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The Woodchuck Model of Hepatitis B Virus Infection
BudC. Tennant and John L Gerin
Abstract Introduction
T
he woodchuck hepatitis virus (WHV) was the first of Blumberg and colleagues' (1965, 1967) identification of the
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the mammalian and avian hepadnaviruses described Australia antigen and the subsequent establishment of its
after discovery of the virus of hepatitis B (HBV). relation to hepatitis B virus (HBV1) (Dane et al. 1970; Okuchi
Woodchucks chronically infected with WHV develop pro- and Murakami 1968; Prince 1968) are among the most impor-
gressively severe hepatitis and hepatocellular carcinoma, tant medical discoveries of modern times and have resulted
which present as lesions that are remarkably similar to those in major advances in understanding the natural history and
associated with HBV infection in humans. The initial viro- control of viral infections of the liver. As a result of HBV
logical studies and studies of pathogenesis utilized wood- infection, a small percentage of adults and a much larger
chucks that had been trapped in the wild and had acquired proportion of infants and young children develop persistent
WHV infection naturally. Research with wild woodchucks infection, becoming chronic carriers and potential sources of
was complicated by lack of knowledge of their backgrounds HBV. Characteristically chronic HBV carriers develop chronic
(e.g., dietary history, exposure to parasites or environmental hepatitis that often progresses to cirrhosis (Alberti et al. 1978,
toxins, and source and duration of WHV infection). Breed- 1979; Dudley et al. 1972; Prince et al. 1970), and there is
ing colonies of woodchucks have been established and main- convincing epidemiological evidence that HBV is an etio-
tained in laboratory animal facilities, and laboratory-reared logical factor in the pathogenesis of primary hepatocellular
woodchucks are superior for experimental studies of patho- carcinoma (HCC1) of humans (Blumberg and London 1982;
genesis or hepatocarcinogenesis.. It is possible to infect Blumberg etal. 1975;Hadziyannis 1981;Macnabetal. 1976;
neonatal woodchucks born in the laboratory with standard- Maupas et al. 1975, 1980; Popper et al. 1982; Prince et al.
ized inocula and produce a high rate of chronic WHV carriers 1975; Sherlock et al. 1970; Szmuness 1978).
that are useful for controlled investigations. WHV has been In at least four members of the family Sciuridae, natu-
shown experimentally to cause hepatocellular carcinoma, rally occurring infection with HBV-like viruses (hepadna-
supporting conclusions based on epidemiological and viruses) has been described (Marion et al. 1980,1983,1986;
molecular virological studies that HBV is an important etio- Minuk et al. 1986; Snyder 1968; Snyder and Ratcliffe 1969;
logical factor in human hepatocarcinogenesis. Chronic WHV Summers et al. 1978; Tennant et al. 1991b). The objective of
carrier woodchucks have become a valuable animal model this article is to summarize the experimental studies that have
for the preclinical evaluation of antiviral therapy for HBV been conducted with the Eastern woodchuck (Marmota
infection, providing useful pharmacokinetic and pharmaco- monax, groundhog), with the woodchuck hepatitis virus
dynamic results in a relevant animal disease model. It also (WHV1) and the use of this species as an animal model for
has been shown that the pattern of toxicity and hepatic injury HBV research.
observed in woodchucks treated with certain fluorinated
pyrimidines is remarkably similar to that observed in humans
that were treated with the same drugs, suggesting the wood- Natural History of WHV Infection
chuck has significant potential for the preclincial assessment
of antiviral drug toxicity. Summers and his colleagues (1978) described WHV infec-
tion in a colony of woodchucks in which high rates of chronic
Key Words: antiviral drug; hepatitis B virus; hepatocellular hepatitis and HCC had been observed (Summers et al. 1978).
carcinoma; nucleoside analogs; woodchuck hepatitis virus The serum from 15% of the woodchucks contained viral
'Abbreviations used in this article: AGSHV, arctic ground squirrel hepatitis
virus; DHB V, duck hepatitis B virus; GSHV, ground squirrel hepatitis virus;
Bud C. Tennant, D.V.M., is James Law Professor of Comparative Medicine HBV, hepatitis B virus; HBX, hepatitis B virus X gene; HCC, hepatocellular
in the Department of Clinical Sciences, College of Veterinary Medicine, carcinoma; NDMA, nitrosodimethylamine; WHcAg, nucleocapsid or core
Cornell University, Ithaca, New York. John L. Gerin, Ph.D., is Director of protein of woodchuck hepatitis virus; WHsAg, exterior envelope of wood-
the Division of Molecular Virology and Immunology, Georgetown Univer- chuck hepatitis virus; WHV, woodchuck hepatitis virus; WHX, woodchuck
sity Medical Center, Rockville, Maryland. hepatitis X antigen.
Volume 42, Number 2 2001 89DNA polymerase activity and viral particles that morpho- addition to woodchucks from Pennsylvania (Snyder 1968;
logically resembled HBV. It was concluded that WHV was Snyder and Ratcliffe 1969), other cases of hepatic neoplasia
a member of the same family of viruses to which HBV have been reported in laboratory-maintained woodchucks
belonged (Summers et al. 1978). originally trapped in New York and Maryland (Bond 1970;
WHV is classified as a member of the genus Long et al. 1975).
