCR assays for the following viruses: canine adenovirus-2 (CAV-2), canine distemper virus, canine herpesvirus-1 (CHV-1), canine parainfuenza virus, canine respiratory coronavirus, infuenza A virus, and West Nile virus. Signalment, clinical, and historical information was recorded and compared between study groups., Results: Viruses were detected by either virus isolation or PCR methods significantly more frequently in conjunctival samples from dogs with conjunctivitis (7/30 [23.3%]) than dogs without conjunctivitis (0/30 [0%]). Canine herpesvirus-1 was isolated from 2 conjunctival samples and detected by use of PCR assay in 5 conjunctival samples. Canine adenovirus-2 was isolated from 1 conjunctival sample and detected by use of PCR assay in 2 conjunctiva samples. Sexually intact dogs and frequent exposure to dogs outside the household were positively associated with viral detection in the conjunctivitis group., Conclusions and Clinical Relevance: Results suggested that CHV-1 and CAV-2 are common etiologic agents of conjunctivitis in domestic dogs. Risk factors for viral conjunctivitis in dogs reflected increased exposure to other dogs and opportunities for contact with infectious secretions.

A young adult male domestic shorthair cat was presented for physical examination, routine vaccinations, and a fecal examination. Physical examination revealed no significant abnormalities. Eggs of the raccoon pancreatic fluke Eurytrema procyonis were detected by fecal flotation. Results of a complete blood count and serum biochemistry panel were normal. Abdominal sonography revealed an enlarged hypoechoic pancreas with a hyperechoic rim, and a distended and thickened pancreatic duct. Serum pancreatic lipase immunoreactivity (PLI) was increased. These findings supported the possibility of fluke-associated pancreatitis. Treatment with praziquantel/pyrantel/febantel was associated with resolution of sonographic abnormalities and normalization of PLI.

In a small controlled study, clevudine, a potent inhibitor of hepadnaviruses, including hepatitis B virus and woodchuck hepatitis virus, suppressed hepatitis delta virus (HDV) viremia in chronically infected woodchucks. Suppression was correlated with the marked reduction of woodchuck hepatitis virus surface antigen in individual animals, consistent with the concept that repression of surface antigen expression may be a useful antiviral strategy for HDV.

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.

L: -FMAU [1-(2-fluoro-5-methyl-beta,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of hepatitis B virus (HBV) and duck hepatitis B virus replication in cell culture and duck hepatitis B virus replication in acutely infected Peking ducks. The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of woodchuck hepatitis virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection.

Acyclovir triphosphate is a potent inhibitor of hepatitis B virus DNA polymerase, but acyclovir treatment provides no benefit in patients with hepatitis B virus infection. This is due in part to the fact that hepatitis B virus, unlike herpes simplex virus, does not code for a viral thymidine kinase which catalyzes the initial phosphorylation of acyclovir. We synthesized 1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing hepatitis B virus replication in 2.2. 15 cells, while acyclovir was inactive. The greater antiviral activity of 1-O-octadecyl-sn-glycero-3-P-acyclovir appeared to be due to liver cell metabolism of the compound to acyclovir monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815-1822, 1997). However, a closely related compound without a hydroxyl group at the sn-2 position of glycerol, 1-O-hexadecylpropanediol-3-P-acyclovir, was more active and selective in 2.2.15 cells in vitro. In this study, we treated woodchucks chronically infected with woodchuck hepatitis virus with increasing oral doses of 1-O-hexadecylpropanediol-3-P-acyclovir and assessed the response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly inhibited viral replication in vivo, as indicated by a 95% reduction in serum woodchuck hepatitis virus DNA levels and by a 54% reduction in levels of woodchuck hepatitis virus replicative intermediates in the liver. Higher doses were somewhat less effective. In contrast, 20 mg of acyclovir/kg twice daily, a 5. 3-fold-higher molar dosage, had no demonstrable activity against woodchuck hepatitis virus. Oral 1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe and effective in chronic woodchuck hepatitis virus infection.

Acute hepadnavirus infections either resolve or progress to chronicity. Factors that influence chronicity as an outcome of hepatitis B virus (HBV) infection in humans can be studied experimentally in the woodchuck model. Accordingly, several woodchuck hepatitis virus (WHV) inocula were characterized. Representative inocula had high titers of infectious virus (approximately 10(7.7)-10(9.5) woodchuck 50% infectious doses per milliliter [WID(50%)/mL] by subcutaneous inoculation), with 1 WID(50%) ranging between 21 and 357 physical virion particles. WHV7P1 (standard high dose, 5 x 10(6) WID(50%)) produced a 72% chronicity rate (i.e., percent chronic of total infected) in neonatal woodchucks (1-3 days old). Comparable doses of WHV8P1 resulted in a lower chronicity rate in neonates (34% chronic) indicating that it represented a strain different from WHV7P1. Neonatal woodchucks were more susceptible to chronic infection by high doses of WHV7P1 (range, 65%-75% chronic) compared with 8-week-old weanlings (33% chronic) and adult woodchucks (0% chronic; i.e., all resolved). High doses of cloned wild-type viruses also induced high rates of chronicity in neonates (70%-80% chronic). Chronicity rates in neonates were decreased for low doses of WHV7P1 (500 WID(50%), 9% chronic) and for high doses of a precore WHeAg-minus mutant WHV8 clone (17% chronic). Thus, both age and viral determinants can influence chronicity as an outcome of experimental WHV infection. Standardized inocula will enable the study of mechanisms that initiate and maintain chronic hepadnavirus infection and also provide a means for developing WHV carriers for therapeutic studies.

