JOURNAL OF VIROLOGY, Sept. 2009, p. 8396–8408
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 17
Detection of Clonally Expanded Hepatocytes in Chimpanzees with
Chronic Hepatitis B Virus Infection?†
William S. Mason,1*‡ Huey-Chi Low,2,3Chunxiao Xu,1Carol E. Aldrich,1Catherine A. Scougall,2,3
Arend Grosse,2,3Andrew Clouston,4Deborah Chavez,5Samuel Litwin,1Suraj Peri,1
Allison R. Jilbert,2,3‡ and Robert E. Lanford5‡
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 191111; School of Molecular and Biomedical Science,
University of Adelaide, Adelaide SA 5005, Australia2; Infectious Diseases Laboratories, Institute of Medical and Veterinary Science,
Adelaide SA 5000, Australia3; University of Queensland Clinical Research Centre, Brisbane QLD 4029, Australia4;
and Department of Virology and Immunology, Southwest Foundation for Biomedical Research and
Southwest National Primate Research Center, 7620 NW Loop 410, San Antonio, Texas 782275
Received 3 April 2009/Accepted 8 June 2009
During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA,
producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as
genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into
clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks
(Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of
>1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139–1144,
2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be
explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue,
we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with
hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected
with HBV for at least 20 years. Fifty clones ranging in size from ?35 to 10,000 hepatocytes were detected using
invPCR in 32 liver biopsy fragments (?1 mg) containing, in total, ?3 ? 107liver cells. Based on searching the
analogous human genome, integration sites were found on all chromosomes except Y, ?30% in known or
predicted genes. However, no obvious association between the extent of clonal expansion and the integration
site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large
clones of hepatocytes.
There are approximately 1012hepatocytes in the human
liver, virtually all of which can be infected during a hepatitis B
virus (HBV) infection. Chronic infection leads to the develop-
ment of hepatocellular carcinomas (HCC) in ?25% of pa-
tients, with tumors typically emerging after several decades.
The tumors are clonal and usually contain integrated HBV
DNA that was acquired at an early step in transformation,
prior to tumor outgrowth. The tumors themselves typically do
not support virus replication and do not contain covalently
closed circular DNA (cccDNA), the virus transcriptional tem-
plate. The fact that the tumors typically contain integrated
HBV DNA suggests that they arise through mutation of ma-
ture hepatocytes and not from hepatic stem cells or hepato-
biliary progenitor cells (37, 44, 62). Though contrary reports
have appeared (3, 21), hepatocytes are currently the only liver
cell type unambiguously established to be susceptible to HBV
infection in most individuals.
With a few exceptions, the virus integration sites found in
HCC samples from different patients have not provided in-
sights into the mechanisms of carcinogenesis (8, 9, 53). Aside
from showing a possible preference for transcriptionally active
regions (36), the sites of HBV integration have appeared
somewhat random in location and, by inference, not directly
related to cellular transformation. Thus, it has been hypothe-
sized that hepatocyte transformation usually results from mu-
tations that are caused by persistent inflammation, leading to
cumulative oxidative damage to the host DNA (18); from the
action of a viral oncogene expressed from integrated DNA (11,
20); or even from hit-and-run mechanisms (19). In brief, the
change or series of changes in host gene expression that lead to
HCC in most human HBV carriers are uncertain. In contrast,
inappropriate activation of N-myc2 due to nearby integration
of woodchuck hepatitis virus (WHV) DNA, or to integration at
the distal win and bn3 loci, is found in the majority of HCC
samples from woodchucks chronically infected with WHV
(4–6, 12, 14, 26, 45, 54, 56), implying that this is a critical step
in the formation of woodchuck HCC. About half of the inte-
gration sites inferred to be responsible for N-myc2 activation
are within a few hundred to a few thousand base pairs from
N-myc2, and about half are in the win locus, which maps 150 to
180 kbp from N-myc2 (14, 45).
The earliest mutations needed to begin the transformation
of hepatocytes into tumor cells, whether in chronically infected
humans or woodchucks, appear to lead, first, to the formation
of “premalignant” lesions, also called foci of altered hepato-
cytes (FAH). FAH appear prior to HCC, generally do not
* Corresponding author. Mailing address: Fox Chase Cancer Cen-
ter, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: (215) 728-
2462. Fax. (215) 728-2412. E-mail: firstname.lastname@example.org.
‡ W.S.M., A.R.J., and R.E.L. contributed equally to this work.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 17 June 2009.
support virus replication, and are the morphological sites from
which HCC is thought to emerge (41, 47, 49, 56). FAH may
contain thousands or tens of thousands of hepatocytes, and
thousands of FAH may be present in HBV- or WHV-infected
livers. Interestingly, some FAH have an obviously malignant
phenotype, whereas others do not, suggesting diverse causes
for their emergence. In the woodchuck, a subset of FAH was
shown to express N-myc2, also a characteristic of HCC in
woodchucks, as noted above, but not of other liver cells (56,
A possibly unifying property of HCC and most FAH may be
a failure to express HBV (or WHV) (16, 41, 55, 56). Thus, it is
possible that many or all FAH begin with clonal expansion of
initially rare mutated hepatocytes which, for diverse reasons,
cannot support virus replication and as a result are no longer
targeted by the antiviral immune response. These cells would
have a lower death rate than surrounding hepatocytes (34, 55)
and would, as a result, undergo clonal expansion. Thus, im-
mune evasion via loss of virus production may be an important
early step in oncogenesis. In fact, even cells that have no
morphological changes and are not preneoplastic should un-
dergo clonal expansion if they are unable to support virus
infection. This could, for instance, explain the decline over
time in virus production and the fraction of infected hepato-
cytes in long-term HBV carriers (34). It may also explain the
observation that woodchucks chronically infected with WHV
contain similar numbers of virus-negative FAH and morpho-
logically normal foci of hepatocytes that are evidently not FAH
but, like FAH, are observed because they are virus negative
(55, 56). The general progression to virus-negative hepatocytes
seen in chronic HBV infections in humans may reflect a much
more extreme emergence of hepatocytes that are not part of
FAH (reviewed in reference 34).
Evidence has been presented that cirrhotic nodules and the
more advanced lesions, dysplastic nodules (previously called
adenomatous hyperplasia), contain integrated HBV DNA and
are clonal in HBV patients (1, 38–40, 60, 61). It remains un-
clear if virus-negative foci, or even FAH, are clonal in the
woodchuck. Whether hepatocyte clones are common in non-
cirrhotic livers of HBV patients is also unknown.
As a first step in determining possible links during chronic
hepadnavirus infection between clonal expansion of hepato-
cytes, a decline in the fraction of infected hepatocytes, and
oncogenesis, we examined the livers of woodchucks chronically
infected with WHV (33). Clonal expansion was measured us-
ing inverse PCR (invPCR) to detect virus-cell junctions cre-
ated by random integration of WHV DNA; thus, clonal expan-
sion of hepatocytes leads to an increase, in the hepatocyte
population, of the frequency of particular virus-cell junctions
that were initially present at only one copy. We estimated that
the livers of 2.4-year-old woodchucks contained greater than
100,000 clones of greater than 1,000 hepatocytes, amounting to
1 to 2% of the liver. The extent of clonal expansion may be
even greater, because many clones may not contain integrated
WHV DNA or may contain virus-cell junctions that were not
detectable using the invPCR assay. The incidence of clones of
?1,000 hepatocytes could not be explained by random death
and regeneration of hepatocytes over the relatively short life
span of the woodchucks (33), implying that some other expla-
nation, such as immune evasion, is needed to rationalize their
emergence (34). The morphological correlates of these clones
have not yet been determined.
While studies using the woodchuck model should lead to a
better understanding of liver disease in HBV carriers, analyses
of disease progression in the woodchuck using well-character-
ized human arrays (52) only reliably detect changes in the
expression of highly conserved genes. We therefore initiated
studies to determine if clonal expansion of hepatocytes was
also a feature of chronic HBV infections of chimpanzees and if
clonal expansion was associated with particular integration
sites in or near growth-promoting genes in host DNA. Like
WHV infection of woodchucks, HBV infection of chimpanzees
usually does not lead to cirrhosis, although recently a rare case
of cirrhosis and HCC was detected in a chimpanzee (R. E.
Lanford, unpublished data). Our main expectation was that
much larger clones of hepatocytes should be detected in the
chimpanzees because of their much longer duration of infec-
tion (2.4 years in the woodchucks versus ?20 years in the
Integrated HBV DNA was detected using an invPCR assay
(33, 48) carried out on DNA extracted from small fragments of
chimpanzee liver collected by needle biopsy. Clonal expansion
of hepatocytes was found; however, the anticipated increase in
hepatocyte clone size compared to the woodchuck (33) was not
Next, all integration sites were mapped where possible to the
human genome, for which more complete gene-mapping data
are available than for the chimpanzee. Of the 209 different
virus-cell junctions that were detected out of a total of 506
virus-cell junctions that were sequenced, 146 could be mapped
unambiguously on human chromosomes, and of these, 58 were
mapped to known or predicted host genes. Most of the rest
were in repeated sequences, so their locations are ambiguous.
For others, the fragment of cell DNA was too short to reliably
map. No association was found between integration sites and
the extent of clonal expansion of hepatocytes.
