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Previous Article | Next Article 
Journal of Virology, April 2001, p. 3811-3818, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3811-3818.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Replication of Naturally Occurring Woodchuck
Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and
after Transmission to Naive Woodchucks
Mengji
Lu,1,*
Gero
Hilken,2
Dongliang
Yang,1
Thekla
Kemper,1 and
Michael
Roggendorf1
Institut für
Virologie1 and Zentrales
Tierlaboratorium,2 Universitätsklinikum
Essen, 45122 Essen, Germany
Received 14 September 2000/Accepted 25 January 2001
 |
ABSTRACT |
Woodchuck hepatitis virus (WHV) mutants with core internal
deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in
vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69
and WH70, were found to harbor WHV CID mutants. Consistent with
previous results, WHV CID mutants from both animals had deletions of
variable lengths (90 to 135 bp) within the middle of the WHV core gene.
In woodchuck WH69, WHV CID mutants represented a predominant fraction
of the viral population in sera, normal liver tissues, and to a lesser
extent, in liver tumor tissues. In primary hepatocytes of WH69, the
replication of wild-type WHV and CID mutants was maintained at least
for 7 days. Although WHV CID mutants were predominant in fractions of
cellular WHV replicative intermediates, mutant covalently closed
circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched
fractions. Analysis of cccDNA-enriched fractions from liver tissues of
other woodchucks confirmed that mutant cccDNA represents only a small
fraction of the total cccDNA pool. Four naive woodchucks were
inoculated with sera from woodchuck WH69 or WH70 containing WHV CID
mutants. All four woodchucks developed viremia after 3 to 4 weeks
postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg)
antibody, lymphoproliferative response to WHcAg, and anti-WHV surface
antigen. Only wild-type WHV, but no CID mutant, was found in sera from
these woodchucks. The WHV CID mutant was also not identified in liver
tissue from one woodchuck sacrificed in week 7 p.i. Three
remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants
in the inocula did not significantly change the course of acute
self-limiting WHV infection. Our results indicate that the replication
of WHV CID mutants might require some specific selective conditions.
Further investigations on WHV CID mutants will allow us to have more
insight into hepadnavirus replication.
| |
INTRODUCTION |
Mutations within the core gene of
hepatitis B virus (HBV) were often found in chronically HBV-infected
patients (3, 5, 8, 21; for a review, see reference
14). HBV core internal deletion (CID) is a common type of
mutation (1, 2, 12, 13, 17, 20, 26, 27, 28). In some
patients, HBV CID mutants emerged in association with severe liver
diseases. For example, HBV CID mutants occurred in renal allograft
recipients who suffered from endstage liver diseases (12, 13,
18). CID mutations lead to the expression of truncated, unstable
core proteins (24, 29). Thus, HBV CID mutants are
defective in replication and require trans-complementation
by wild-type virus (22, 29). This fact explains why HBV
CID mutants always co-occurred with wild-type virus in patients
examined so far. However, our knowledge about the replication of HBV
CID mutants in the host is limited. It is also not known whether the
presence of HBV CID mutants influences infection in naive hosts, since
HBcAg harbors major epitopes of host cellular immune responses within
the deleted region (7).
Woodchuck hepatitis virus (WHV), a virus genetically closely related to
HBV, causes acute and chronic infection in its natural host, the
woodchuck (Marmota monax) (9, 10, 11, 25). Particularly, chronically WHV-infected woodchucks develop
hepatocellular carcinoma at high frequency (10, 23).
Recently, WHV CID mutants were found in chronically WHV-infected
woodchucks (4). WHV CID mutants show similar
characteristics to HBV CID mutants: (i) they occur often in chronically
WHV-infected woodchucks but always coexist with wild-type WHV, and (ii)
CIDs are located in the middle of the WHV core gene and lead to
truncation of the core protein. Truncated WHV core proteins appear to
be unstable, as do truncated HBV core proteins (24, 29).