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2'deoxy-2'fluoro-1-beta-D-arabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAU-treated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients.

In vivo transfection of Eastern woodchucks (Marmota monax) with recombinant woodchuck hepatitis virus (WHV) DNA is effective in inducing virus infection for the study of replication, pathogenicity, and oncogenicity of wild-type and mutated WHV. The one drawback to this procedure is the need for preparation of large amounts of WHV DNA. Reduction of the amount of WHV DNA in the transfection protocol necessary to induce infection would save considerable time and resources. Therefore, we conducted a titration of WHV DNA, ranging from 50 micrograms to 50 pg of DNA, in adult woodchucks to determine the minimum infectious dose of recombinant WHV DNA. As little as 50 ng of transfected WHV DNA induced productive infection in adult woodchucks. Thus, transfection with large amounts of recombinant WHV DNA appears to be unnecessary.

Fialuridine (FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil) is toxic to liver, heart, muscle, and nerve in clinical trials for chronic viral hepatitis (CH). Mitochondrial toxicity was hypothesized. To address pathophysiologic mechanisms, we examined mitochondrial changes in FIAU-treated woodchucks (WC) with CH from woodchuck hepatitis virus infection. WC (with and without CH from woodchuck hepatitis virus infection) were treated with FIAU (1.5 mg/kg/day) for 12 weeks. WC were killed. Liver, heart, skeletal muscle, and kidney samples underwent DNA extraction and were analyzed ultrastructurally (transmission electron microscopy). Myocardium, skeletal muscles, and liver samples were analyzed histologically. Abundance of hepatic, myocardial, muscle, and kidney mtDNA decreased in FIAU-treated WC, but the magnitude varied. mtDNA decreased 55% in heart, 65% in kidney, 74% in liver, and 87% in muscle (p < 0.02 for each tissue: FIAU-treated versus FIAU-untreated). Cellular damage was characterized ultrastructurally by mitochondrial enlargement, cristae dissolution, and lipid droplets. Lipid droplets found in the heart, diaphragm, biceps, and liver were sufficient to identify FIAU-treated WC (p < 0.05 each). Widespread mitochondrial damage to many tissues resulted from chronic FIAU treatment and occurred irrespective of CH. It manifested with mtDNA depletion, intracytoplasmic lipid droplets, and destroyed mitochondrial cristae. Defective mtDNA replication with mtDNA depletion seems central to the subcellular pathophysiology of altered energy metabolism and multiorgan failure in FIAU toxicity.

It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3'-Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl-dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP-ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP-ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis.

Cryptosporidium oocysts were found in the feces of a 6-month-old female cat with persistent diarrhea. The oocysts disappeared from the feces immediately after treatment with paromomycin (165 mg/kg of body weight, PO, for 5 days), and the diarrhea eventually resolved.

We analyzed the DNA ploidy and the nuclear size of hepatocytes within hepatocellular carcinoma, putative preneoplastic (clear cell and basophilic foci) and adjacent non-neoplastic liver in 30 woodchucks neonatally infected with the woodchuck hepatitis virus. In livers from control woodchucks, in clear cell foci and in most chronic portal hepatitis, the hepatocytes were diploid, with less than 10% tetraploid cells. Aneuploid peaks were found in 50% of the livers with chronic active hepatitis, in 63% of basophilic foci and in 90% of hepatocellular carcinoma. Within the same tumor, aneuploid peaks with different DNA indices were observed frequently, indicating heterogeneity of tumor. S-phase was always elevated, indicating an increased rate of proliferation. Aneuploid cells had nuclei that were larger than those of control liver cells. In some basophilic foci and in some livers with chronic active hepatitis, abnormal DNA was demonstrated before the development of hepatocellular carcinoma, suggesting that these may be populations of hepatocytes at risk of neoplastic transformation.