MATERIALS AND METHODS
Chimpanzees. Chimpanzees were housed at the Southwest National Primate
Research Center (SNPRC) at the Southwest Foundation for Biomedical Re-
search. The animals were cared for in accordance with the Guide for the Care
and Use of Laboratory Animals, and all protocols were approved by the Insti-
tutional Animal Care and Use Committee. Only a limited number of chronically
HBV-infected chimpanzees were available for this study, since most animals are
exposed to HBV as adults and clear the infection. All of these animals were
positive for HBV surface antigen (HBsAg) and antibodies to HBV core antigen
(anti-HBcAg) by commercial serological assays (Abbott Laboratories). Informa-
tion on exposure to HBV was not available for some animals, since they were
HBV positive at first testing. The histories of the animals were varied, since they
span a period of 43 years and include housing at several different facilities. The
histories relevant to this study are briefly described below. No inclusion criteria
were imposed; all HBsAg-positive animals at SNPRC were included in the study.
Chimpanzee 4x0136 had no history of exposure to HBV and was HBsAg
positive when first tested in 1983. Experimental treatments included exposure to
hepatitis delta virus (HDV) in 1988, treatment with antibody to HBsAg in 1990,
and treatment with antisense to HBV in 1994. Immunizations included pre-S
peptide in 1985 and recombinant HBcAg in 1995.
Chimpanzee 4x0139 was exposed to HBV in 1979 and to HDV in 1984.
Experimental treatments included exposure to recombinant plasmid encoding
interleukin 12 in 1999, lamivudine therapy in 2002, and a single dose of pegylated
alpha interferon in 2006. Immunizations included canary pox virus encoding all
three forms of HBsAg (pre-S1, pre-S2, and S) in 1999, plasmid encoding HBcAg
in 2001, and HBsAg pre-S2 protein in 2001.
VOL. 83, 2009CLONAL EXPANSION OF HEPATOCYTES 8397
Chimpanzee 4x0222 was exposed to HBV in 1984 following immunization with
anti-idiotypic antibodies to HBsAg.
Chimpanzee 4x0327 had no record of HBV exposure. It was obtained from a
private owner and was positive for HBsAg at first testing in 1986. Experimental
therapies included antibody to HBsAg in 1990, antisense HBV oligonucleotides
in 1994, antibody to HBsAg in 2001, and a small-molecule antiviral in 2005.
Chimpanzee 4x0328 was acquired from the same private owner as 4x0327 and
was also positive for HBsAg at first testing in 1986. These two animals have
identical sequences in the HBV core gene and may have been exposed to the
same source or one may have exposed the other. Experimental therapies in-
cluded antibody to HBsAg in 1990, 1994, and 2001 and treatment with the same
small-molecule antiviral as 4x0327 in 2005.
Chimpanzee 4x0230 was exposed to HBV in 1980 following immunization with
HBsAg in 1979. Experimental therapy included 18 doses of alpha interferon in
Chimpanzee 4x0506 had no history of HBV exposure and was HBsAg positive
at first testing in 1983. Experimental treatments included an undefined exposure
to HDV prior to 1983, immunization with extracts from cells infected with an
HBV-carrying retroviral vector in 1995, an HBV DNA vaccine in 1997, and
antibody to HBsAg in 1998.
Chimpanzee 4x0509 had no history of exposure to HBV and was positive for
HBsAg upon first testing in 1984. This animal is a sibling of 4x0506 and likely had
a common HBV exposure. Experimental therapies included an undefined expo-
sure to HDV prior to 1984, antibody to HBsAg in 1991, immunization with
extracts from cells infected with an HBV-carrying retroviral vector in 1995, and
an HBV DNA vaccine in 1997.
Chimpanzee 4x0313 was used as an HBV-negative control animal for these
studies, and samples prior to and 8 weeks after hepatitis C virus (HCV) infection
were included for histology and HBcAg staining (Table 1). A liver biopsy spec-
imen collected in a subsequent study of transient HBV infection in this chim-
panzee was used as a positive control (see Fig. 8A).
The chimpanzees live in group housing in the chimpanzee village at SNPRC
with indoor/outdoor access. The housing is air conditioned and heated and has
color TV. The animals have significant contact with an animal caretaker each
day. A full time Ph.D. primate behaviorist and staff provide an environmental
enrichment program for the animals and visit each animal daily. The animals are
fed Teklad Primate Diet two times per day, as well as fresh fruit and other treats
daily. Cages are cleaned two times per day. SNPRC maintains a large staff of
veterinarians, and one is on call 24 h per day to provide care for the animals.
The liver biopsy tissues were collected under sedation. Similar to humans,
primates recover rapidly from liver needle biopsies and do not appear to expe-
rience significant discomfort. Although recovery is uneventful, animals are mon-
itored closely for several hours. Liver tissues were fixed with 10% formalin in
phosphate-buffered saline or with ethanol-acetic acid (EAA) (3:1), dehydrated,
embedded in paraffin, and sectioned at 5 ?M onto glass slides. Liver tissues were
also quick-frozen and stored at ?80°C for subsequent nucleic acid extraction.
Assays for serum HBsAg, anti-HBc, and HBV DNA in chronically infected
chimpanzees. HBV DNA in serum was quantified by real-time PCR. The DNA
was purified using the Qiagen DNA Mini Kit essentially as described by the
manufacturer. Briefly, samples were incubated with the proteinase K digestion-
lysis buffer, bound to the silica gel membrane of a QiaAmp Spin Column, washed
with ethanol buffers, and eluted with water. HBV DNA was then quantified by
a real-time, 5? exonuclease PCR (TaqMan PCR Core Reagent Kit; Applied
Biosystems) using a primer/probe combination that recognized a portion of the
HBV surface gene (30). The primers and probe were selected using Primer
Express software (Applied Biosystems, Foster City, CA). The fluorogenic probe
was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine and
was obtained from Synthegen (Houston, TX). The primers and probe used were
as follows: forward primer (5?-CCGTCTGTGCCTTCTCATCTG-3?; 1551 to
1571), reverse primer (5?-AGTCCAAGAGTYCTCTTATGYAAGACCTT-3?;
1674 to 1646), and Fam-Tamra probe (5?-CCGTGTGCACTTCGCTTCACCT
CTGC-3; 1577 to 1602), numbered according to reference 24 (accession no.
AF222323). The primers and probe were used at 10 pmol per 50-?l reaction
mixture. The PCRs included a denaturation step of 10 min at 95°C and then 40
cycles of amplification using the universal TaqMan PCR standardized condi-
tions: 15 s at 95°C for denaturation and 1 min at 60°C for annealing and
extension. Standard curves for HBV DNA copy number equivalents were de-
rived from plasmid DNA. Serum HBsAg levels were determined by end point
dilution using an enzyme-linked immunosorbent assay purchased from Abbott
TABLE 1. Chimpanzees chronically infected with HBV
(GE/ml of serum)c
% in situ
2.8 ? 103
?0.020.30 Severe portal and
Mild portal and lobular
Moderate portal and
Mild portal and lobular
Mild portal and lobular
Mild portal and lobular
Moderate portal and
Mild portal and lobular
4x0139F 30 292.1 ? 108
70 10050 0.20
2.9 ? 102
3.2 ? 106
1.6 ? 103
3 0.04 0.009h
9.1 ? 105
1.4 ? 103
1.0 ? 108
aAge is given for the time of the biopsy used in virus-cell DNA junction analyses.
b? indicates the minimal duration of infection, since the animal was HBV positive at the time of the first testing and complete histories were not available.
cHBV DNA and HBsAg values and histologic activity are from the time of the biopsy used in virus-cell DNA junction analyses.
dPeriodic acid Schiff diasterase (PAS-D) staining of Kupffer cells that increase in size due to the uptake of cell remnants.
eFibrosis scores based on the Ishak staging system (25) (0, no fibrosis; 1, fibrous expansion of some portal areas, with or without short fibrous septa; 2, fibrous
expansion of most portal areas, with or without short fibrous septa).
fChimpanzee 4x0313 was a control animal biopsied prior to and 8 weeks after infection with HCV.
gND, not done.
hChimpanzee 4x0327 had 1 in situ hybridization-positive hepatocyte present out of 11,322 total hepatocytes scanned.
iF, female; M, male.
8398 MASON ET AL.J. VIROL.
Laboratories and purified HBsAg standards. Anti-HBsAg and anti-HBc were
determined by enzyme-linked immunosorbent assay (Abbott Laboratories).