Our findings provide an opportunity to study CID mutants in the
woodchuck model system. In the present study, we screened chronically
WHV-infected woodchucks for WHV CID mutants. WHV CID mutants were found
in 2 of 14 woodchucks. We studied the replication of WHV CID mutants in
liver samples and primary hepatocyte cultures prepared from these
woodchucks. Further, we examined the infection of naive woodchucks with
virus stocks containing WHV CID mutants to clarify the possible
influence of CID mutants on the course of WHV infection and
WHV-specific immune responses.
| |
MATERIALS AND METHODS |
Woodchucks, sera, and liver samples of woodchucks.
Naive and
chronically WHV-infected woodchucks were purchased from North Eastern
Wildlife (Ithaca, N.Y.). All woodchucks were tested for WHV surface
antigen (WHsAg), anti-WHV core antigen (anti-WHcAg), and anti-WHsAg to
determine their status. WHV DNA was detected in serum samples of these
woodchucks by spot blot hybridization with a full-length WHV genome
probe. Liver and hepatocellular carcinoma (HCC) samples from woodchucks
were taken after euthanasia, immediately frozen in liquid nitrogen, and
stored at 
80°C.
Isolation of DNA from woodchuck sera and liver samples.
Sera
(100 µl) were subjected to digestion with proteinase K (0.15 M NaCl,
10 mM Na-EDTA, 1% sodium dodecyl sulfate [SDS], 50 mM Tris-HCl, 20 mg of proteinase K per ml, pH 8.2) and phenol-chloroform extraction.
WHV DNA was precipitated with ethanol by standard procedures.
Total DNA from liver samples of chronically WHV-infected woodchucks was
extracted with the QIAamp Tissue Kit (Qiagen, Hilden, Germany)
according to the manufacturer's instructions. Briefly, about 25 mg of
frozen liver samples was ground to powder in liquid nitrogen by using a
mortar and pestle, lysed in 180 µl of lysis buffer, and digested with
proteinase K. Samples were then mixed with 210 µl of ethanol and
applied to a QIAamp spin column. DNA was bound to the column, washed
twice, and eluted by buffers supplied with the kit.
To extract WHV covalently closed circular DNA (cccDNA), 100 mg of
frozen liver samples was ground to powder in liquid nitrogen by using a
mortar and pestle and was homogenized with 1 ml of ice-cold Tris-EDTA
buffer, pH 8.0. After adding 1 ml of 4% SDS, the mixture was vortexed
vigorously to shear cellular DNA. Cellular DNA, proteins, and viral
protein-bound DNA were precipitated by adding 0.5 ml of KCl, 2.5 M, to
the mixture. The precipitates were sedimented by centrifugation in a
Sorvall RT 6000B at 3,000 rpm at 4°C for 10 min. Supernatants were
collected and extracted with phenol. Nucleic acids were recovered by
ethanol precipitation.
Encapsidated WHV DNA was extracted as follows. Frozen liver samples
(100 mg) were ground to powder in liquid nitrogen by using a mortar and
pestle and were homogenized with 1 ml of ice-cold Tris-EDTA buffer, pH
8.0. Homogenate was mixed with 50 µl of 10% NP-40 and incubated on
ice for 5 to 30 min. Nuclei and cell debris were removed by
centrifugation. Supernatants, after adding magnesium acetate (final
concentration, 6 mM) and DNase I (50 µg/ml), were incubated at 37°C
for 30 min. Then additional reagents were added to adjust to 10 mM
EDTA, 10% SDS, 0.1 M NaCl, and 0.5 mg of pronase per ml. The mixtures
were incubated at 37°C for 60 min and extracted with phenol. Nucleic
acids were recovered by ethanol precipitation.
Amplification of WHV core gene and pre-S1/pre-S2 region by PCR,
cloning, and sequence analysis of PCR products.