Orthohepadnavirus, family Hepadnaviridae (Gust et al. The hepatic neoplasms associated with naturally acquired
1986; Melnick 1982; Robinson et al. 1982). The genetic WHV infection characteristically were well-differentiated
organization of WHV is similar to HBV and to other mam- HCCs with hyperchromatic nuclei and prominent nucleoli
malian hepadnaviruses. Numerous 22-nm filaments and (Snyder and Summers 1980). In most cases, chronic, active
spherical particles are found in the serum of infected wood- hepatitis was present in the non-neoplastic liver, with abun-
chucks and are composed of the envelope protein of the virus. dant portal mononuclear cell infiltration extending beyond
Complete virions are 42 to 45 nm in diameter and are com- the limiting plate. There also was scattered parenchymal
posed of the exterior envelope (WHsAg1), the nucleocapsid hepatocellular necrosis, bile duct proliferation, and in some
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or core protein (WHcAg1), and, within the nucleocapsid, the there was evidence of early fibrosis (Popper et al. 1981; Roth
DNA genome (Ganem and Varmus 1987; Tiollais et al. et al. 1985; Snyder and Summers 1980; Snyder et al. 1982).
1985). The replicative cycle of WHV appears to be identical Progression of neoplasia from foci of altered hepatocytes, to
to that of HBV (Ganem and Varmus 1987; Summers 1987; small neoplastic nodules and to frank HCC, was described,
Tiollais et al. 1985). Transition of viral DNA to RNA during and some HCCs contained significant numbers of infiltrating
the life cycle of hepadnaviruses has similarities to that of hematopoietic cells (Popper et al. 1981). Trabecular, pseudo-
retroviruses (Ganem and Varmus 1987; Summers 1987). glandular, and pelioid histological patterns have been
Integration of viral DNA into the host genome is not, how- observed in HCCs of woodchucks, similar to that of humans
ever, essential for replication of hepadnaviruses as is the case (Roth et al. 1985). Metastasis of HCC outside the liver,
with retroviruses. The integration of WHV DNA sequences which occurs in humans and experimental animal models
into host cell genomic DNA may have an important role in with some frequency, has not been reported in woodchucks
hepatocarcinogenesis (described below). by most investigators, although pulmonary metastases have
The woodchuck habitat extends from northern Georgia, been observed (Roth et al. 1985).
Alabama, and Mississippi in the southern United States, west
to Oklahoma, Kansas, Nebraska, and North and South Dakota,
north to Quebec and Labrador, and across Canada to British Other Naturally Occurring Hepadnavirus
Columbia and the Yukon Territory, including a region in Infections of Animals
southeastern Alaska. A comprehensive, seroepidemiological
study of WHV infection has not been performed, and the Since the description of WHV, closely related hepadna-
prevalence of WHV infection throughout most of this range viruses have been described in several other mammalian and
remains unknown. WHV infection is hyperendemic in the avian species (Table 1). The morphology and genetic orga-
mid-Atlantic states, and the woodchucks originally studied nization of these hepadnaviruses are similar (Ganem and
by Summers and colleagues (1978) were from Pennsylvania. Varmus 1987; Tiollais et al. 1985). The ground squirrel
Of the woodchucks from Pennsylvania, New Jersey, and hepatitis virus (GSHV1) was described in California ground
Maryland, 23% were test positive for WHsAg, and an addi- squirrels (Spermophilus beecheyi) (Kodama et al. 1985;
tional 36% were positive for anti-WHs antibody for 59% Marion et al. 1980, 1986; Weiser et al. 1983). Persistent
prevalence rate of infection (Tyler et al. 1981). Others have GSHV infection is associated with chronic hepatitis and with
confirmed high rates of WHV infection in the mid-Atlantic HCC, although the frequency of HCC is believed to be less
states (Wong et al. 1982). In contrast, the rate of WHV than that associated with chronic WHV infection and develops
infection in central New York State has been estimated to be at an older age (Cullen and Marion 1996; Marion et al. 1986).
approximately 2% based on the presence of antibody to the A similar hepadnavirus has been described in the arctic
WHV surface anitgen antibody (Wong et al. 1982), and the ground squirrel (Spermophilus parryi) and named the arctic
rate of persistent WH surface antigenemia is less than 0.2%. ground squirrel hepatitis virus (AGSHV1). Infection with
Although the number of woodchucks tested is small, no sero- AGSHV was associated with a remarkably high rate of HCC
logical evidence of WHV infection has been found in Vermont, (Testut et al. 1996). Infection with a putative hepadnavirus
Massachusetts, or Iowa (Lutwick et al. 1982; Summers 1981; has been described in Eastern gray squirrels (Spermophilus
Young and Sims 1979). carolinensis) in Pennsylvania (Feitelson et al. 1986). Lesions
Hepatocellular neoplasms were described in woodchucks of hepatitis were reported, but hepatic tumors were not
from the Philadelphia Zoological Garden many years ago in observed. Evidence also has been reported for hepadnavirus
the form of multiple hepatic "adenomas" that ranged from infection in Richardson's ground squirrels (Spermophilus
0.3 to 1.0 cm in diameter (Fox 1912; Ratcliffe 1933). Two richardsonii) originating in Alberta (Minuk et al. 1986;
woodchucks were later described with primary hepatic neo- Tennant et al. 1991b). The hepatic lesions including HCC
plasms, one from the Washington Zoological Park and one were remarkably similar to those described in woodchucks,
trapped in Bethesda, Maryland (Habermann et al. 1954). In California ground squirrels, and Arctic ground squirrels.
90 ILAR JournalTable 1 Hepatitis B viruses (hepadnaviruses) of animals
Virus Scientific Name Host
Genus: Orthohepadnavirus
Hepatitis B virus (HBV)a Human Homo sapiens
Woodchuck hepatitis virus (WHV) Woodchuck, groundhog Marmota monax
California ground squirrel hepatitis virus (GSHV) California ground squirrel Spermophilus beecheyi
Arctic ground squirrel hepatitis virus (AGSHV) Arctic ground squirrel Spermophilus parryii
Woolly monkey hepatitis B virus (WMHBV) Woolly monkey Lagothrix labotricha
Genus: Avihepadnavirus
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Duck hepatitis B virus (DHBV) Domestic duck, Pekin duck Anas domesticus
Heron hepatitis B virus (HHBV) Grey heron Ardea cineria
Snow goose hepatitis B virus (SGHBV) Snow goose Anser caerulescens
a
Naturally acquired HBV infection also has been demonstrated in the chimpanzee, gorilla, gibbon, and orangutan.