Determination of the prevalent HBV core gene sequence in the livers of
chimpanzees chronically infected with HBV. DNA was extracted from multiple
?1-mg fragments of liver cut from biopsy cores and containing ?800,000 cells. In
brief, the fragments were placed in 400 ?l of 0.05 M Tris-HCl, pH 7.6, 0.1 M
NaCl, 0.01 M EDTA, 0.5% (wt/vol) sodium dodecyl sulfate, 1 mg proteinase K
per ml at 55°C for 2 h with periodic vortexing, by which time the liver fragment
had been digested. The mixture was extracted with an equal volume of phenol-
chloroform (1:1), and DNA was precipitated from the aqueous phase, after the
addition of 1 ?l of 20 mg dextran/ml, by the addition of 2 volumes of 100%
The substrate for integration into host DNA is predominantly double-stranded
linear viral DNA (DSL DNA) (15, 59), formed as an aberrant by-product of
hepadnavirus DNA synthesis (46). Integrations occur near the ends of DSL
DNA, apparently via nonhomologous end joining. Some integrations also appear
to occur via recombination in the cohesive-overlap region of relaxed circular
(RC) DNA, possibly after conversion of RC DNA into a fully double-stranded
DNA by strand displacement synthesis through the cohesive overlap (59). An
even lower level of integration may occur via recombination elsewhere in the
Detection of integrants by invPCR has been carried out by assaying for virus-
cell junctions occurring near one of the ends of DSL DNA and/or in the cohesive
overlap of RC DNA. In the present study, we assayed for virus-cell junctions
between cell DNA and sequences near the left end of DSL DNA, i.e., the end
located just upstream of the HBV core gene and corresponding to the 5? end of
viral pregenomic RNA (46). As a preliminary step necessary to define restriction
endonuclease cleavage sites and to design PCR primers for invPCR, the se-
quence of the HBV strain present in each chimpanzee was determined by PCR
amplification of the 5? end of the pregenome at nt ?1820 and the viral core gene
using the primers 5?-GGGGGAGGAGATTAGGTTAA-3? (1744 to 1763)
and5?-TTATGAGTCCAAGGAATACT-3? (2472 to 2453) (numbered according
to reference 24; accession no. AF222323), followed by direct sequencing.
InvPCR for detection of virus-cell junctions and clonal expansion of hepato-
cytes. Following extraction of liver DNA as described above, high-molecular-
weight DNA was purified away from low-molecular-weight viral DNA by elec-
trophoresis in 1% low-melt agarose gels in E buffer (0.04 M Tris-HCl, 0.02 M
sodium acetate, 1 mM EDTA, pH 7.2, 0.5 ?g/ml ethidium bromide). This step
was performed to reduce cccDNA, which interferes with the invPCR assay (59).
The section of the gel containing DNA with a size of 10 to 20 kbp or larger was
excised and equilibrated overnight at 4°C with NEBuffer 3 (New England Bio-
labs; 0.05 M Tris-HCl, 0.1 M NaCl, 0.01 M MgCl2, 0.001 M dithiothreitol, 0.01%
[wt/vol] Triton X-100, pH 7.9). The fluids were discarded, and the gel slice was
melted for 20 min at 72°C and then cooled to 37°C. BglII was added, and the
DNA was digested for 1 h at 37°C. BglII cleaves the HBV present in the
chimpanzees (Table 1) at nucleotide (nt) 2425 (but not between that location and
the 5? end of DSL DNA at nt ?1820) and at an unknown location in the host
DNA that depends upon the site of integration (Fig. 1). The DNA was then
purified using a QIAquick PCR purification kit, as described by the manufac-
turer. Control experiments revealed ?50% recovery of DNA from the QIAquick
columns using fragments of lambda DNA ranging in size from 0.5 to 2.4 kbp.
InvPCR assays for HBV DNA integrated near BgIIl in the host DNA were
then carried out as previously described (33, 48). The BglII-digested DNA was
incubated with T4 DNA ligase to circularize the BglII restriction fragments and
then digested with BsaWI, which cleaves HBV DNA at nt 2331, to create linear
DNA fragments in which chimpanzee DNA, including the virus-cell junction, was
flanked by HBV DNA (Fig. 1). Subsequent digestion with BsaBI, which cleaves
at nt 2430, was carried out to reduce the background signal from any remaining
viral cccDNA. Though amplification of a full-length cccDNA did not occur under
our PCR conditions, cccDNAs with internal deletions, or that religate due to rare
cleavages at BglII star sites (sites with an imperfect match to the BglII recogni-
tion site), can cause significant background in the assay. The DNA was next
serially diluted in 96-well trays (8 rows by 12 columns), and the diluted DNA was
subjected to nested PCR (33, 48). In general, the first dilution was distributed to
the first row (12 wells) in a plate, the next row contained a further threefold
dilution, and so on, with the eighth row serving as a DNA-negative control. The
outer primers for chimpanzees 4x0327 and 4x0328 were F1 (5?-CTCGCAGAC
GAAGGTCTCAAT-3?; 2390 to 2410) and R1a (5?-AGATCACGGACCGAAG
GAAAGAA-3?; 1991 to 1969); the inner primers were F2 (5?-CCGCGTCGCA
GAAGATCT-3?; 2413 to 2430) and R2a (5?-GTCAGCAGGCAAAAACGAG
AGTAA-3?; 1968 to 1945) (Fig. 1). The outer primers for the remaining
chimpanzees (Table 1) were F1 and R1b (5?-AGATCACGGACCGACGGAA
AGAA-3?; 1991 to 1969); the inner primers were F2 and R2b (5?-TCAGAAG
GCAAAAAAGAGAGTAAC-3?; 1967 to 1944).
The PCR products were subjected to agarose gel electrophoresis, and the
bands were cut out of the gels for subsequent sequencing (48). Automated
sequencing of virus-cell junctions was carried out using the appropriate R2
primer. Sequence alignments were carried out using the GCG program FASTA
in order to locate virus-cell junctions. Cell sequences adjacent to integrated HBV
DNA were then screened using Sequencher (Gene Codes Corporation) to iden-
tify virus-cell junctions present in hepatocytes that had undergone clonal expan-
sion. The chromosome and nucleotide locations of chimpanzee DNA sequence
homologues in the human genome were determined using a BLAST search of
the database reference genomic sequences (refseq_genomic). The coding poten-
tial at this location in the human genome was evaluated using the Affymetrix
Integrated Genome Browser (March 2006 build). The reported transcriptional
activities in liver of genes in which integration occurred (see Table S1 in the
supplemental material) was derived from the Genomics Institute of the Novartis
Research Foundation BioGPS website and database (http://biogps.gnf.org).
Out of 826 sequences that were determined, 320 appeared to be derived from
cccDNA, while 506 were from virus-cell junctions. A summary of the locations in
HBV DNA of the virus-cell junctions detected in liver fragments from the eight
HBV-infected chimpanzees is presented in Fig. 2. Of the virus-cell junctions
shown here, representing distinct integration events, 120 out of 176 mapped near
to but downstream of the left end of DSL DNA, 40 out of 176 mapped with in
the cohesive-overlap domain, and 16 out of 176 mapped upstream of the cohe-
Estimates of cell numbers per milligram of liver were determined using a
PCR-amplified fragment of the chimpanzee alpha interferon receptor 1 gene
(accession no. NC_006488) (primers 5?-GTAACTTCAGGGTCTCCTTCTT-3?
[4909 to 4930] and 5?-CCTTGGGGCAATCATGTTAT-3? [5438 to 5419]) as a
control for quantitative PCR (qPCR). The primers used for quantifying chim-
panzee DNA by real-time qPCR were 5?-TTGCTACCCTTTGGCTGCAT-3?
(5099 to 5118) and 5?-AAAGGGGAGACAGCTGAGAA-3? (5318 to 5299).
Histology, histochemistry, and in situ hybridization. As noted above, liver
biopsy specimens were fixed in buffered formalin or EAA (28), paraffin embed-
FIG. 1. Procedure for invPCR detection of virus-cell junctions cre-
ated by integration of HBV DNA. A hypothetical integration of the
left end of DSL DNA (at nt 1820) into host DNA is shown at the top.
To detect integrations in or near this position, high-molecular-weight
liver DNA was cleaved with BglII restriction endonuclease, which cuts
HBV DNA at nt 2425 and host DNA at an unknown location. The
DNA was then circularized by incubation with T4 DNA ligase and cut
with BsaWI restriction endonuclease (followed by BsaBI digestion to
reduce background from cccDNA) to produce linear molecules in
which the virus-cell junction was flanked by viral sequences. These
fragments were diluted in 96-well PCR trays and amplified by nested
PCR, using virus specific primers as described in the text.
VOL. 83, 2009 CLONAL EXPANSION OF HEPATOCYTES8399
ded, and sectioned (5 ?m). Sections of formalin- and EAA-fixed chimpanzee
liver tissue were imported into Australia under a permit from the Convention on
International Trade in Endangered Species of Wild Fauna and Flora. Formalin-
fixed sections were used for hematoxylin and eosin staining, periodic acid Schiff
after diastase digestion, Gordon and Sweets staining of reticulin, and immuno-
staining of HBcAg. For HBcAg detection, slides were deparaffinized in EZ-
DeWax (BioGenex; HK 585) for 10 min and rinsed with water. Antigen retrieval
was performed in a microwave pressure cooker for 15 min at 1,000 W and 15 min
at 300 W in citrate buffer (antigen retrieval solution; BioGenex; HK 086-9K).
The cooled slides were rinsed with water and phosphate-buffered saline and
treated sequentially with peroxidase suppressor, universal block, and avidin (all
reagents from a Pierce 36000 Immuno Histo Peroxidase Detection Kit). The
primary antibody was prepared in rabbits against purified HBV core particles.