All mammalian hepadnaviruses possess a gene, termed X, that encodes a protein capable of transactivating virus gene expression. The X gene overlaps the polymerase and precore genes as well as two newly identified open reading frames (ORFs) termed ORF5 and ORF6. In this investigation, we examined whether ORF5, ORF6, and the X gene were important for the replication of woodchuck hepatitis virus (WHV) in susceptible woodchucks. First, we investigated whether proteins were produced from ORF5 and ORF6 by in vitro translation of appropriate viral transcripts, searched for antibodies against the putative proteins in the sera of animals infected with wild-type virus, and looked for an antisense WHV transcript, necessary for expression of a protein from ORF6, in the livers of acutely or chronically infected woodchucks. All such experiments yielded negative results. Next, we used oligonucleotide-directed mutagenesis to introduce termination codons into ORF5 and ORF6 at two locations within each ORF. Adult woodchucks in groups of three were transfected with one of the four mutant genomes. All of these woodchucks developed WHV infections that were indistinguishable from those of animals transfected with the wild-type WHV recombinant. Polymerase chain reaction amplification and direct DNA sequencing confirmed that reversion of the mutants to a wild-type genotype did not occur. Taken together, these data indicate that ORF5 and ORF6 are not essential for virus replication and are unlikely to represent authentic genes. Finally, we generated five WHV X-gene mutants that either removed the initiation codon for protein synthesis or truncated the carboxyl terminus of the protein by 3, 16, 31, or 52 amino acids. Groups of three adult woodchucks were transfected with one of the five X-gene mutants. Only the mutant that possessed an X gene lacking 3 amino acids from the carboxyl terminus was capable of replication within the 6-month time frame of the experiment. In contrast, all seven woodchucks transfected with wild-type WHV DNA developed markers consistent with viral infection. Thus, it is likely (P < 0.01) that the WHV X gene is important for virus replication in the natural host.

The nucleotide sequence of an isolate of woodchuck hepatitis virus (WHV) from the serum of a woodchuck trapped in New York state (WHVNY) was compared with the sequences of previously published isolates. The nucleotide sequence of WHVNY was closest to that of an isolate originating from New Jersey: the two genomes shared a 15 nucleotide in-frame deletion in the region where the presurface and polymerase genes overlap (nucleotides 3260-3274) and differed by 54 point mutations (1.6% of genome). Amino acid differences ranged from 0.4% in the surface gene to 5.7% in the X gene. Three isolates from woodchucks that originated in Pennsylvania and Maryland did not contain the deletion and differed from WHVNY by 102 to 106 point mutations (3.0% to 3.2% of nucleotides). Amino acid changes ranged from 0.5% in the core gene to 5.7% in the X-gene. Thus, WHVNY differed little from previous isolates. Next, the genomes from 102 independent clones of WHVNY were compared to ascertain the extent of sequence variation among WHV genomes in a chronically infected animal. A total of 98 clones had genomes of unit length while 2 clones had genomes shorter than unit length and 2 clones had genomes longer than unit length. The clones not of unit length possessed deletions or inverted duplications of sequence. The rate of mutation in the viral genes was 2.65 mutations per 10,000 nucleotides in the precore domain, 1.27 per 10,000 in the X-gene, 0.98 per 10,000 in the presurface gene, and 3.77 per 10,000 at the 5' end of the core gene.(ABSTRACT TRUNCATED AT 250 WORDS)

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

The immunosuppressive agent cyclosporine was given to adult woodchucks during acute experimental infection with woodchuck hepatitis virus (WHV). All 17 woodchucks given WHV alone or with a vehicle resolved the infection (i.e., zero chronicity), but when cyclosporine was given throughout the incubation and acute phases of infection (0-12 or 14 weeks; n = 12), the rate of chronic infection increased to 92%. When cyclosporine was given only during the incubation period (0-4 weeks; n = 10) or only during the acute phase of infection (2-12 weeks; n = 9), the rates increased to 50% and 55%, respectively. However, when the drug was given after the acute phase (8-18 weeks; n = 9), the chronic infection rate (11%) did not differ from that in untreated and vehicle controls. Immune responses inhibited by cyclosporine are important in resolution of acute WHV infection and occur mainly during the first 8 weeks. Immunosuppression of these responses for even short intervals during incubation (e.g., 0-4 weeks) increases the risk of chronicity.

Retrospective analysis of necropsy findings of 705 woodchucks was performed to determine the prevalence and morphology of immune-mediated glomerulonephritis, its relationship to woodchuck hepatitis virus (WHV) infection, and the presence of major WHV antigens. Twenty-six woodchucks had glomerular lesions. Renal tissue of the 26 animals was evaluated histologically and immunohistochemically for immune-mediated glomerulonephritis. Of these 26 animals, immune-mediated glomerulonephritis was diagnosed in six, all of which were chronic WHV carriers. Membranous glomerulonephritis was identified in three animals, two of which also had mesangial proliferation. Host immunoglobulin was present within the mesangium and along capillary loops in all three. Woodchuck hepatitis virus core antigen (WHcAg) was present along capillary loops of two of these animals, one membranous and one mixed, and in the mesangium of all three. Woodchuck hepatitis virus surface antigen (WHsAg) deposition was similar to WHcAg deposition but was only present along capillaries in those animals with mixed nephritis. The remaining three animals had mesangial proliferation. WHsAg and host immunoglobulin deposition were predominately mesangial; WHcAg was not detected. Transmission electron microscopy showed thickening of the capillary loop basement membranes and subepithelial electron-dense deposits in animal one, and deposits in the mesangium in animal six.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.