Infection with the duck hepatitis B virus (DHBV1) has Summers and colleagues (1980) described the experi-
been reported in domestic Pekin ducks (Anas domesticus) mental infection of 4- to 8-mo-old woodchucks with serum
(Mason et al. 1980; Omata et al. 1983). DHBV has a world- from chronic WHV carriers. They described successful pro-
wide distribution. From Germany, avian hepadnaviruses also ductive infection, but infection was self-limited, and no
have been described in grey herons (Ardea cinerea) woodchucks became chronic carriers. Attempts by others to
(Sprengel et al. 1988) (heron hepatitis B virus) and in snow infect juvenile or adult woodchucks experimentally resulted
geese (Anser caerulescens) (Chang etal. 1999). Much of the in acute WHV infection (Millman et al. 1984; Tyler et al.
current understanding of hepadnavirus replication is based 1986; Wong et al. 1982) but, with one exception, did not
on research using DHBV in vivo and in vitro (Marion et al. cause chronic WHV infection. Morphological and molecu-
1987). HCC has been described infrequently in association lar virological studies of the liver have shown that virtually
with DHBV infection (Omata et al. 1983), and the hepato- 100% of hepatocytes become infected after experimental
carcinogenicity of DHBV in ducks remains questionable. WHV infection (Kajino et al. 1994). Although replicative
Hepatic neoplasms have not been associated with heron hepa- forms were cleared rapidly during recovery, covalently
titis B virus infection (Sprengel et al. 1988). closed circular WHV DNA persisted in three of 10 wood-
chucks after evidence of WHV replication had ceased
(Kajino etal. 1994). Clearance of experimental WHV infec-
Experimental Hepadnavirus Infection of tion in adult woodchucks is associated with robust humoral
Woodchucks and cell-mediated immune responses (Guo et al. 2000;
Menne et al. 1997; Shanmuganathan et al. 1997). Treatment
The original observations of WHV infection were made with with immune suppressive doses of cyclosporine A signifi-
woodchucks trapped in the native habitat and subsequently cantly increases the rate of chronic WHV infection (Cote et
maintained in the laboratory. Such woodchucks, in which al. 1991, 1992).
WHV infection was naturally acquired, proved to be valu- When adult humans are infected with HBV, less than 5%
able sources of virus and hepatic tissue for histological and become chronic carriers (Seeff et al. 1987; Tassopoulos et al.
molecular analyses; however, their limitations for experi- 1987). HBV infection early in life, however, results in high
mental purposes were soon recognized. It was impossible to rates of chronic infection (Blumberg and London 1982;
know when and for how long trapped woodchucks had been Ganem and Varmus 1987). The high rates of chronic WHV
infected with WHV or to be certain about their nutritional infection observed in woodchucks from hyperendemic areas
history and/or exposure to environmental factors, which (Popper et al. 1981; Snyder and Summers 1980; Tyler et al.
might have influenced the course of WHV infection. Impor- 1981) suggested that, as in humans, infection in woodchucks
tantly, hepatic lesions caused by nematodes such as Ackertia early in life (or possibly vertical transmission) must be
marmotae and Capillaria sp. were common in wild wood- necessary to account for the high WHV carrier rates.
chucks (Cohn et al. 1986) and could complicate the interpre- To utilize the advantages of the woodchuck as an experi-
tation of experimental results. mental animal model, it became necessary to breed and rear
Volume 42, Number 2 2001 91woodchucks in laboratory animal facilities. The result allowed explanation may relate to the unusual circannual reproduc-
knowledge of the experimental woodchucks' genetic back- tive cycle of the woodchuck. For at least 8 mo of the year,
ground, definition of their diet, and, possibly, prevention of the testicles of male woodchucks are abdominal and produce
their natural exposure to WHV infection and other diseases little or no testosterone, resulting effectively in functional
that are endemic in wild woodchuck populations. To meet castration (Baldwin et al. 1985).
this requirement, a breeding colony of WHV-negative wood- With one exception, the diseases caused by WHV infec-
chucks was established at Cornell University in 1979. The tion are confined to the liver. Immune-mediated glomerulo-
colony now serves as the source of woodchucks for experi- nephritis has been reported with increased frequency in
mental studies of WHV infection and hepatocarcinogenesis. woodchucks chronically infected with WHV, and it appears
Woodchucks born in the laboratory animal setting are inocu- to be similar etiologically to the glomerulonephritis caused
lated at birth with diluted serum from standardized infectious by WHV (Peters et al. 1992).
pools obtained from chronic WHV carrier woodchucks (Cote Investigation of hepadnavirus replication has been
et al. 2000a,b; Gerin et al. 1989; Popper et al. 1987). After impeded by the limited availability of tissue culture systems.