HBV core protein was expressed using baculovirus, and core particles were
purified from the medium of infected insect cells by pelleting them on a sucrose
cushion, banding on a cesium chloride gradient, and then banding on a sucrose
gradient (2, 31). The slides were incubated for 1 h at room temperature with the
rabbit anti-core antibody diluted 1:200 in universal block containing biotin, for
0.5 h with biotinylated goat anti-rabbit immunoglobulin G, and for 0.5 h with
avidin-biotin complex. The slides were developed with diaminobenzidine, coun-
terstained with Mayer’s hematoxylin, and mounted with Pierce mounting me-
Sections of EAA-fixed chimpanzee liver were used for detection of HBV DNA
by in situ hybridization essentially as previously described (27), incorporating a
prehybridization step in 50% deionized formamide, 0.5 mg/ml tRNA, and 0.5
mg/ml carrier DNA without labeled probe for 60 min at 37°C. Full-length HBV
DNA (pBluBac45HBV1.3; GenBank V01460 ) and control (pBlueBac4.5;
Invitrogen) probes were labeled with digoxigenin-UTP by nick translation
(Roche), denatured, and used at 2.5 ng/?l of hybridization mixture. Following in
situ hybridization, the sections were counterstained with hematoxylin.
Computational modeling of the liver. To determine the association between
cumulative hepatocyte turnover and clonal expansion due solely to random death
and regeneration of hepatocytes, we used the program Comp10, which simulates
a liver of 1 million hepatocytes (35). Each hepatocyte at the start is uniquely
identified and present only once. In each cycle of the simulation, 2.5% of
hepatocytes were selected at random to die and another 2.5% were selected at
random to divide to restore liver mass. It should be noted that a cycle is not a day;
it is simply a computational cycle. Similar results were generated when 0.5%
rather than 2.5% was used per cycle; that is, the results were not sensitive to
changes in hepatocyte turnover rates. Because of the large sample population (1
million hepatocytes), differences between runs were minimal and did not alter
the conclusions, which were based on the distributions of hepatocyte clone sizes.
The program and Fortran code are available upon request to S. Litwin or W.
Mason; the code and a more detailed explanation of the program are presented
Serum HBV DNA and HBsAg levels decline over time in
HBV-infected chimpanzees. The group of animals used in this
study represented all of the chronically HBV-infected animals
at SNPRC; no selection criteria were imposed. All of the chim-
panzees in this study were positive for HBV DNA (i.e., virus)
except for 4x0313, which served as an HBV-negative control
(Table 1). Levels of HBV DNA varied from undetectable (less
than 100 to 1,000 genome equivalents [GE] per ml of serum) to
greater than 1 ? 108GE per ml at the time of biopsy in 2006,
when virus-cell junctions were analyzed. Similarly, all animals
except 4x0313 were positive for HBsAg. Serum HBsAg levels
spanned a 350-fold range, from 0.2 to 70 ?g per ml.
We next examined historical and more recent serum samples
to determine if the serum HBV DNA levels were similar be-
fore and after the 2006 biopsy. Remarkably, we detected spon-
taneous clearance or multiple-log-unit reductions in the levels
of HBV DNA in four of the nine animals (4x0136, 4x0327,
4x0506, and 4x0509) (Fig. 3), with one other animal (4x0222)
having less than 1,000 GE per ml during the entire time frame
examined. Levels of HBsAg in 2003 and 2006 were consistent
with the findings on HBV DNA. All animals remained positive
for HBsAg, and none developed anti-HBsAg antibodies.
InvPCR of chimpanzee liver DNA reveals clonal expansion
of hepatocytes. DNA for invPCR was extracted from four
?1-mg fragments of liver cut from each needle biopsy speci-
men. The high-molecular-weight fraction, containing DNA of
?10 to 20 kbp, was partially purified by preparative gel elec-
trophoresis. This procedure reduces the amount of cccDNA in
the preparations, which otherwise competes with detection of
virus-cell junctions by invPCR, primarily through the genera-
tion of short, easily amplified viral-DNA fragments (59). The
purified high-molecular-weight DNA was then cleaved with
BglII, and virus-cell junction fragments were self-ligated to
produce a circular DNA, as shown in Fig. 1. Cleavage with
BsaWI was then carried out to generate a linear DNA in which
the virus-cell junction was flanked by viral DNA. The DNA was
then serially diluted in 96-well microtiter trays, with each di-
lution distributed through 12 wells, and subjected to nested
PCR with the HBV-specific primers (F1 and R1a or R1b, and
F2 and R2a or R2b) that span virus-cell junctions. The PCR
products generated in individual wells were resolved by aga-
rose gel electrophoresis, cut out of the gel, and sequenced to
identify virus-cell junctions.
Representative results of gel electrophoresis of the products
of nested PCRs are shown in Fig. 4. Figure 4A shows a highly
repeated virus-cell junction fragment present in a fragment of
liver from chimpanzee 4x0136. After correction for DNA
losses, this fragment corresponded to a clone size of ?10,000
FIG. 2. Locations of virus-cell junctions detected by invPCR. The
cumulative fraction of virus-cell junctions is plotted versus the site on
the HBV DNA genome representing the virus-cell junction site. The
vast majority of virus-cell junctions mapped either between the R2
sequencing primer and the left end of DSL DNA (Fig. 1) or within the
cohesive-overlap domain of HBV, which extends to nt 1603. Approx-
imately 10% (16/176) mapped upstream of this region. In general,
those junctions located more than 500 to 600 nt from the R2 primer
were detectable because of internal deletions between R2 and the
8400 MASON ET AL.J. VIROL.
hepatocytes. Figure 4B shows a similar analysis of a liver frag-
ment from chimpanzee 4x0328. No highly repeated bands were
apparent; however, sequencing of the various PCR products
revealed two clones of ?130 hepatocytes and one of ?400
hepatocytes. The results from analysis of four ?1-mg liver
fragments from each of the eight chimpanzees are summarized
in Fig. 5. As shown, the clones ranged in size from ?30 to 50
hepatocytes, the lower-range cutoff of the assay, up to 10,000,
with a median size of 200, and with 10 clones of ?1,000 hepa-
Since the chimpanzees had been chronically infected with
HBV for at least 20 years at the time of biopsy, random death
and regeneration within the hepatocyte population could ex-
plain the clonal expansion of some hepatocytes with integrated
viral DNA and the loss of others, particularly if the liver is
viewed as a closed system in which essentially all hepatocyte
replacement occurs by division of mature hepatocytes. This
was calculated, as described in Materials and Methods, for a
simulated liver of 1 million hepatocytes (roughly the size of the
liver fragments that were analyzed), which were considered to
be initially unique with respect to viral DNA integration sites.
FIG. 3. Decline in viremia in chronically HBV-infected chimpanzees over time. Virus titers (GE/ml) were determined by qPCR assays for virus
DNA genomes in chimpanzee sera, as described in Materials and Methods. The year of the liver biopsy used for invPCR was 2006 (2006a for
chimpanzee 4?0327; 2006b represents a later biopsy in 2006). Œ, not detected, ?100 GE/ml.
FIG. 4. Gel electrophoresis of invPCR products indicating exten-
sive clonal expansion. Following BglII cleavage, circularization, linear-
ization with BsaWI (Fig. 1), and incubation with BsaBI, DNAs were
diluted in 96-well trays. The indicated fractions of the total DNA
extracted from each liver fragment from chimpanzees 4x0136 (A) and
4x0328 (B) were distributed in 12 wells and amplified by nested PCR.
The products were detected by agarose gel electrophoresis and ex-
tracted for sequencing. Lane M, HaeIII-cut phage ?X DNA.
FIG. 5. Summary of clone sizes detected by serial dilution and
nested PCR of inverted virus-cell junctions. The distribution of clone
sizes detected in the livers of the eight chronically HBV-infected chim-
panzees is shown. Ten clones of ?1,000 hepatocytes were detected in
an analysis of 32 liver biopsy specimens (4 each from eight chimpan-
zees) containing in total ?3E7 liver cells. On average, ?0.1% of
hepatocytes may be present as clones of 1,000 or more hepatocytes
detected by invPCR.
VOL. 83, 2009 CLONAL EXPANSION OF HEPATOCYTES8401
Figure 6 shows the expected hepatocyte clone size distribu-
tions, assuming that all integrants were detectable. As can be
seen, in this model, no clones of ?1,000 hepatocytes were
predicted after cumulative hepatocyte turnover equivalent to
replacement of the entire liver 91 times (Fig. 6B). Even after
273 turnovers (Fig. 6D), only 1.44% of the clones with greater
than 200 hepatocytes would be expected to be greater than
1,000 hepatocytes in size. The fact that 40% of the clones
greater than 200 hepatocytes in size were also greater than
1,000 in size in our experiments suggests that the clonal ex-
pansion, particularly the appearance of clones of ?1,000 hepa-
tocytes in size, is not attributable solely to random death and
regeneration in the hepatocyte population. It should be noted
that random death and regeneration must still play a role in the
emergence of hepatocyte clones.
An alternative, but not exclusive, explanation for clonal ex-
pansion is that integration of virus DNA near particular host
genes gives the respective hepatocytes a selective growth or
survival advantage. To determine if the integration sites clus-
tered at hot spots or, for clonally expanded hepatocytes, were
adjacent to genes associated with growth or survival, we at-
tempted to map the flanking cell DNA for all 209 distinct
integration sites, detected by invPCR, to the human genome.