The WHV core gene
(nucleotides [nt] 2021 to 2587) and pre-S1/pre-S2 region (nt 2992 to
338) were analyzed by PCR as described previously (4). The
primers were designed according to the WHV genome sequence published by
Galibert et al. (9): wc1, 5' TGG GGC CAT GGA CAT AGA
TCC TTA 3' (nt 2015 to 2038), and wc2, 5' CAT TGA ATT CAG
CAG TTG GCA GAT GG 3' (nt 2570 to 2597), for the WHV core gene;
ps1, 5' CAG CTA GTG CAA CAT AAT CC 3' (nt 2976 to 2995), and
ps2, 5' CCT GTA ATC CTG CGA GGA GT 3' (nt 338 to 319), for
the WHV pre-S region. PCR products were visualized on ethidium
bromide-stained agarose gels. Each sample that yielded more than one
band was further examined. DNA fragments of interest were purified from
the gel with a QIAquick gel extraction kit (Qiagen). These fragments
were subjected to cloning into pCRII-Vector (Invitrogen, Leek, The
Netherlands). Sequencing of plasmids was performed by a commercial
service (MWG Biotech, Munich, Germany).
Nested PCR was used for the amplification of serially diluted DNA
preparations extracted from serum samples of woodchuck WH69. The first
PCR was run with WhpreC, 5' TAA ATG CAT GCG ACT TCT GTA ACC A 3'
(nt 1907 to 1931), and wc3, 5' TTA TGT ACC CAT TGA AGA TCA
GCA G 3' (nt 2605 to 2581). The second PCR was performed with wc1
and wc2. Both PCRs were run over 30 cycles of 1 min at 94°C, 1 min at
50°C, and 2 min at 72°C.
Liver perfusion on woodchucks and primary woodchuck hepatocyte
cultures.
Liver perfusion was carried out according to the
following protocol. Anesthetized woodchucks received an intravenous
injection of 2 ml of heparin (104 U/ml). After opening the
peritoneum, 400 ml of calcium-free preperfusion solution (Spinner
minimal essential medium supplemented with 2 mM glutamine, 0.05%
glucose, 20 mM HEPES [pH 7.4], insulin, 5 mM sodium pyruvate, and 50 IU of penicillin-streptomycin per ml) were pumped into the liver
through a portal vein. Then 400 ml of collagenase medium (Williams
medium supplemented with 0.4 mg of collagenase per ml, 3 mM
CaCl2, 2 mM glutamine, 0.05% glucose, 20 mM HEPES [pH
7.4], 12 IU of insulin per ml, 5 mM sodium pyruvate, and 50 IU of
penicillin-streptomycin per ml) was pumped through a portal vein with a
flow rate of 20 ml/min. Liver tissues were dissected from the abdominal
cavity. Hepatocytes were separated from liver tissue with forceps and a
scalpel and were stirred in 100 ml of collagenase medium for an
additional 30 min at 37°C, 5% CO2. Cell suspensions were
filtered through gauze to remove tissue fragments and passed through a
70-µm filter. Hepatocytes were separated from other cells by repeated
centrifugation at 50 × g.
Primary woodchuck hepatocytes were seeded in 60-mm plates at a density
of 106 per well. Plates were coated with collagen type 1 before use. Hepatocytes were maintained for 7 days in Williams medium
supplemented with 2 mM glutamine, 0.05% glucose, 20 mM HEPES (pH 7.4),
hydrocortisone, 12.5 µg of inosine per ml, 12 IU of insulin per ml, 5 mM sodium pyruvate, 50 IU of penicillin-streptomycin per ml, and 1%
dimethyl sulfoxide. Medium was changed at days 1, 3, and 5.
Analysis of WHV replication intermediates in woodchuck primary
hepatocytes.
WHV replication intermediates, encapsidated WHV DNA,
and cccDNA were extracted as described in the previous section.
Extracted DNA was subjected to Southern blot hybridization or PCR.
Immunohistochemical staining of HCC tissue sections with
anti-WHcAg antibody.