Praziquantel was used successfully for treatment of a small number of dogs and 1 cat infected with Paragonimus kellicotti. To further evaluate the usefulness of this drug in treating such infections, 7 cats and 7 dogs were inoculated orally with metacercariae (12 and 20 to 22, respectively) obtained from crayfish, then were treated after the infections became patent; 2 cats and 2 dogs served as noninfected controls. Beginning 1 week before infection, and continuing weekly thereafter, physical, hematologic, and fecal examinations were performed on each animal; thoracic radiography was performed every other week. By postinoculation week 6, all dogs given metacercariae had patent infection diagnosed on the basis of positive results of fecal examination. By postinoculation week 7, 5 cats had confirmed patent infection, but 2 cats given metacercariae never had patent infection or had signs of infection. Clinical signs of infection were minor and included increased respiratory tract noise, slight inducible cough, or mild dyspnea. Transient eosinophilia was detected in dogs around postinoculation week 3. Pretreatment radiography revealed cavitated lesions in cats only; pleural lines and patchy infiltrates in cats and dogs; or pneumothorax in dogs only. The treatment regimen consisted of 23 mg of praziquantel/kg of body weight given every 8 hours for 3 days; 1 infected cat and dog were not treated. By 11 days after treatment, eggs had disappeared from the feces of infected animals, and marked resolution of lung lesions was evident radiographically.(ABSTRACT TRUNCATED AT 250 WORDS)

The factors involved in the establishment of persistent hepadnavirus infection are poorly understood. Recent findings demonstrate that the sequence of the genome of hepatitis B virus (HBV) is variable in infected individuals and that, in some cases, virus mutants predominate. Our objectives in the present study were to analyze the variability of woodchuck hepatitis virus (WHV) genomes in an infected animal and to determine whether sequence heterogeneity played a critical role in the ability of WHV to induce chronic infection. We cloned and determined the complete nucleotide sequence of three supercoiled genomes from an animal that became infected after inoculation with a standardized WHV serum pool (i.e., the WHV7 virus pool). We found that there were four nucleotide substitutions among the three genome sequences as well as a 73-nucleotide deletion in one of the recombinants. DNA transfection experiments revealed that only one of the three recombinants was capable of independent replication. These data suggest that a significant proportion of replicative templates in woodchucks that are infected with WHV are defective virus genomes. Next, we compared the outcome of acute infection after inoculation with a serum pool containing a uniform population of replication competent virus (i.e., the WHV7R pool) with a serum pool composed of WHV genomes of variable sequence. The WHV7R serum pool originated from a woodchuck that became a chronic carrier after in vivo transfection of the liver with the infectious WHV7 recombinant. Neonatal woodchucks were inoculated with 5 x 10(6) WHV genome equivalents of either the WHV7 pool or the WHV7R pool. All animals in the study became acutely infected with WHV. Of the animals infected with the WHV7 serum pool, 65% became chronic carriers, while 80% of the animals infected with the WHV7R serum pool developed chronic infection. Thus, infection of woodchucks with a serum pool containing defective virus resulted in a rate of chronic WHV infection that was similar to, or even lower than, a rate from a pool containing only wild-type virus. This suggests that the presence of defective virus in the inoculum is not a prerequisite for the establishment of persistent hepadnavirus infections.