The human genome map was used because the full chimpanzee
map is not yet available. As shown in Table 2, out of 209
TABLE 2. Distribution of HBV DNA integration sites
No. of virus-
2.19 ? 108
2.37 ? 108
1.94 ? 108
1.87 ? 108
1.78 ? 108
1.67 ? 108
1.55 ? 108
1.42 ? 108
1.15 ? 108
1.31 ? 108
1.31 ? 108
1.29 ? 108
9.55 ? 107
8.72 ? 107
8.11 ? 107
7.99 ? 107
7.75 ? 107
7.45 ? 107
5.58 ? 107
5.94 ? 107
3.39 ? 107
3.44 ? 107
1.48 ? 108
2.28 ? 107
aTheoretical integration sites on human chromosomes were identified as
described in Materials and Methods. A more detailed summary of the integra-
tion sites is presented in Table S1 in the supplemental material. The results are
normalized in the right-hand column to integrations per 108bp to illustrate that
the distribution by chromosome did not show any significant preference. This
normalization would be incorrect by a factor of 2 for the sex chromosomes in
males (XY) and possibly females (XX) if the Barr body is refractory to integra-
tion. The lack of detection of Y-specific integrants may be related in part to the
fact that six of eight chimpanzees that were studied were females. In addition,
some of the integrants that could not be uniquely mapped may have integrated
bClone size ? 1,000 hepatocytes.
cOut of 10 clones that were greater than 1,000 hapatocytes in size, integration
sites in 6 could be assigned to specific chromosomes (see Table S1 in the
FIG. 6. Predicted clone size distributions resulting from random
death and regeneration of hepatocytes. (A to D) The numbers and
clone sizes of hepatocytes associated with different amounts of total
cumulative liver destruction were calculated using the computer pro-
gram Comp10 as described in Materials and Methods, assuming that
all hepatocytes had an equal probability of dying or dividing to main-
tain liver cell mass. In this simulation, the fates of all hepatocyte
lineages in a liver that initially contained 1 ? 106distinguishable and
distinct hepatocytes were followed during a period in which hepato-
cytes were destroyed and replaced by division of other hepatocytes at
a rate of 2.5% per simulation cycle. The fraction of initial hepatocyte
lineages (i.e., the complexity of the hepatocyte population) at the end
of the simulation is shown in parentheses. (D) Ninety-six out of 3,642
clones were ?1,000 hepatocytes in size; 25 out of 1,741 that were ?200
hepatocytes in size were also ?1,000 hepatocytes (25/1,741 ? 1.44%).
(E) Effect of liver turnover on the genetic complexity of the hepatocyte
population, assuming that, in the absence of turnover, all hepatocytes
were genetically distinguishable. In this simulation, the complexity was
the total number of genetically distinct hepatocytes divided by the total
number of hepatocytes. Complexity at time zero was assumed to be 1.
8402 MASON ET AL.J. VIROL.
distinct integrants, 146 could be mapped to a single human
chromosome. Most of the other integrations were associated
with host DNA that was repeated one or more times, so that a
correct integration site could not be assigned. Integrations
were found on all but the Y chromosome; failure to detect Y
integrants may reflect the limited number of integrations that
were mapped and the fact that only two of the eight HBV
carrier chimpanzees were male. Of the 146 that were mapped,
58 mapped to known or predicted genes, 4 to exons, and the
remainder to introns (see Table S1 in the supplemental mate-
rial). Integration in only 6 out of 10 of the clones greater than
1,000 hepatocytes in size could be mapped to a definite chro-
mosome, and of these, only 2 mapped to a known gene.
In brief, neither selective outgrowth of clones of ?1,000
hepatocytes due to integration of HBV DNA near (i.e., within
a few hundred thousand nucleotides of) a known oncogene nor
nonselective outgrowth due to random death and proliferation
appeared to explain the emergence of these large clones of
hepatocytes that takes place during chronic HBV infection of
Another possibility is that clones of hepatocytes emerge that
have lost the ability to express HBV and are therefore no
longer targets of the host immune response. For instance,
suppose that all hepatocytes had an equal probably of dividing
to maintain liver size but that hepatocytes that did not support
infection died at only half the rate (e.g., 0.05% per day) of
hepatocytes that did support infection (e.g., 0.1% per day). In
this situation, over a period of ?5 years the hepatocytes with a
survival advantage would expand 1,000-fold relative to those
without this advantage (34). Therefore, selective survival may
explain the emergence of hepatocyte clones. To see if this
possibility was at least feasible, we asked if there was a signif-
icant accumulation of uninfected hepatocytes (i.e., hepatocytes
in which virus was not detected by conventional histological
assays) during the course of chronic HBV infection in chim-
panzees, as is also seen in human infections. We assume that
most or all hepatocytes were infected following the initial ex-
posure to HBV.
Histological analyses of chimpanzee liver biopsy specimens.
Chronically HBV- and HCV-infected chimpanzees at the
Southwest Foundation for Biomedical Research have been
monitored for progression of liver disease for more than a
decade. In most instances, sections from formalin-fixed, paraf-
fin-embedded liver tissue at multiple time points were avail-
able for analyses. In general, HCV-infected chimpanzees
present with no fibrosis and limited histological activity (not
shown), while several of the HBV-infected chimpanzees had
an Ishak staging score of 2 (25) (Table 1), denoting fibrosis in
most portal areas (Fig. 7). A single HBV-infected chimpanzee
(4x0230) progressed to cirrhosis (Ishak score, 6) and HCC in
2008, both of which are rare in HBV-infected chimpanzees
(R. E. Lanford, unpublished data). Histological activity in
many instances included mild to moderate portal and lobular
hepatitis for HBV-infected livers (Table 1 and Fig. 7) and little
to no inflammatory changes in the acutely HCV-infected chim-
panzee (4x0313) (Table 1). To some degree, the differences in
staging of liver disease may have been due to the duration of
infection, since most of the HBV-infected chimpanzees had
been infected for greater than 25 years and only a few HCV-
infected chimpanzees were in their second decade of chronic
Immunostaining for HBcAg was performed on the formalin-
fixed tissues from multiple time points where available. Con-
trol tissues collected in a separate study of acute-resolving
HBV infection revealed intense cytoplasmic and nuclear stain-
ing in essentially 100% of the hepatocytes at 8 weeks postin-
fection (Fig. 8A), while the preinfection tissue from the same
animal was negative (not shown). HBcAg stains from the
chronically HBV-infected animals displayed several different
patterns. In general, the percentage of positive hepatocytes
was consistent with both the serum HBV DNA and HBsAg
levels and the liver HBV DNA levels (Table 1 and Fig. 3).
FIG. 7. Histological analysis of liver tissue from chimpanzees with chronic HBV infection. Liver tissue was analyzed for histological changes
using hematoxylin and eosin (A, B, and C) and Gordon and Sweets reticulin (D, E, and F) staining. Histological activity included chimpanzee
4x0327 with mild to moderate portal and lobular hepatitis (A) and mild fibrosis (Ishak score, 1) (D); chimpanzee 4x0230 with mild portal and
lobular hepatitis and large-cell dysplasia (B) and moderate fibrosis (Ishak score, 2) (E); and chimpanzee 4x0509 with moderate portal and lobular
hepatitis and large-cell dysplasia (C) and moderate fibrosis (Ishak score, 2) (F). Bars ? 200 ?M.
VOL. 83, 2009CLONAL EXPANSION OF HEPATOCYTES8403
Several of the animals, although positive for serum HBsAg and
HBV DNA at low levels, did not display significant staining
of hepatocytes for HBcAg: 4x0136 (Fig. 8B), 4x0222 (Fig.
8D), and 4x0506 (Fig. 8H). Both 4x0136 and 4x0506 had rare
HBcAg-positive hepatocytes, mostly staining in the nucleus,
and had areas with lymphocytic infiltrates.
Of particular interest, biopsy specimens from several ani-
mals exhibited distinct foci of HBcAg-negative hepatocytes
surrounded by hepatocytes that were uniformly HBcAg posi-
tive (Fig. 8C). The foci were particularly well defined in 4x0327
(Fig. 8E and F) in biopsy specimens taken at multiple times
during 2005, while biopsy specimens taken in 2006, the time of
FIG. 8. HBcAg immunostaining of tissue sections of liver biopsy specimens from chimpanzees chronically infected with HBV. Immunostaining to
detect HBcAg in sections of formalin-fixed liver was carried out as described in Materials and Methods. Except as noted, the biopsy specimen that was
stained was the same one collected in 2006 and used for invPCR. (A) Liver biopsy tissue collected from chimpanzee 4x0313 with acute HBV infection
at 8 weeks postinoculation (Lanford et al., unpublished). (B) Chimpanzee 4x0136 2006 biopsy specimen. (C) Chimpanzee 4x0139 2006 biopsy specimen.