Excised liver tissues were immediately fixed
in formalin (4% formaldehyde in phosphate-buffered saline, pH 7.4) for
24 h and then embedded in paraffin. Sections of paraffin-embedded
tissue were prepared. Polyclonal rabbit antibodies raised to WHcAg were used to detect WHcAg expression in liver tissue. Liver sections were
incubated with diluted antibodies (1:100) and stained with DAKO
EnVision+ System (DAKO Corporation, Carpinteria, Calif.) according to
the manufacturer's instructions. For a control, a parallel section of
liver was treated in the same manner, except WHcAg-specific antibodies
were replaced by normal rabbit serum.
| |
RESULTS |
WHV CID mutants occurred in chronically WHV-infected
woodchucks.
According to a previous study, WHV CID occurs in
chronically WHV-infected woodchucks at a frequency of about 10%
(4). For further studies on the replication of CID
mutants, we screened 14 wild-caught woodchucks with chronic WHV
infection to identify individuals carrying WHV CID mutants. WHV DNA was
extracted from serum samples from these woodchucks and subjected to PCR
for amplification of the WHV core gene (nt 2021 to 2587). WHV CID
mutants were found in two woodchucks, WH69 and WH70 (Fig.
1A). The ratios of WHV wild type to CID
mutants were about 1:10 in WH69 and 2:1 in WH70. WHV CID persisted in
the following 6 months until the sacrifice of WH69 and WH70. The WHV
titers in WH69 and WH70 were at about 108 to
109 genome equivalents/ml, as estimated by spot blot
hybridization, indicating that the replicative activity of WHV CID
mutants in vivo was comparable with that of the WHV wild type. PCR
amplification of the WHV pre-S1/pre-S2 region (nt 2992 to 338) was
performed with samples from the same 14 woodchucks. No deletion mutant
of WHV pre-S was found in these woodchucks (data not shown).
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FIG. 1.
Identification of WHV CID mutant genomes in serum
samples of chronically WHV-infected woodchucks WH69 and WH70. (A) PCR
with DNA-extracted serum samples from woodchucks WH69 and WH70. WH8 was
a virus stock containing only wild-type WHV. PCR fragments
corresponding to the wild-type WHV core gene and deletion mutants are
marked wild type and CID, respectively. (B) PCR amplification of WHV
wild type and CID mutants in defined ratios of 1:10, 1:1, and 10:1.
Templates of WHV wild type and CID mutants were mixed in defined ratios
and adjusted to different copy numbers.
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To exclude the possibility that PCR led to a selective amplification of
wild-type or mutant sequences, cloned fragments containing the
wild-type WHV core gene sequence and CID mutations were mixed in
different ratios (1:10, 1:1, and 10:1). PCRs were performed with
initial template numbers ranging from 5 × 107 to
5 × 103 copies per reaction mixture. In all cases,
the wild-type-to-CID ratios in final PCR products remained
approximately the same as those in the initial reaction mixtures (Fig.
1B). PCR using linearized plasmids as templets gave the same results
(not shown). Therefore, our PCR protocol is appropriate to
quantitatively analyze the presence of WHV CID mutants.
WHV population in sera from woodchucks WH69 and WH70.
The
presence of WHV CID mutants in WH69 and WH70 provided an opportunity to
study the replication and infection of such mutants in woodchucks.
Prior to further experiments, the CID mutants were characterized in
detail. PCR products of the WHV core gene from WH69 were cloned into
pCR2.1 vector, and 10 randomly chosen clones were subjected to further
sequence analysis. Only one of these clones from WH69 contained the
wild-type sequence of the WHV core gene (Fig.
2A). Eight other clones contained a WHV
CID mutation of 135 bp at nt 2264 to 2398. Another WHV CID mutation of
132 bp at nt 2269 to 2401 was identified in the remainder. Both WHV CID
mutations have been found in other woodchucks in a previous study
(4). To conform the results of cloning, we analyzed the ratio of WHV wild type to CID mutants by PCR with diluted DNA preparations from serum samples of WH69. Nested PCR was necessary for
the amplification of very small numbers of WHV genomes down to a single
copy in diluted samples. Totals of 15 and 7 of 20 individual reactions
with 1:3 × 106- and 1:107-diluted
samples, respectively, were positive (Fig. 2B). At the dilution of
1:3 × 106, 1 wild type, 12 CID mutants, and 2 mixtures of wild type and CID mutant were detected. Seven CID mutants
were identified in nested PCR with 107-diluted samples.