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inoculation, woodchucks are monitored using specific sero- However, full-length clones of the genomes of HBV (Will et
logical markers of WHV infection (WHV DNA, WHsAg, al. 1982), WHV (Miller et al. 1990), DHBV (Sprengel et al.
anti-WH core antibody, and anti-WH surface antibody) (Cote 1984), and GSHV (Seeger et al. 1984, 1987) have been
etal. 1993; Wong etal. 1982). shown to be infectious; and injection directly into the hepatic
The rate of chronic WHV infection after neonatal inocu- parenchyma of the homologous host species results in pro-
lation is 60% or greater (Cote et al. 2000a,b; Gerin et al. ductive hepadnavirus infection. During transfection experi-
1989; Popper et al. 1987; Tennant et al. 1988). Survival ments to determine the viral gene or genes responsible for
analyses have been made of chronic WHV carriers experi- host range restriction, it was found that woodchucks could be
mentally infected at birth with WHV, of woodchucks that infected with GSHV (Seeger et al. 1987, 1991). The chip-
recovered from neonatal WHV infection by clearing WH munk (Eutamias species) also has been shown to be suscep-
viremia and developing anti-WHs antibody, and of control tible to GSHV infection (Trueba et al. 1985).
woodchucks not infected with WHV but born and raised HCC in woodchucks chronically infected with WHV
under similar laboratory conditions. All WHV carriers were infection has been reported to occur more frequently (Gerin
dead by 56 mo of age, and the lifetime risk of HCC was et al. 1989; Popper et al. 1981, 1987) than in California
100% (Gerin et al. 1989; Popper et al. 1987). The median ground squirrels chronically infected with GSHV; and in
time to death from HCC in WHV carriers was 29 mo. In GSHV infection, HCC develops at an older age (Marion et
contrast, 42% of the woodchucks with resolved WHV infec- al. 1986). Because woodchucks could be infected with both
tion and 62% of uninfected controls were alive at 56 mo of hepadnaviruses, it could be determined whether the apparent
age. Although the rate of HCC in WHV carriers was signifi- differences in oncogenicity of GSHV and WHV in their
cantly higher than that of woodchucks in which WHV infec- respective natural hosts were the result of differences in host
tion was resolved, 17% of the woodchucks that recovered response or of viral genetic differences. When neonatal
from neonatal WHV infection developed HCC (Gerin et al. woodchucks were experimentally infected with WHV or
1989). HCC was not observed in the uninfected, laboratory- GSHV, the rates of chronic infection for each virus were
reared, control woodchucks. These results provide compel- similar, and at 2 yr of age, no difference was noted in the
ling direct experimental evidence for the carcinogenicity of severity of chronic hepatitis (Seeger et al. 1991). By 2 yr,
WHV and, by analogy, for other mammalian hepadnaviruses hepatic neoplasms had developed in 13 of 16 chronic WHV
(HBV, GSHV, and AGSHV) in which naturally acquired carriers. In sharp contrast, only one of 16 chronic GSHV
hepadnavirus infection has been associated with HCC. The carriers at that age had developed a single, grossly identifi-
rate of HCC in woodchucks with experimentally induced able hepatic tumor nodule (5 mm diameter), which was clas-
chronic WHV infection was similar to that observed in sified as a hepatic adenoma. On the basis of these observa-
woodchucks with naturally acquired chronic WHV infection tions, it was concluded that the difference between GSHV
(Popper et al. 1981; Roth et al. 1985; Snyder and Summers and WHV in oncogenic capacity that had been observed in
1980), and the presence of preneoplastic foci of altered their respective natural hosts must be due to genetic differ-
hepatocytes with progressive aneuploid change (Cullen et al. ences between the viruses, although additional differences in
1994; Mi et al. 1994) also was similar (Abe et al. 1988; host factors could not be excluded.
Toshkov et al. 1990). It would be useful to infect California ground squirrels
In contrast to humans and experimental rats, the sex of with WHV, but this may not be possible. Attempts to trans-
experimental woodchucks does not influence their rate of feet California ground squirrels with WHV DNA have not
infection with HCC. This result was unexpected because yet been successful (Seeger et al. 1987). It should, however,
both in humans infected with HBV and in laboratory rodents be possible to assess differences in the viral genetic determi-
in which HCC is induced by chemical hepatocarcinogenesis, nants of oncogenicity using WHV/GSHV chimeric hepadna-
males tend to have higher rates of HCC than females. The virus constructs, and such investigations are under way.
92 1LAR JournalExperimental Infection of Ducks with DHBV These now-classical investigations demonstrated that the
relative risk of HCC in chronic HBV carriers was 100 times
DHBV was identified originally in Pekin ducks in China higher than in noncarrier individuals (Beasley 1982, 1988;
(Omata et al. 1983, 1984), where it caused persistent infec- Beasley and Lin 1978; Beasley et al. 1981). A similar high
tion. Subsequently, DHBV infection was demonstrated in relative risk of HCC in HBV carriers also has been reported
domestic duck populations in the United States (Mason et al. in the United States (Prince and Alcabes 1982).
1980, 1983), and it probably has a worldwide distribution. A second line of molecular evidence comes from the
In contrast to mammalian hepadnaviruses, chronic DHBV demonstration of covalent integration of truncated HBV
infection is characteristically not associated with significant DNA sequences in the cellular DNA of hepatic tumors of
histological changes in the liver (Cullen et al. 1989; Marion most HBV carriers (Brechot et al. 1981; Chen et al. 1982;
et al. 1984) or with mild changes. As mentioned previously, Shafritz 1982). These observations suggest that HBV has a
DHBV appears not to be a primary cause of HCC in ducks. direct oncogenic role similar to that of other tumor-producing
Experimental studies of DHBV infection (Fukuda et al. viruses in which integrated viral DNA or, in the case of
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1987; Jilbert et al. 1988,1996; Marion et al. 1987; Mason et retroviruses, proviral DNA causes malignant transformation
al. 1983; Vickery and Cossary 1996) have shown age to be a by insertional mutagenesis (Chen et al. 1982; Shafritz 1982).
critical determinant of the outcome of hepadnavirus infection Finally, the comparative medical evidence strongly
as it is in humans and other mammals. Congenitally infected supports an etiological role of hepadnaviruses in hepato-
ducklings or ducklings infected at an early age develop per- carcinogenesis. At least four members of the family Sciuridae
sistent DHBV infection. Infection of older ducks is followed have been described in which persistent hepadnavirus infec-
typically by resolution of infection, often with inflammatory tion has been closely associated with development of HCC.