The arrow indicates a distinct focus of HBcAg-negative hepatocytes surrounded by uniformly HBcAg-positive hepatocytes. (D) Chimpanzee 4x0222 2006
biopsy specimen. (E) Chimpanzee 4x0327 2005 biopsy specimen. (F) Another field from the chimpanzee 4x0327 2005 biopsy specimen. (G) Chimpanzee
4x0328 2006 biopsy specimen. (H) Chimpanzee 4x0506 2006 biopsy specimen. (I) Chimpanzee 4x0509 2006 biopsy specimen. The 2005 biopsy specimen
is shown for chimpanzee 4x0327 because the core staining and viremia were dramatically reduced in the animal by 2006.
8404 MASON ET AL.J. VIROL.
biopsy for viral-host junction analysis, were mostly negative for
HBcAg staining, consistent with the decrease in HBV DNA at
that time (not shown). A biopsy specimen taken from this
animal in 2004 had only two discernible HBcAg-negative he-
patocyte foci, while the biopsy specimens from 2005 had
HBcAg-negative hepatocyte foci in nearly every field. A second
animal that subsequently cleared HBV DNA during the study
period, 4x0509, also exhibited well-defined areas of negative
hepatocytes (Fig. 8I), but this biopsy specimen also contained
lymphocytic infiltrates and areas of lobular disarray. The ani-
mal rapidly cleared a high-level viremia the year following the
biopsy for this study (Fig. 3). Other animals, 4x0139 (Fig. 8C)
and 4x0328 (Fig. 8G), displayed intense nuclear and cytoplas-
mic HBcAg staining throughout most of the section, which
tended to correlate with a stable, high-level viremia. However,
even these animals exhibited areas that did not stain for
HBcAg (Fig. 8C), but they were not as well defined as those in
other animals. Thus, a correlation existed between a progres-
sive loss of HBV DNA in the serum during the study period
and the presence of HBcAg-negative foci in the liver, consis-
tent with the progressive emergence of mutated or epigeneti-
cally altered hepatocytes that were refractory to HBV infec-
Liver tissue from each chimpanzee was also analyzed using
in situ hybridization for the lobular distribution of hepatocytes
containing detectable levels of HBV DNA (Table 1 and Fig. 9).
Different patterns of HBV DNA were detected in individual
chimpanzees. In general, the percentage of hepatocytes con-
taining detectable levels of cytoplasmic HBV DNA was lower
than the percentage containing detectable levels of HBV core
antigen (compare chimpanzee 4x0328 in Fig. 8G and 9C).
However, like the distribution of HBcAg-positive hepatocytes,
the percentages of HBV DNA-positive hepatocytes varied
widely between animals and between individual hepatocytes
within each chimpanzee. For example, chimpanzee 4x0230 had
high levels of HBV DNA detected in 5 to 10% of hepatocytes,
while all other hepatocytes contained undetectable amounts of
HBV DNA; chimpanzees 4x0136, 4x0327, 4x0328, and 4x0506
had low or undetectable HBV DNA, while chimpanzee 4x0509
had HBV DNA in ?50% of hepatocytes.
In summary, as in HBV-infected humans, there is significant
accumulation of apparently uninfected virus-free hepatocytes
during chronic HBV infection in chimpanzees.
The invPCR assay detected proliferation of hepatocytes to
produce clones of hundreds to thousands of hepatocytes in the
livers of chronically HBV-infected chimpanzees. The results
were not dissimilar to our findings in chronically infected
woodchucks (33). This was surprising, in view of the fact that
the chimpanzees had been infected ?10 times as long as the
woodchucks at the time that liver biopsy specimens were ana-
lyzed. Why larger hepatocyte clones were not found in the
chimpanzees than in the woodchucks is unclear. A priori, it
might be concluded that this difference existed because liver
disease is more severe in the woodchuck. However, we are not
aware of any evidence that this is the case. The rapid occur-
rence of HCC in chronically infected woodchucks (29) might
be an indicator of a more active hepatitis, but it more likely
reflects the novel mechanism of HCC in the woodchuck, re-
sulting from enhancer insertion with activation of N-myc2 (13,
50) or, occasionally, C-myc (22, 23). Another possibility for the
similar clone sizes in the two species is that there are anatomic
constraints on clonal expansion, at least in the noncirrhotic
liver characteristic of infected woodchucks and chimpanzees.
In contrast, more extensive clonal expansion appears to occur
in cirrhotic nodules that arise in chronically infected humans
(1, 42, 61), with clone sizes reaching 105to 106hepatocytes or
While various explanations can be proposed, and may exist,
for clonal expansion of nontransformed hepatocytes, the actual
FIG. 9. Detection of HBV DNA in chimpanzee liver tissue by in situ hybridization. EAA-fixed liver tissue collected in 2006 was analyzed for
the presence of HBV DNA by in situ hybridization using a digoxigenin-labeled HBV DNA probe with counterstaining with hematoxylin. HBV
DNA was detected at variable levels in the cytoplasm of hepatocytes. The percentages of hepatocytes containing HBV DNA varied widely between
animals and between individual hepatocytes within the liver of each animal. (A) Chimpanzee 4x0136, ?0.02%. (B) Chimpanzee 4x0327, 0.009%.
(C) Chimpanzee 4x0328, ?0.02%. (D) Chimpanzee 4x0230, 5 to 10%. (E) Chimpanzee 4x0506, ?0.02%. (F) Chimpanzee 4x0509, ?50%. Bars ?
VOL. 83, 2009 CLONAL EXPANSION OF HEPATOCYTES8405
reasons remain uncertain. At present, we believe that an im-
mune evasion model plays a major role. Hepatocytes that did
not support viral infection would have a survival advantage
over hepatocytes that produce high titers of virus, as seen, for
example, at the peaks of transient infections, since they would
no longer be targeted by antiviral cytotoxic T lymphocytes
(CTLs). A survival advantage, even without a growth advan-
tage, would lead to clonal expansion of the affected hepato-
cytes (34). Considerable evidence exists that is consistent with
In humans (7, 17), woodchucks (33), and, as illustrated here,
chimpanzees (Fig. 8), a common feature of long-term infec-
tions is the emergence of large numbers of hepatocytes that no
longer support hepadnavirus replication and antigen expres-
sion. In the woodchuck, these are found both in FAH, which
are considered preneoplastic, and in foci of morphologically
normal hepatocytes (32, 41, 55, 56, 58). This also appears to be
the case for FAH in humans (16). In chimpanzees (Fig. 8), as
in humans (34, 51), it is also clear that chronic infections can be
associated with infection of only 10 to 50% or even fewer
hepatocytes, despite the fact that virus is still being produced
and, at least in naïve hosts, all hepatocytes would normally be
assumed to be susceptible to infection (e.g., as in Fig. 7A). Our
inference is that significant numbers of hepatocytes in long-
term carriers are no longer virus susceptible, even if a clear
focal organization is not evident.
However, there is an alternative or at least coexisting factor
that would cause clonal expansion of normal hepatocytes and
could contribute to the observation of clonal expansion via
invPCR. Random death and regeneration would lead to clonal
expansion (Fig. 6A to D) and genetic narrowing of the hepa-
tocyte population (Fig. 6E) as long as hepatocytes in the adult
liver are an essentially closed or at least partially closed pop-
ulation that is maintained mostly by self-renewal. This process
could fortuitously lead to the emergence of minor populations
of hepatocytes that did not support virus replication, irrespec-
tive of whether this allowed them to avoid antiviral CTLs. As
noted previously, the extent of clonal expansion of hepatocytes
in the infected woodchucks could not be explained entirely by
this model of random death and regeneration in the hepato-
cyte population (33). Whether this is also true in the HBV-
infected chimpanzees is less clear. A simple mathematical
analysis (Fig. 6) suggested that, as in the woodchucks, some-
thing other than random killing and division within the hepa-
tocyte population must be responsible for the clonal expansion
that we observed (Fig. 5). The data summarized in Table S1 in
the supplemental material do not support the notion that the
site of HBV DNA integration is a factor in clonal expansion.
The results shown in Fig. 7 to 9 are consistent with, but not
proof of, an immune evasion model. Thus, immune evasion
still seems a plausible option.
It is important, however, to keep in mind that there are no
data that conclusively distinguish between (i) the generation of
apparently virus-resistant hepatocytes and neoplastic progres-
sion via mutation and clonal expansion of mature hepatocytes
and (ii) the alternative possibility that FAHs, foci of virus-
negative hepatocytes, and/or HCCs all arise as a result of
blocked differentiation and transformation of hepatocyte pro-
genitor cells (43). The latter alternative rests, in part, upon the
concept that mature hepatocytes become senescent during
chronic HBV infection because of persistent CTL killing and
compensatory regeneration; ultimately, this is assumed to mit-
igate their ability to grow into tumors. For instance, at a rate of
turnover of 1% per day, hepatocytes equivalent to 73 livers
would have died over a 20-year period of chronic infection,
which amounts, in the absence of any contribution from hepa-
tocyte progenitor cells, to all surviving hepatocytes being, on
average, the result of 146 serial mitotic events. For compari-
son, assuming a 0.1% daily turnover in a normal liver, which is
likely an overestimate, hepatocytes equivalent to only 7.3 livers
would have died, with an average history among surviving
hepatocytes of 14.6 rounds of mitosis. Nonetheless, the validity
of the senescence argument is uncertain, in part because the
amount of turnover in the normal liver, which seems to occur
via self-renewal, is unknown and in part because it would be
expected that progenitor cells would proliferate and differen-
tiate to form mature hepatocytes as an ongoing process if older
hepatocyte lineages have a reduced capacity to replicate. Thus,
at least in theory, senescence should not occur as an observable
Thus, current data and concepts of liver maintenance seem
most consistent with the conclusion that clonal expansion of
normal-appearing hepatocytes, as well as of the altered hepa-
tocytes that form FAH, is the explanation for the hepatocyte
clones observed by invPCR. Likewise, immune evasion seems
a possible contributor in both cases. By inference, chronic
infection would lead indirectly to extensive evolution and re-
population of the liver with hepatocytes which, as a result of
mutation or epigenetic changes, are no longer able to support
The present study supports but does not prove this im-
mune evasion model. Clonal expansion of chimpanzee hepa-
tocytes could not be solely attributed either to random death
and regeneration or to integration of viral DNA at particu-
lar regions of host DNA. Identification of the morphological
correlates of these clones may provide a more definitive
explanation of their origin.