Abstract- In a retrospective postmortem survey of laboratory woodchucks (Marmota monax), four neoplastic and one hyperplastic skin lesions were identified. These included two lipomas and single cases each of squamous cell carcinoma, apocrine gland adenoma and sebaceous gland hyperplasia. Three animals were male, the other two female. Animals ranged from 2 to 7 years of age. Four animals were negative for markers of woodchuck hepatitis virus (WHV). The other, a 7-year-old female with an axillary lipoma, was a chronic carrier. Four neoplasms were discrete masses; the digital squamous cell carcinoma was more extensive, infiltrative and interfered with prehension. The low incidence of skin tumours (0.8 per cent) parallels that described in other laboratory rodent species. Résumé- Une étude rétrospective portant sur des autopsies de marmottes de laboratoire (Marmota monax) a permis la mise en évidence de 4 lésions néoplasiques et d'une lésion hyperplasique de la peau. Il s'agissait de deux lipomes ainsi que de cas isolés d'épthélioma spinocellulaire, d'adénome apocrine et d'hyperplasie sébacée. Trois animaux étaient des mäles et deux, des femelles. L'äge variait de 2 à 7 ans. Quatre animaux étaient négatifs au marqueur de l'hépatite virale de la marmotte (WHV). Le cinquième, une femelle de 7 ans présentant un lipome axillaire, était un porteur chronique. Quatre des néoplasies étaient des masses discrètes; l'épithélioma spinocellulaire du doigt était plus extensif, infiltrant et gênait la préhension. La faible incidence des tumeurs cutanées (0,8%) est identique à celle que l'on décrit chez d'autres rongeurs de laboratoire. Zusammenfassung- In einer retrospektiven postmortalen Untersuchung von Labor-Murmeltieren (Marmota monax) wurden vier neoplastische und eine hyperplastische Hautveränderung festgestellt. Darunter waren zwei Lipome und einzelne Fälle von jeweils einem Plattenepithelkazinom, einem apokrinen Adenom und einer Talgdrüsenhyperplasie. Drei Tiere waren männlich, die beiden anderen weiblich. Das Alter der Tiere lag zwischen 2 und 7 Jahren. Vier der Tiere reagierten negativ auf Murmeltier-Hepatitis-Virus-Marker (WHV). Ein weiteres 7 Jahre altes weibliches Tier mit einem Lipom im Achselbereich war chronischer Träger. Vier der Neubildungen stellten diskrete Massen dar; das Plattenepithelkarzinom im Fußbereich war ausgedehnter, infiltrativ wachsend und beeinträchtigte die Greifbewegungen. Das geringe Vorkommen von Hauttumoren (0,8%) entspricht demjenigen, das bei anderen Nagern beschrieben wurde. Resumen  En un estudio retrospectivo postmortem de marmotas de laboratio (Marmota monax) se identificaron cuatro lesiones neoplásicas cutáneas y una lesión cutánea de tipo hiperplásico. Entre estas se encontraban dos lipomas y casos individuales de carcinoma de células escamosas, adenoma de glándulas apocrinas e hiperplasia de glándulas sebáceas. Tres animales eran machos y los dos restantes eran hembras. La edad de los animales oscilaba entre los dos y los siete años. Cuatro animales fueron negativos para maracadores del virus de la hepatitis de la marmota. El otor, una hembra de 7 años de edad con un lipoma axilar, era un portador crónico. Cuatro neoplasias eran masas bien delimitadas mientras que el carcinoma de células escamosas digital era aprehensión. Esta baja incidencia de neoplasias cutánea en la marmota (0,8%) es semejante a la descrita en otros roedores de laboratorio.

Abstract- Large, fluctuant, fluid-filled subcutaneous masses containing hundreds of Taenia crassiceps cysticerci were detected in 13 woodchucks trapped in central New York and Maryland. The axillary region (7 of 13 animals) and adjacent lateral thoracic wall (4 of 13 animals) were most frequently affected. One animal had a focal parasitic granuloma in the lung and another had numerous cysticerci present within the hepatic parenchyma. Histologically, parasite sections were surrounded by varying degrees of fibrosis and mild scattered to coalescing lymphocytic aggregates were present in the adjacent subcutaneous tissues. There was minimal infiltration of subcutaneous lesions into the underlying musculature. Scanning electron microscopy of the parasites indicated the presence of numerous developing buds in the area of the posterior bladder. Scanning and transmission electron microscopy indicated that cysticerci were covered with a dense coating of microtriches. Taenia crassiceps lesions were found in 13 (2.6 per cent) of 501 trapped woodchucks, and in none of 293 colony-born woodchucks. No relationship between Taenia crassiceps infection and woodchuck hepatitis virus infection could be established. Résumé- Des masses sous-cutanées volumineuses, fluctuantes, remplies de liquides, contenant des centaines de cysticerques de Taenia crassiceps furent découvertes chez 13 marmottes capturées au centre de l'Etat de New-York et dans le Maryland. La région axillaire (7 des 13 animaux) et la paroi thoracique latérale adjacente (4 des 13 animaux) étaient les plus fréquemment atteintes. Un animal avait un granulome parasitaire focal dans un poumon et un autre avaient de nombreux cysticerques dans le parenchyme hépatique. D'un point de vue histologique, les sections de parasite étaient entourées par une fibrose d'importance variable, et des aggrégats lymphocytaires modérés, dispersés ou coalescents, étaient présents dans les tissus sous-cutanés adjacents. Il y avait une infiltration minimale des lésions sous-cutanées dans les muscles sous-jacents. La microscopie électronique à balayage montra la présence de nombreux bourgeons en développement dans la zone postérieure de la vessie. La microscopie électronique à balayage et par transmission indiqua que les cysticerques étaient couverts par un enduit dense de microtriches. Des lésions dûes àTanenia crassiceps furent découvertes chez 2.6% (13 sur 501) des marmottes capturées, et 0% (0 sur 293) des marmottes nées en captivité. Aucune relation entre l'infestation par Taenia crassiceps et l'hépatite infectieuse virale de la marmotte ne put être établie. Zusammenfassung- Bei 13 im Zentrum von New York und Maryland gefangenen Waldmurmeltieren wurden große, fluktuierende, flüssigkeits-gefüllte subcutane Massen festgestellt, die Hunderte von Taenia crossiceps cysticerci enthielten. Am häufigsten waren der Axillarbereich (boi 7 von 13 Tieren) und die angrenzende seitliche Brustwand (bei 4 von 13 Tieren) betroffen. Ein Murmeltier hatte ein fokales parasitäres Granulom in der Lunge, ein anderes zahlreiche Cysticerci im Leberparenchym. Im histologischen Bild zeigten sich von verschieden starken Fibrosen umgebene Parasitenausschnitte und leicht verstreute bis konfluierende Lymphozytenaggregate im angrenzenden subcutanen Gewebe. Die darunterliegende Muskulatur wurde nur minimal von den subcutanen Läsionen infiltriert. Unter dem Rasterelektronenmikroskop zeigten die Parasiten zahlreiche sich entwickelnde Knospen im Bereich der hinteren Blase. Im Rasterelektronenmikroskop und im Transmissions-Elektronenmikroskop waren die Cysticeri mit einer dichten Schicht aus Mikrotrichen bedeckt. Taenia crassiceps-Läsionen wurden bei 2.6% (13 von 501) der gefangenen und bei 0% (0 von 293) der koloniegeborenen Waldmurmeltiere nachgewiesen. Ein Zusammenhang zwischen der Taenia crassiceps - Infektion und einer Waldmurmeltier-Hepatitis-Virusinfektion konnte nicht festgestellt werden. Resumen  En 13 marmotas capturados en Nueva York y en Maryland se detectaron masas subcutáneas de gran tamaño, llenas de líquido y que contenían cientos de cisticercos de Taenia crassiceps. Las zonas más afectadas fueron la región axilar (7 de 13 animales) y la pared torácica adyacente (4 de 13 animales). Un animal presentaba un granuloma parasitario focal en un pulmón y otro presentaba numerosos cisticercos en el parénquima hepático. Histológicamente, los parásitos aparecían rodeados por una reacción fibrosa de diferente intensidad y en el tejido subcutáneo próximo se observaban agregados linfocitarios. La musculatura subyacente no aparecía afectada. Estudios de microscopía electrónica de transmisión y de barrido demostraron que los cisticercos estaben recubiertos por una densa capa de microtricos. Lesiones producidas par Taenia crassiceps se detectaron en el 2,6% (13 de 501) de los capturados y en el 0% (0 de 293) de los procedentes de un criadero. No se pudo demostrar relación alguna entre la infestación con Taenia crassiceps y la infección con el virus de la hepatitis de las marmotas.