changes in the liver. As in the woodchuck, adult DHBV How hepadnaviruses actually cause HCC, however, is not
infection results in the infection of almost all hepatocytes completely understood.
with the expression of DHBV DNA and of viral proteins. Two general mechanisms have been proposed to explain
Immune clearance of DHBV infection occurs rapidly and hepadnavirus-associated hepatocarcinogenesis (Tennant
apparently without massive hepatocellular necrosis (Jilbert 1992). One hypothesis is that hepadnaviruses have a direct
et al. 1992). molecular role (Buendia 1994; Buendia and Pineu 1995). In
Much of what is known about the replication of hepadna- this model, integration of hepadnaviral nucleic acid sequences
viruses has been learned using the DHBV system both in into host cell DNA is considered a critical mutagenic event,
vitro and in vivo (Summers and Mason 1982). The role of which causes an alteration in expression of genes that regu-
covalently closed circular DNA as the template for viral late the cell cycle (protooncogenes and tumor suppressor
transcription was first demonstrated in the duck system genes). Ultimately, these changes result in neoplastic transfor-
(Summers 1987). The molecular structure (Newbold et al. mation of hepatocytes. Hepadnavirus integration is thought to
1995), the mechanism of replenishment of the cccDNA pool, initiate cell transformation in a manner comparable with that
and the control of this pathway by surface antigen molecule caused by chemical hepatocarcinogens (Farber and Sarma
(Summers et al. 1990; 1991) all have been investigated pri- 1987; Pitot and Dragan 1991).
marily using DHBV. The duck also has been valuable in Support for the molecular hypothesis comes from detec-
antiviral drug development (Tsiquaye et al. 1996). tion of integrated hepadnaviral sequences in the cellular DNA
of most primary hepatic tumors from HBV carriers (Brechot
et al. 1981; Chen et al. 1982; Shafritz 1982). Similar integra-
Mechanisms of Hepatocarcinogenesis tions of viral nucleic acids have been observed in other mam-
Associated with Hepadnavirus Infection malian cancers caused by DNA tumor viruses (Johnson and
Williams 1982). Integrations in apparently non-neoplastic
There are three lines of evidence that indicate HBV is an hepatic tissue of HBV carriers suggest integration precedes
important etiological factor in human hepatocarcinogenesis. formation of hepatic neoplasms. Although occasional inte-
The first is the epidemiological evidence demonstrating the grations have been identified in or near a potential oncogene,
close relation between the prevalence of HBV infection and the large majority of HBV integrations in human HCC appear
that of HCC (Maupas et al. 1980; Szmuness 1978). Signifi- to be random.
cantly higher rates of HCC are observed in individuals with Integrated WHV sequences also have been found in most
chronic HBV infection than in case controls both in the hyper- HCCs from WHV-infected woodchucks (Korba et al. 1989).
endemic regions of Africa (Blumberg et al. 1975; Maupas et al. Characteristically, only portions of the viral genome are inte-
1980; Prince et al. 1975) and Asia (Asano et al. 1982; Chien grated, and the sequences are often rearranged (Ganem and
et al. 1981; Hann et al. 1982; Okuda et al. 1982) and in the Varmus 1987; Schodel et al. 1980). Molecular cloning and
United States and Great Britain, where the rates of HBV analyses of integrated WHV DNA and associated flanking
infection and HCC were relatively low (Omata et al. 1979; sequences of cellular DNA initially demonstrated that inte-
Tabor et al. 1977; Viola et al. 1981). Compelling evidence grations occurred at multiple sites within the woodchuck
of the etiological role of HBV infection in hepatocarcinogenesis genome (Fuchs et al. 1989; Kaneko et al. 1986; Korba et al.
comes from prospective epidemiological studies in Taiwan. 1989; Mitamura et al. 1982; Ogston et al. 1982; Rogler and
Volume 42, Number 2 2001 93Summers 1984; Rogler et al. 1987). Multiple integrations The physiological function, if any, of N-myc2 remains
were sometimes observed in a single tumor mass. One unknown (Fourel et al. 1992). A distinctive feature of
explanation for multiple integrations was that several hepatocarcinogenesis in woodchucks with chronic WHV
hepadnavirus integrations could have accumulated in a single infection appears to be the coupling of viral integration into
hepatocyte and been propagated clonally by the transformed the myc family of protooncogenes. Viral integrations appear to
hepatocyte. The alternative was that an initial integration of be preferentially associated with the N-myc2 locus (Flajolet
hepadnaviral DNA was followed by secondary rearrange- et al. 1997; Wei et al. 1998). Insertion of WHV enhancer
ments of integrated viral sequences (Rogler 1991). Integra- sequences either upstream or downstream of the N-myc2 cod-
tions of WHV DNA have been reported in apparently ing domain result in increased transcription either of normal
nontumorous liver of woodchucks (Fuchs et al. 1989; Korba N-myc2 RNA or of a hybrid N-myc2fWHV transcript that is
et al. 1989; Mitamura et al. 1982; Ogston et al. 1982; Rogler initiated at the normal N-myc2 start site. Transcriptional
and Summers 1984; Rogler et al. 1987). These reports, activation by enhancer insertion appears to be a common
combined with the unique integration patterns of individual mechanism (Wei et al. 1992). A liver-specific regulatory
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hepatic tumors, suggests that integration occurs early in element in the WHV genome has been identified that appears
hepatocarcinogenesis and before clonal expansion. Evidence to control cis activation of N-myc2 (Fourel et al. 1996).
of integrations in early neoplastic nodules has been described Downstream integration of WHV DNA also has been associ-
before development of frank HCC (Rogler 1991; Yang et al. ated with activation of N-myc2 (Fourel et al. 1994).