We are grateful to Jesse Summers (University of New Mexico), John
Taylor, and Christoph Seeger (FCCC) for helpful discussions during
the course of this work. We are also grateful for technical assistance
from the facilities for DNA sequencing and oligonucleotide synthesis
of the Fox Chase Cancer Center, to Kathleen Brasky at SNPRC for
veterinary support, and to Gene Hubbard and Edward Dick in the
pathology laboratory at SNPRC for preparation and evaluation of liver
W.S.M. was supported by grants from the National Institutes of
Health (5R01AI018641; CA06927) and by an appropriation from the
Commonwealth of Pennsylvania. A.R.J. was supported by the National
Health and Medical Research Council of Australia (Project Grant
453507). This investigation was conducted in part in facilities con-
structed with support from Research Facilities Improvement Program
grants (C06 RR016228 and C06 RR012087) from the NIH National
Center for Research Resources and using resources at the Southwest
National Primate Research Center (P51 RR13986). R.E.L., W.S.M.,
and A.R.J. were also supported in part by grant AI067455 awarded to
1. Aoki, N., and W. S. Robinson. 1989. State of hepatitis B viral genomes in
cirrhotic and hepatocellular carcinoma nodules. Mol. Biol. Med. 6:395–408.
2. Beames, B., and R. E. Lanford. 1993. Carboxy-terminal truncations of the
HBV core protein affect capsid formation and the apparent size of encap-
sidated HBV RNA. Virology 194:597–607.
8406 MASON ET AL.J. VIROL.
3. Blum, H. E., L. Stowring, A. Figus, C. K. Montgomery, A. T. Haase, and
G. N. Vyas. 1983. Detection of hepatitis B virus DNA in hepatocytes, bile
duct epithelium, and vascular elements by in situ hybridization. Proc. Natl.
Acad. Sci. USA 80:6685–6688.
4. Bruni, R., I. Conti, U. Villano, R. Giuseppetti, G. Palmieri, and M. Rapic-
etta. 2006. Lack of WHV integration nearby N-myc2 and in the downstream
b3n and win loci in a considerable fraction of liver tumors with activated
N-myc2 from naturally infected wild woodchucks. Virology 345:258–269.
5. Bruni, R., E. D’Ugo, R. Giuseppetti, C. Argentini, and M. Rapicetta. 1999.
Activation of the N-myc2 oncogene by woodchuck hepatitis virus integration
in the linked downstream b3n locus in woodchuck hepatocellular carcinoma.
6. Bruni, R., E. D’Ugo, U. Villano, G. Fourel, M. A. Buendia, and M. Rapicetta.
2004. The win locus involved in activation of the distal N-myc2 gene upon
WHV integration in woodchuck liver tumors harbors S/MAR elements.
7. Burrell, C. J., E. J. Gowans, R. Rowland, P. Hall, A. R. Jilbert, and B. P.
Marmion. 1984. Correlation between liver histology and markers of hepatitis
B virus replication in infected patients: a study by in situ hybridization.
8. Dejean, A., L. Bougueleret, K. H. Grzeschik, and P. Tiollais. 1986. Hepatitis
B virus DNA integration in a sequence homologous to v-erb-A and steroid
receptor genes in a hepatocellular carcinoma. Nature 322:70–72.
9. Dejean, A., and H. de The. 1990. Hepatitis B virus as an insertional mutagene
in a human hepatocellular carcinoma. Mol. Biol. Med. 7:213–222.
10. Delaney, W. E., and H. C. Isom. 1998. Hepatitis B virus replication in human
HepG2 cells mediated by hepatitis B virus recombinant baculovirus. Hepa-
11. Feitelson, M. A., and J. Lee. 2007. Hepatitis B virus integration, fragile sites,
and hepatocarcinogenesis. Cancer Lett. 252:157–170.
12. Flajolet, M., A. Gegonne, J. Ghysdael, P. Tiollais, M. A. Buendia, and G.
Fourel. 1997. Cellular and viral trans-acting factors modulate N-myc2 pro-
moter activity in woodchuck liver tumors. Oncogene 15:1103–1110.
13. Flajolet, M., P. Tiollais, M. A. Buendia, and G. Fourel. 1998. Woodchuck
hepatitis virus enhancer I and enhancer II are both involved in N-myc2
activation in woodchuck liver tumors. J. Virol. 72:6175–6180.
14. Fourel, G., J. Couturier, Y. Wei, F. Apiou, P. Tiollais, and M. A. Buendia.
1994. Evidence for long-range oncogene activation by hepadnavirus inser-
tion. EMBO J. 13:2526–2534.
15. Gong, S. S., A. D. Jensen, C. J. Chang, and C. E. Rogler. 1999. Double-
stranded linear duck hepatitis B virus (DHBV) stably integrates at a higher
frequency than wild-type DHBV in LMH chicken hepatoma cells. J. Virol.
16. Govindarajan, S., A. Conrad, B. Lim, B. Valinluck, A. M. Kim, and P.
Schmid. 1990. Study of preneoplastic changes in liver cells by immunohis-
tochemical and molecular hybridization techniques. Arch. Pathol. Lab. Med.
17. Gowans, E. J., C. J. Burrell, A. R. Jilbert, and B. P. Marmion. 1985. Cyto-
plasmic (but not nuclear) hepatitis B virus (HBV) core antigen reflects HBV
DNA synthesis at the level of the infected hepatocyte. Intervirology 24:220–
18. Hagen, T. M., S. Huang, J. Curnutte, P. Fowler, V. Martinez, C. M. Wehr,
B. N. Ames, and F. V. Chisari. 1994. Extensive oxidative DNA damage in
hepatocytes of transgenic mice with chronic active hepatitis destined to
develop hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 91:12808–
19. Hessein, M., G. el Saad, A. A. Mohamed, A. M. el Kamel, A. M. Abdel Hady,
M. Amina, and C. E. Rogler. 2005. Hit-and-run mechanism of HBV-medi-
ated progression to hepatocellular carcinoma. Tumori 91:241–247.
20. Hildt, E., and P. H. Hofschneider. 1998. The PreS2 activators of the hepatitis
B virus: activators of tumour promoter pathways. Recent Results Cancer
21. Hsia, C. C., R. P. Evarts, H. Nakatsukasa, E. R. Marsden, and S. S. Thor-
geirsson. 1992. Occurrence of oval-type cells in hepatitis B virus-associated
human hepatocarcinogenesis. Hepatology 16:1327–1333.
22. Hsu, T., T. Moroy, J. Etiemble, A. Louise, C. Trepo, P. Tiollais, and M.
Buendia. 1988. Activation of c-myc by woodchuck hepatitis virus insertion in
hepatocellular carcinoma. Cell 55:627–635.
23. Hsu, T. Y., G. Fourel, J. Etiemble, P. Tiollais, and M. A. Buendia. 1990.
Integration of hepatitis virus DNA near c-myc in woodchuck hepatocellular
carcinoma. Gastroenterol. Jpn. 2:43–48.
24. Hu, X., H. S. Margolis, R. H. Purcell, J. Ebert, and B. H. Robertson. 2000.
Identification of hepatitis B virus indigenous to chimpanzees. Proc. Natl.
Acad. Sci. USA 97:1661–1664.
25. Ishak, K., A. Baptista, L. Bianchi, F. Callea, J. De Groote, F. Gudat, H.
Denk, V. Desmet, G. Korb, R. N. MacSween, et al. 1995. Histological grading
and staging of chronic hepatitis. J. Hepatol. 22:696–699.
26. Jacob, J. R., A. Sterczer, I. A. Toshkov, A. E. Yeager, B. E. Korba, P. J. Cote,
M. A. Buendia, J. L. Gerin, and B. C. Tennant. 2004. Integration of wood-
chuck hepatitis and N-myc rearrangement determine size and histologic
grade of hepatic tumors. Hepatology 39:1008–1016.
27. Jilbert, A. R. 2000. In situ hybridization protocols for detection of viral DNA
using radioactive and nonradioactive DNA probes. Methods Mol. Biol. 123:
28. Jilbert, A. R., T.-T. Wu, J. M. England, P. D. L. M. Hall, N. Z. Carp, A. P.
O’Connell, and W. S. Mason. 1992. Rapid resolution of duck hepatitis B
virus infections occurs after massive hepatocellular involvement. J. Virol.