Normal reference values for total serum protein, albumin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), gamma glutamyl transferase (GGT), alkaline phosphatase (AP), and total bilirubin were established in 48 clinically healthy woodchucks. To validate the use of these biochemical tests in the woodchuck for assessment of liver injury, carbon tetrachloride was administered to produce hepatocellular necrosis and the common bile duct was surgically occluded to produce cholestasis. Biochemical tests were performed prior to experimental treatment and thereafter in surviving woodchucks for a period of 6 weeks. There were marked increases in the serum activities of AST, ALT, and SDH following carbon tetrachloride administration and all 3 enzymes appeared to be useful markers of acute hepatocellular injury. The predominate biochemical abnormalities in woodchucks with bile duct obstruction were hyperbilirubinemia, hypercholesterolemia and increased serum AP and GGT activities. The increase of GGT occurred earlier following bile duct obstruction and the magnitude of increase was greater than that of AP, suggesting that GGT would be the preferred serum enzyme test in the woodchuck for assessment of cholestatic liver injury.

A modified laparoscopic technique for liver biopsy in dogs was developed, using an otoscope to observe the liver and to guide the biopsy needle. The procedure was used to obtain liver biopsy specimens from 7 control dogs and 22 treated dogs during studies of the hepatic effects of long-term anticonvulsant drug therapy. In addition, biopsy specimens were obtained from 10 clinical patients with diffuse liver disease. Diagnostic liver specimens were obtained from all dogs with minimal complications. The procedure used basic principles of needle biopsy technique under visual control. It enabled the operator to observe the liver, select a biopsy site, and obtain diagnostic samples quickly, with considerably less expense than by use of fiberoptic laparoscopy. The results of this study indicate that the modified laparoscopic approach is a safe, practical method for liver biopsy in dogs.

Woodchuck hepatitis virus (WHV) DNA was cloned from viral particles obtained from the serum of a woodchuck with a naturally acquired infection. The complete nucleotide sequence of the virus genome was determined and found to be 3323 base pairs long. Transfection experiments demonstrated that the recombinant WHV DNA was infectious in each of 18 woodchucks tested and established a chronic carrier state in 1 of 13 neonates and 3 of 5 adult animals. WHV DNA from serum particles from the chronically infected neonate was cloned and the nucleotide sequence of three independent recombinants was compared directly with that of the input recombinant DNA. The consensus sequence of the three progeny genomes was identical to that of the parental DNA sequence. Therefore, transfection of woodchuck livers with recombinant WHV DNA induces active virus replication and gene expression and yields progeny genomes that are faithful copies of the input virus genome.