1993, 1996). Recently, Buendia and her colleagues have reported that
Mammalian hepadnaviruses do not contain oncogenes transgenic mice carrying the N-myc2 gene under the control
similar to those found in transforming retroviruses (Ganem of WHV regulatory sequences are highly predisposed to
and Varmus 1987; Tiollais et al. 1985). Up-regulation of cancer of the liver (Renard et al. 2000). Seventy percent devel-
most of the well-characterized protooncogenes has not been oped either HCC or hepatocellular adenomas. A transgenic
demonstrated (Moroy et al. 1986). Increased expression (5- founder that carried the unmethylated WHV/N-myc2 trans-
to 50-fold) of c-myc was observed, however, in three of a gene sequence died at the age of 2 mo with a large liver
series of nine woodchuck HCCs (Hsu et al. 1988; Moroy et tumor, demonstrating the high oncogenic capacity of the
al. 1986, 1989), and truncation and rearrangement of the woodchuck N-myc retroposon. Mutations or deletions of the
gene were demonstrated. In one tumor, no direct linkage (3-catenin gene similar to those of HCCs from humans and
between WHV DNA integration and c-myc activation was mice (De La Coste et al. 1998) were present in 25% of the
demonstrated. In that case, rearrangement and activation of hepatic tumors of the N-myc2 transgenic animals, and tumor
c-myc appeared to be similar to that observed in Burkitt's latency (time to tumor) was significantly reduced (Renard et
lymphoma and in acute B- and T-cell leukemias in which al. 2000). When N-myc2 transgenic mice were crossed with
chromosomal translocations are present (Moroy et al. 1986). p53 null mice, the absence of one p53 allele markedly accel-
In the other two integrations, c-myc activation was the result erated the onset of liver cancer, providing direct experimental
of insertional mutagenesis (Hsu et al. 1988), and WHV DNA evidence for synergy in multistage hepatocarcinogenesis
insertions interrupted different regions of the c-myc locus. between activation of N-myc2 and diminished expression of
The position and orientation of the WHV and c-myc sequences p53 (Renard et al. 2000).
and the use of the c-myc promoter excluded involvement of Like the woodchuck, the California ground squirrel pos-
the hepadnaviral promoter in c-myc activation (i.e., promoter sesses a functional, transcriptionally active N-myc2 locus in
insertion). In both cases, WHV sequences analogous to one the brain (Quignon et al. 1996). However, increased N-myc2
of the HB V enhancers were present, suggesting an enhancer expression is unusual in HCCs from California ground squirrels
insertion mechanism (Hsu et al. 1988). In a large series of infected with GSHV. Amplification of C-myc expression,
woodchuck HCCs, activation of c-myc was observed in 10% however, is more frequent in ground squirrel hepatic neo-
of the tumors (Dejean and deThe 1990). plasms than in those of woodchucks (Transy et al. 1992).
Buendia and her colleagues now have demonstrated that HCCs from woodchucks experimentally infected either with
N-myc messenger RNA was overexpressed in 60% of wood- WHV or with GSHV have been analyzed (Hansen et al.
chuck HCCs examined, and this transcript was not detected 1993). The propensity for WHV genomic DNA to integrate
in normal woodchuck liver. Insertions of WHV nucleic acid in or near the N-myc2 locus in HCCs from chronic WHV
sequences were located adjacent to cellular N-myc sequences carriers was confirmed, whereas in woodchuck HCCs associ-
in 20% of the tumors studied. Woodchucks have two N-myc ated with GSHV infection, such integrations were exceptional.
loci. One N-myc locus was homologous to the N-myc gene Seven of 17 (41%) WHV-induced tumors had rearrange-
of other mammalian species; the other was an intronless gene ments of the N-myc2 allele. Only one of 16 GSHV-associated
with the characteristic structure of a retrotransposon and was tumors (6%), however, had such N-myc2 rearrangements.
identified as N-myc2 (Fourel et al. 1990). N-myc2 was Based on the observations in GSHV-induced HCCs from
mapped to the X chromosome. Expression of N-myc2 is ground squirrels and from woodchucks, it was concluded
highly restricted, and the brain is the only normal tissue of that the differences in hepadnavirus insertion and in N-myc
the woodchuck in which N-myc2 RNA was detected (Fourel activation between woodchucks and ground squirrels were due
et al. 1992). primarily to viral genetic differences and not to differences
94 ILAR Journalin the respective host species (Hansen et al. 1993). A similar hepatocellular injury, regeneration, and cirrhosis that pre-
conclusion was reached by Buendia and her colleagues cede development of HCC (Johnson and Williams 1987).
(Quignon et al. 1996). Experimental evidence supporting the role of hepatic
Transfection of a nonmalignant, SV-40 T antigen- inflammation and hepatocellular injury as a key event in
transformed mouse hepatic cell line with HBV DNA pro- hepadnavirus-associated HCC comes from the work of
duced cells with a malignant phenotype that grew in soft agar Chisari and his colleagues with transgenic mice described
and were tumorigenic in nude mice. The hepatitis B virus X above (Chisari 2000; Chisari et al. 1989; Dunsford et al.
gene (HBX1) transcript was expressed at a much higher level 1990; Moriyama et al. 1990). In the lines of transgenic mice
than that observed in vivo (Hohne et al. 1990). In subsequent they have studied, a direct relation was found between the
studies, it was concluded that overexpression of the HBX expression and retention of HBsAg in hepatocytes and the
was required for malignant transformation of the cell line severity of hepatitis. A close correlation also was observed
(Seifer et al. 1991). HBX is known to promote hepato- between the severity of hepatitis in transgenic mice and
carcinogenesis in c-myc transgenic mice (Terradillos et al. development of HCC (Chisari et al. 1989; Dunsford et al.