29. Korba, B. E., F. V. Wells, B. Baldwin, P. J. Cote, B. C. Tennant, H. Popper,
and J. L. Gerin. 1989. Hepatocellular carcinoma in woodchuck hepatitis
virus-infected woodchucks: presence of viral DNA in tumor tissue from
chronic carriers and animals serologically recovered from acute infections.
30. Lanford, R. E., D. Chavez, A. Barrera, and K. M. Brasky. 2003. An infectious
clone of woolly monkey hepatitis B virus. J. Virol. 77:7814–7819.
31. Lanford, R. E., and L. Notvall. 1990. Expression of hepatitis B virus core and
precore antigens in insect cells and characterization of a core-associated
kinase activity. Virology 176:222–233.
32. Li, Y., H. Hacker, A. Kopp-Schneider, U. Protzer, and P. Bannasch. 2002.
Woodchuck hepatitis virus replication and antigen expression gradually de-
crease in preneoplastic hepatocellular lineages. J. Hepatol. 37:478–485.
33. Mason, W. S., A. R. Jilbert, and J. Summers. 2005. Clonal expansion of
hepatocytes during chronic woodchuck hepatitis virus infection. Proc. Natl.
Acad. Sci. USA 102:1139–1144.
34. Mason, W. S., S. Litwin, and A. R. Jilbert. 2008. Immune selection during
chronic hepadnavirus infection. Hepatol. Int. 2:3–16.
35. Mason, W. S., C. Xu, H. C. Low, J. Saputelli, C. E. Aldrich, C. Scougall, A.
Grosse, R. Colonno, S. Litwin, and A. R. Jilbert. 2008. The amount of
hepatocyte turnover that occurred during resolution of transient hepadna-
virus infections was lower when virus replication was inhibited with ente-
cavir. J. Virol. 82:1778–1789.
36. Murakami, Y., K. Saigo, H. Takashima, M. Minami, T. Okanoue, C. Bre-
chot, and P. Paterlini-Brechot. 2005. Large scaled analysis of hepatitis B
virus (HBV) DNA integration in HBV related hepatocellular carcinomas.
37. Newsome, P. N., M. A. Hussain, and N. D. Theise. 2004. Hepatic oval cells:
helping redefine a paradigm in stem cell biology. Curr. Top. Dev. Biol.
38. Ochiai, T., Y. Urata, T. Yamano, H. Yamagishi, and T. Ashihara. 2000.
Clonal expansion in evolution of chronic hepatitis to hepatocellular carci-
noma as seen at an X-chromosome locus. Hepatology 31:615–621.
39. Paradis, V., D. Dargere, F. Bonvoust, L. Rubbia-Brandt, N. Ba, P. Bioulac-
Sage, and P. Bedossa. 2000. Clonal analysis of micronodules in virus C-
induced liver cirrhosis using laser capture microdissection (LCM) and
HUMARA assay. Lab. Investig. 80:1553–1559.
40. Piao, Z., Y. N. Park, H. Kim, and C. Park. 1997. Clonality of large regen-
erative nodules in liver cirrhosis. Liver 17:251–256.
41. Radaeva, S., Y. Li, H. J. Hacker, V. Burger, A. Kopp-Schneider, and P.
Bannasch. 2000. Hepadnaviral hepatocarcinogenesis: in situ visualization of
viral antigens, cytoplasmic compartmentation, enzymic patterns, and cellular
proliferation in preneoplastic hepatocellular lineages in woodchucks.
J. Hepatol. 33:580–600.
42. Robinson, W. S., L. Klote, and N. Aoki. 1990. Hepadnaviruses in cirrhotic
liver and hepatocellular carcinoma. J. Med. Virol. 31:18–32.
43. Roskams, T. 2006. Liver stem cells and their implications in hepatocellular
and cholangiocarcinoma. Oncogene 25:3818–3822.
44. Roskams, T. A., N. D. Theise, C. Balabaud, G. Bhagat, P. S. Bhathal, P.
Bioulac-Sage, E. M. Brunt, J. M. Crawford, H. A. Crosby, V. Desmet, M. J.
Finegold, S. A. Geller, A. S. Gouw, P. Hytiroglou, A. S. Knisely, M. Kojiro,
J. H. Lefkowitch, Y. Nakanuma, J. K. Olynyk, Y. N. Park, B. Portmann, R.
Saxena, P. J. Scheuer, A. J. Strain, S. N. Thung, I. R. Wanless, and A. B.
West. 2004. Nomenclature of the finer branches of the biliary tree: canals,
ductules, and ductular reactions in human livers. Hepatology 39:1739–1745.
45. Seeger, C., and W. S. Mason. 1999. Woodchuck and duck hepatitis B viruses,
p. 607–621. In R. Ahmed and I. Chen (ed.), Persistent viral infections. John
Wiley and Sons Ltd., Chichester, West Sussex, England.
46. Staprans, S., D. D. Loeb, and D. Ganem. 1991. Mutations affecting hepad-
navirus plus-strand DNA synthesis dissociate primer cleavage from translo-
cation and reveal the origin of linear viral DNA. J. Virol. 65:1255–1262.
47. Su, Q., and P. Bannasch. 2003. Relevance of hepatic preneoplasia for human
hepatocarcinogenesis. Toxicol. Pathol. 31:126–133.
48. Summers, J., A. R. Jilbert, W. Yang, C. E. Aldrich, J. Saputelli, S. Litwin, E.
Toll, and W. S. Mason. 2003. Hepatocyte turnover during resolution of a
transient hepadnaviral infection. Proc. Natl. Acad. Sci. USA 100:11652–
49. Thorgeirsson, S. S., and J. W. Grisham. 2003. Overview of recent experi-
mental studies on liver stem cells. Semin. Liver Dis. 23:303–312.
50. Ueda, K., Y. Wei, and D. Ganem. 1996. Activation of N-myc2 gene expression
by cis-acting elements of oncogenic hepadnaviral genomes: key role of en-
hancer II. Virology 217:413–417.
51. Volz, T., M. Lutgehetmann, P. Wachtler, A. Jacob, A. Quaas, J. M. Murray,
M. Dandri, and J. Petersen. 2007. Impaired intrahepatic hepatitis B virus
productivity contributes to low viremia in most HBeAg-negative patients.
VOL. 83, 2009 CLONAL EXPANSION OF HEPATOCYTES 8407
52. Wang, F., P. W. Anderson, N. Salem, Y. Kuang, B. C. Tennant, and Z. Lee. Download full-text
2007. Gene expression studies of hepatitis virus-induced woodchuck hepa-
tocellular carcinoma in correlation with human results. Int. J. Oncol. 30:33–44.
53. Wang, J., F. Zindy, X. Chenivesse, E. Lamas, B. Henglein, and C. Brechot.
1992. Modification of cyclin A expression by hepatitis B virus DNA integra-
tion in a hepatocellular carcinoma. Oncogene 7:1653–1656.
54. Wei, Y., G. Fourel, A. Ponzetto, M. Silvestro, P. Tiollais, and M. A. Buendia.
1992. Hepadnavirus integration: mechanisms of activation of the N-myc2
retrotransposon in woodchuck liver tumors. J. Virol. 66:5265–5276.
55. Xu, C., T. Yamamoto, T. Zhou, C. E. Aldrich, K. Frank, J. M. Cullen, A. R.
Jilbert, and W. S. Mason. 2007. The liver of woodchucks chronically infected
with the woodchuck hepatitis virus contains foci of virus core antigen-negative
hepatocytes with both altered and normal morphology. Virology 359:283–294.
56. Yang, D., E. Alt, and C. E. Rogler. 1993. Coordinate expression of N-myc 2
and insulin-like growth factor II in pre-cancerous altered hepatic foci in
woodchuck hepatitis virus carriers. Cancer Res. 53:2020–2027.
57. Yang, D., R. Faris, D. Hixson, S. Affigne, and C. E. Rogler. 1996. Insulin-like
growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver
epithelial cells. J. Virol. 70:6260–6268.
58. Yang, D. Y., and C. E. Rogler. 1991. Analysis of insulin-like growth factor II
(IGF-II) expression in neoplastic nodules and hepatocellular carcinomas of
woodchucks utilizing in situ hybridization and immunocytochemistry. Carci-
59. Yang, W., and J. Summers. 1999. Integration of hepadnavirus DNA in
infected liver: evidence for a linear precursor. J. Virol. 73:9710–9717.
60. Yasui, H., O. Hino, K. Ohtake, R. Machinami, and T. Kitagawa. 1992. Clonal
growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules.
Cancer Res. 52:6810–6814.
61. Yeh, S. H., P. J. Chen, W. Y. Shau, Y. W. Chen, P. H. Lee, J. T. Chen, and
D. S. Chen. 2001. Chromosomal allelic imbalance evolving from liver cirrho-
sis to hepatocellular carcinoma. Gastroenterology 121:699–709.
62. Zhou, H., L. E. Rogler, L. Teperman, G. Morgan, and C. E. Rogler. 2007.
Identification of hepatocytic and bile ductular cell lineages and candidate
stem cells in bipolar ductular reactions in cirrhotic human liver. Hepatology
8408 MASON ET AL.J. VIROL.