Advanced chronic hepatic disease was observed in 5 dogs that had received anticonvulsant drug therapy for 2 to 3 years. Clinical signs included anorexia, weakness, and restlessness, and 2 dogs also had ascites. There were remarkable increases in the serum activities of alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase. The total serum bile acid concentration was high in 3 of 4 dogs that were tested. Sulfobromophthalein excretion was delayed in all dogs. Histologic examination of liver specimens from 4 of the dogs demonstrated macronodular or micronodular cirrhosis.

Hepatic function tests were performed on 48 dogs that had been given primidone, phenytoin, or a combination of anticonvulsant drugs for 6 months or longer. Except for histories of seizures, 44 of the dogs were healthy at the time the tests were performed. Abnormal test results were observed most frequently in dogs given only primidone and in dogs given combinations of anticonvulsant drugs. The test results that were abnormal most often were those for alanine transaminase and alkaline phosphatase activities, and sulfobromophthalein excretion. The dosage of anticonvulsant drug was found to modify certain test results. Statistically significant positive correlations were found between the dosage of primidone and serum alanine transaminase activity and between the dosage of phenytoin and serum alkaline phosphatase activity. Four of the dogs were examined because of signs of weakness and anorexia and 2 also had ascites. Three of the 4 dogs were euthanatized 2 to 49 days after admission with clinical signs compatible with hepatic failure, and cirrhosis of the liver was confirmed at necropsy. The fourth dog died at home and was not necropsied. Four of the remaining 44 dogs that apparently were healthy at the time of examination had abnormalities in hepatic biochemical test results that were comparable with those in the 4 dogs with clinical illness. We concluded that, although results of hepatic biochemical tests frequently may be abnormal in dogs given anticonvulsant drugs long-term, severe hepatic injury is observed less often.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma disappearances of sulfobromophthalein (BSP) and indocyanine green (ICG) given in single-dose IV injections to cats were measured. The BSP was given at a dose of 5 mg/kg of body weight; ICG was given at a dose of 1.5 mg/kg of body weight. The half-life, disappearance rate (percent per minute) and clearance (ml/min/kg) were determined during the initial phase of plasma dye elimination. The half-life for BSP was 2.2 +/- 0.7 minutes, and for ICG, 3.8 +/- 0.9 minutes. The disappearance rate for BSP was 34.7 +/- 10.1, and for ICG, 19.2 +/- 4.5. The clearance for BSP was 26.3 +/- 23.1 and for ICG, 8.6 +/- 4.1. The 30-minute percentage retention for BSP was 0.6 +/- 0.8% and for ICG, 7.3 +/- 2.9%. In the cat, ICG was cleared from the plasma less rapidly than BSP. There were no significant differences between males and females for these values. For both BSP and ICG, a significantly shorter half-life, faster disappearance rate, and lower 30-minute percentage retention were observed in the cat, compared with values observed in the dog given similar dye dosages. Clearance curves for BSP and ICG in the cat were similar to those in the dog, indicating that the 2 species may have similar volumes of dye distribution and excretory mechanisms. The results of the present study indicate that the 30-minute plasma level following a 5 mg/kg dose of BSP in normal cats is often too low for measurement, whereas the 30-minute plasma concentration of ICG following a 1.5 mg/kg dose is adequate. For the 30-minute dye retention test in the cat, ICG may be a preferred diagnostic agent.

Methyltestosterone (MT) or ethinyl estradiol (EE) was administered to adult rabbits for 20 weeks beginning with initial daily doses of 0.4 mg/kg MT and 0.015 mg/kg EE for three weeks, then these dosages were doubled at 3-week intervals to a maximum dosages 6.4 mg/kg and 0.24 mg/kg, respectively. Within 2 weeks, the serum gamma-glutamyltransferase activity of MT and EE treated rabbits was significantly greater than controls and increased progressively throughout the treatment period. Aspartate aminotransferase activity was also increased at 2 weeks and remained so for 17 weeks. Serum alkaline phosphatase was elevated at 2 weeks but thereafter was normal indicating that this enzyme is of no value in detecting steroid-induced hepatic dysfunction. Elevated serum bile acid concentration and prolonged BSP clearance indicated marked hepatic excretory dysfunction at higher dose levels. Histologic abnormalities were observed in the livers of both MT and EE treated rabbits. These lesions were more severe in the EE group in which there was marked bile duct proliferation, mononuclear cell infiltration of portal areas, and perilobular fibrosis. The studies indicate that the rabbit is susceptible to development of hepatic injury when receiving 17 alpha-alkyl substituted steroids and may be a useful animal model for investigations of the pathogenesis of steroid-induced cholestatic liver injury.