Downloaded from https://academic.oup.com/ilarjournal/article-abstract/42/2/89/727383 by guest on 17 April 2020
1997) and may promote hepadnavirus-induced hepato- 1990; Sell et al. 1991). Increased free radical production
carcinogenesis (Seeger and Mason 2000). within the liver was associated with oxidative DNA damage
The X gene appears to be essential for normal replication in this model, similar to that suggested in woodchucks
of hepadnaviruses in vivo (Chen et al. 1993). The wood- (Bannasch et al. 1995) and that observed in other mice (Faux
chuck hepatitis X (WHX1) protein is coexpressed with et al. 1992).
WHcAg in the liver of chronic WHV carrier woodchucks
(Feitelson et al. 1993; Jacob et al. 1997). Feitelson and
colleagues demonstrated that when WHX was cotranslated Interaction of Hepadnaviruses and
in vitro with p53, WHX/p53 complexes developed, and simi- Chemical Carcinogens in
lar WHX/p53 complexes were demonstrated in the livers of Hepatocarcinogenesis
chronic WHV carriers (Feitelson et al. 1997). Combined
with observations in HBV transgenic mice (Ghebranious and Aflatoxin is among the most potent hepatocarcinogens
Sell 1998; Ueda et al. 1995), the observations of Feitelson et known and is a frequent contaminant of the diets of popula-
al. suggest that binding of WHX to p53 may prevent entry of tions with high incidence rates of HBV infection and HCC.
p53 into the nucleus, diminish tumor suppressor activity, and The possible interaction between hepadnavirus infection and
represent an important mechanism by which the hepadna- aflatoxin has been investigated using the woodchuck model
virus X-gene product could promote hepatocarcinogenesis (Bannasch et al. 1995; Rivkina et al. 1996; Tennant et al.
(Feitelson et al. 1997). Mutations of p53 also may alter its 1991a). When administered early in life to woodchucks
tumor suppressor activity, but such mutations were found experimentally infected at birth with WHV, aflatoxin B, did
primarily in the less differentiated hepatic tumors, suggest- not appear to increase the rate of chronic WHV infection or
ing the mutations altered tumor progression at a later stage of the rate of development of HCC (Tennant et al. 1991a).
development (Hsia et al. 2000; Ueda et al. 1995). When administered beginning at 1 yr of age to established
The second hypothesis regarding the role of hepadnavirus chronic WHV carriers, the time to tumor was moderately but
in hepatocarcinogenesis is that hepatic injury caused by viral significantly reduced by aflatoxin Bj treatment (Bannasch et
infection and related hepatocellular regeneration provide an al. 1995). A similar synergistic interaction between HBV
environment that enhances fixation of spontaneous muta- and aflatoxin has been reported in the tree shrew (Yan et al.
tions, rearrangements, or chromosomal translocations that 1996).
are responsible for malignant transformation of hepatocytes. It has been shown that HBV transgenic mice are more
Such a role for hepadnaviruses in hepatocarcinogenesis would susceptible to chemical hepatocarcinogenesis than controls
be comparable to the processes of promotion and/or progres- (Dragani et al. 1990). When transgenic mice that expressed
sion recognized in multistage chemical hepatocarcinogenesis HBsAg were exposed to nitrosodiethylamine (NDMA1) or
(Farber and Sarma 1987; Hanahan and Weinberg 2000; Pitot aflatoxin, they developed hepatic adenomas and HCC more
and Dragan 1991; Tennant 1992). rapidly and more extensively than unexposed transgenic
Support for this model comes from the observation that controls or normal mice receiving either of these hepato-
chronic liver injury associated with cirrhosis frequently pre- carcinogens. These results demonstrated a significant syner-
cedes the development of HCC in HBV-infected people gistic interaction between chemical hepatocarcinogens and
(Kalayci et al. 1991; Zhou et al. 1999). Chronic hepatitis C the chronic hepatocellular injury induced by overexpression
virus infection results in progressively severe chronic hepati- of HBsAg in transgenic mice (Sell et al. 1991). A similar
tis and cirrhosis and is frequently associated with develop- synergistic effect of HBX expression in transgenic mice and
ment of HCC. A significant increase in the rate of HCC has NDMA in hepatocarcinogenesis has also been reported
been observed in inherited forms of chronic liver disease in (Slagle et al. 1996).
humans including hemochromatosis and alpha-1-antitrypsin Another possible hepatocarcinogenic mechanism related
deficiency (Schafer and Sorrell 1999). These inherited dis- to hepatic inflammation has been suggested by studies of
eases also are associated with development of progressive nitric oxide (NO) production in chronic viral hepatitis. In the
Volume 42, Number 2 2001 95liver, NO biosynthesis from arginine is catalyzed by Use of the Woodchuck Model in
inducible NO synthetase. Endotoxin, y-interferon, and other Development of Antiviral Therapy
cytokines (Billiar et al. 1992a,b; Cullen et al. 1989; Curran et
al. 1990, 1991; Geller et al. 1993; Hortellano et al. 1992; Woodchucks with experimentally induced chronic WHV
Knowles et al. 1990; Wood et al. 1993) can increase the infection are now frequently used in the preclinical assess-
activity of this enzyme significantly. Under certain circum- ment of antiviral drugs being developed for treatment of
stances, NO may have a protective effect against experi- chronic HBV infection. Nucleoside analogs (Chu et al. 1998;
mental hepatic injury (Curran et al. 1991). Under other Hostetler et al. 2000; Korba et al. 2000a,b,c) and immune
conditions, hepatic production of NO may contribute to response modifiers have been tested (Gangemi et al. 1996;
development of hypotension in septic shock (Kilbourn et al. Tennant et al. 1996b). Before testing in woodchucks, the
1990). drugs are screened for antiviral activity against HBV in the
Woodchucks chronically infected with WHV excrete 2.2.15 cell system, a HepG2 cell line engineered to produce
more nitrate in the urine and more NDMA than uninfected HBV constitutively (Korba and Gerin 1992). Acyclovir and
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control woodchucks (Liu et al. 1991). Similar increased NO azidothymidine, which had no selectivity in 2.2.15 cells, had
production has been observed in HBV transgenic mice no antiviral effect in the woodchuck model of HBV infection.