Primidone, phenytoin, or phenytoin and primidone in combination were given to healthy Beagle dogs for 6 months. Serum biochemical changes in dogs given primidone alone or phenytoin and primidone in combination for the entire 6-month test period included increased activities of alanine aminotransferase, alkaline phosphatase (AP), and gamma-glutamyltransferase, and decreased concentrations of albumin and cholesterol. Changes in dogs given phenytoin alone were limited to increased AP activity and decreased albumin concentration. Sulfobromophthalein excretion and conjugated bile acid concentration were within normal limits. All dogs given primidone alone or phenytoin alone remained clinically healthy throughout the treatment period. Three of 8 dogs given both drugs in combination became clinically ill after 9, 14, and 15 weeks of treatment, and were euthanatized. Two of the dogs developed clinical jaundice. In addition to the serum biochemical abnormalities observed in clinically healthy dogs, these dogs developed hyperbilirubinemia, delayed sulfobromophthalein excretion, and increased conjugated bile acid concentrations. Histologic examination of the liver showed intracanalicular casts of bile pigment typical of intrahepatic cholestasis in all 3 dogs. Histologic findings characteristic of treated dogs included hepatocellular hypertrophy attributable to hyperplasia of the smooth endoplasmic reticulum. Single-cell necrosis and multifocal lipidosis were observed in individuals of all treatment groups. Electron microscopy of the liver showed dilated bile canaliculi and damaged sinusoidal epithelium in dogs given both drugs. The elevated serum AP activity, associated with anticonvulsant drug therapy, was found to be exclusively the liver isoenzyme by cellulose acetate electrophoresis. The hepatic AP was localized to primarily the canalicular membranes by enzyme histochemistry. There was a statistically significant positive correlation between the AP activities of liver and serum. The results of this study indicate that long-term administration of anticonvulsant drugs to dogs is associated with clinical, serum biochemical, and histologic evidence of hepatic dysfunction. High drug dosage contributed most to abnormal serum biochemical test results, and combining phenytoin with primidone was responsible for more severe electron microscopic lesions of the liver of surviving dogs and for the death of 3 dogs.

Congenital portosystemic venous shunt causing signs of hepatic encephalopathy was diagnosed in 7 cats. The left gastric vein served as the shunt in four of these. Increases in blood ammonia and postprandial serum bile acids were the most consistent serum biochemical abnormalities. Excessive variation in red blood cell shape was a common but nonspecific hematologic finding. The jejunal-mesenteric venous injection of contrast material was the preferred method of portography to diagnose portosystemic shunts. Two cats were treated successfully by partial surgical occlusion of their shunts.

Sulfobromophthalein (BSP) and indocyanine green (ICG) were given in single-dose IV injections to dogs and plasma disappearances were measured. The BSP was given at a dose of 5 mg/kg of body weight, and ICG was given at doses of 1.0 and 1.5 mg/kg. The half-life for BSP was 4.2 +/- 0.8 minutes, and for ICG at the 1.0 mg/kg dose, 9.0 +/- 2.0 minutes, and at the 1.5 mg/kg dose, 8.4 +/- 1.9 minutes. The disappearance rate (percentage per minute) for BSP was 17.0 +/- 3.2, and for ICG at the small dose level, 8.1 +/- 1.7, and at the large dose level, 8.7 +/- 2.2. The clearances (ml/min/kg) for BSP were 15.4 +/- 6.2 and for ICG at the small dose level, 3.7 +/- 0.7, and at the large dose level, 3.9 +/- 0.7. The 30-minute percentage retentions for BSP were 1.9 +/- 1.1 and for ICG at the small dose level, 14.7 +/- 5.0, and for ICG at the large dose level, 11.4 +/- 3.0. All of the differences between BSP and ICG means for these values measured were statistically significant (P less than 0.01). There were no significant differences between sexes or between the ICG doses for any of the values (P greater than 0.05).

Hepatic imaging with 99mTc-sulfur colloid was used to diagnose primary hepatocellular carcinoma (PHC) in woodchucks infected with woodchuck hepatitis virus (WHV). Based on imaging results, 6 of 12 WHV-infected woodchucks had space-occupying hepatic lesions, and all 6 had PHC. Of the remaining woodchucks, 2 did not have PHC, 2 had discrete tumors (less than 1 cm diameter), 1 had miliary small tumors, and 1 had tumors located near the great vessels. Hepatic imaging was a valuable technique for diagnosis of PHC in WHV-infected woodchucks.

The livers of 16 woodchucks with naturally acquired chronic infection with woodchuck hepatitis virus were examined both grossly and histologically in 14 biopsy specimens and seven necropsy specimens. Fifteen woodchucks had lesions characteristic of chronic hepatitis; ten of these had chronic active hepatitis, four had chronic persistent hepatitis, and one had cirrhosis with nodular regeneration. In one woodchuck there was massive hepatic necrosis attributed to infection with an unclassified protozoan. Thirteen woodchucks had primary hepatocellular carcinoma. Metastasis to the lung was observed in only one woodchuck. These results were compared to liver lesions in 149 woodchuck hepatitis virus-negative woodchucks. Chronic hepatitis comparable to that associated with woodchuck hepatitis virus infection was not observed in woodchuck hepatitis virus-negative woodchucks although in one, a single, small hepatocellular adenoma was found.