(Chisari 2000). Nitrate and NDMA are derived from NO Adenine-5'-arabinoside monophosphate, which had moder-
produced from L-arginine. Urinary nitrate excretion also is ate antiviral activity in vitro, had significant antiviral activity
increased in a human patient with chronic HBV infection in woodchucks in vivo, and activity was increased by specific
(Nguyen et al. 1992). liver targeting (Enriquez et al. 1995). Most nucleoside ana-
In primary hepatocyte cultures from normal woodchucks, logs with intermediate antiviral activity in vitro against HBV
NO synthesis can be induced by endotoxin, and hepatocytes had intermediate antiviral activity in vivo. One exception
cultured from chronic WHV carriers produce significantly was l,-2/deoxy-2'fluoro-l-(3-D arabinofuranosyl-5-iodo-
more NO and nitrosamine than hepatocytes from uninfected uracil, which had modest in vitro activity. In woodchucks,
controls (Liu et al. 1992). SV-40 T antigen-transformed however, l,-2'deoxy-2'fluoro-l-P-D arabinofuranosyl-5-iodo-
uracil had potent antiviral activity but was highly toxic, as
woodchuck hepatocyte cell lines have the capacity to pro-
had been observed in humans (Tennant et al. 1998). The in
duce NO in response to endotoxin (Liu et al. 1993) utilizing
vitro activity of l-(2-fluoro-5-methyl-p\L-arabinofuranosyl)
the L-arginine-NO pathway and to produce NDMA. WHsAg
uracil (L-FMAU, clevudine) was only moderate but was
purified from woodchuck serum can induce NO production
highly potent in vivo in WHV carrier woodchucks (Chu et al.
in woodchuck hepatocyte cultures (Liu et al. 1994). NO
1998; Peek et al. 2000). Lobucavir also had intermediate in
reacts with O2 to produce NO2, which exists in equilibrium
vitro activity in the 2.2.15 cell system but, at the dosage
with N2O3 and N2O4. N 2 O 3 and N2O4 are potent nitrosating
studied, had potent antiviral activity against WHV (Tennant
agents capable of reacting with water to form nitrite and nitrate
et al. 1996a).
(Marietta et al. 1988) or, in neutral solution, of nitrosating
dialkylamines to form nitrosamines. NO could act as a car- Lamivudine, which had a very high selective index in
cinogen indirectly by causing formation of hepatocarcinogenic vitro, was a potent antiviral drug in woodchucks with favor-
nitrosamines such as NDMA, which is a strong alkylating able pharmacokinetics (Rajagopalan et al. 1996) and was
agent. Point mutations can be induced by NDMA by methy- without toxicity at doses of 5 or 15 mg/kg/day for 28 days
lation of guanine to 7-methyl and to O6~methylguanine, or by (Korba et al. 2000b; Peek et al. 1997). The absence of effect
methylation of adenine to 3-methyladenine (Archer 1989; of lamivudine on hepatic cccDNA was postulated to be due
Magee 1989). to the absence of cell division (Moraleda et al. 1997).
NO also has the capacity to act as a direct mutagen (Wink Lamivudine has been shown in the woodchuck model to
et al. 1991). In vitro, NO can deaminate deoxynucleosides, act synergistically both with alpha-interferon (Korba et al.
deoxynucleotides, or intact DNA and contribute to 2000a) and with famciclovir (Korba et al. 2000c). Similarly,
deamination-related mutations. It has been predicted based high antiviral potency of the closely related emtricitabine has
on studies of the rate of deamination of guanine to xanthine been shown in WHV carrier woodchucks (Korba et al.,
in vitro that guanine/cytosine —> adenine/thymine transitions 2000d). Extended lamivudine treatment of woodchucks with
could be a frequent form of mutation induced by NO. Adeno- chronic WHV infections delayed the development of HCC
sine deamination to hypoxanthine would be expected to result and significantly extended survival in one study (Peek et al.
in adenine/thymine —» guanine/cytosine transitions, and 1997). In another study, no effect of lamivudine was observed
removal of the xanthine formed by guanine deamination on hepatocarcinogenesis. Although treatment was begun at
would result in depurination and transversions (Nguyen et al. an older age, the duration of treatment was not as long and
1992). The demonstration of increased NO formation in the the apparent effect of treatment on viral load was not as great
hepadnavirus-infected liver suggests that NO could have an (Mason et al. 1998). A very high rate of polymerase gene
important role in hepatocarcinogenesis. mutations was detected in both humans and woodchucks
96 ILAR Journalafter long-term lamivudine therapy (Baumert et al. 1998; synthesis and release of cGMP and nitric oxide biosynthesis by hepato-
Brechot et al. 1981; Peek et al. 1997; Zhou et al. 1999). cytes. AmJPhysiol262:C1077-C1082.
Billiar TR, Hoffman RA, Curran RD, Langrehr JM, Simmons RL. 1992b.
A role for inducible nitric oxide biosynthesis in the liver in inflammation
and in the allogeneic immune response. J Lab Clin Med 120:192-197.
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