These results indicated that the ratio of WHV wild type to CID mutants
ranged between 1:7 and 1:8, comparable with results gained by other
approaches.
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FIG. 2.
Heterogeneity of WHV CID mutants in woodchuck WH69. (A)
Analysis of the WHV population in WH69 by the cloning of the PCR
product of the WHV core region. The numbering of the nucleotide
positions is according to Galibert et al. (9). The
positions of CIDs are indicated by broken lines and the nucleotide
positions. The numbers on the broken lines indicate the respective
length of deletions. Nr, number. (B) Analysis of the WHV population in
WH69 by nested PCR with diluted preparations of extracted serum DNA.
Samples at dilutions of 1:3 × 106 and
1:107 were subjected to nested PCR. The numbers of WHV wild
type (wt), CID mutants, and mixtures of both types (wt+CID) are
indicated. Diluted samples resulting in negative PCR are indicated as
blank.
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The majority of cloned PCR products derived from WH70 had the wild-type
WHV core sequence. A CID mutation of 90 bp between nt 2268 and 2357 was
found. These results were concordant with our previous finding that
genetically different CID mutants coexist together with wild-type WHV
in chronically WHV-infected woodchucks.
WHV CID mutants in normal and HCC tissues.
To study the
replication of WHV CID mutants in liver tissues, woodchuck W69 was
sacrificed. An HCC was found at the left lobus of the liver. In a
previous work, WHV replication was found to occur in HCC tissues from
chronically WHV-infected woodchucks. It is of interest whether WHV wild
type and CID mutants may unevenly distribute in normal and HCC tissues.
Thus, biopsies from normal liver tissue and HCC tissue were taken to
examine the WHV replication and the presence of WHV CID mutants. WHV
replication intermediates were detected in total DNA fractions from HCC
and liver tissues by Southern blot hybridization (Fig.
3A). An immunohistochemical staining of
the WHcAg showed that WHcAg was expressed in cancerous tissues (Fig.
3B). These results indicated that WHV replication also occurred within
HCC tissues. The presence of WHV CID mutants in normal and HCC tissues
was examined by WHV core-specific PCR. The ratio of WHV wild type to
CID mutants in normal tissues was comparable with that in serum samples
(Fig. 3C). In contrast, WHV wild type in HCC tissues appeared to be the
major part of viral populations. Thus, WHC CID mutants replicated
preferentially in normal tissues of WH69 and to a lesser extent in HCC.
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FIG. 3.
(A) Detection of WHV replication intermediates in normal
woodchuck liver tissues (lane 1) and woodchuck HCC tissues from WH69
(lanes 2 through 4). (B) The immunohistological staining of HCC section
with anti-WHcAg antibody. Magnification, ×1,000. (C) WHV core-specific
PCR with DNA extracted from normal liver tissues (lane 1) and three
different parts of HCC tissues from WH69 (lanes 2 through 4).
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HBV CID mutants were reported to occur in patients with HCC (16,
29). To examine the occurrence of WHV CID mutants in HCC
tissues, total DNA was extracted from HCC tissues from seven additional
woodchucks and was subjected to Southern blot analysis and PCR.
Different amounts of WHV replication intermediates were detected in all
HCC tissues by Southern blotting (data not shown). WHV core-specific
PCR with the same samples revealed that WHV CID mutants were present in
five HCC samples (Fig. 4). Three
woodchucks, DW499, DW15-8, and DW3, were found to harbor WHV CID
mutants in sera and normal liver samples (4). The ratio of
WHV CID mutants to wild type was comparable in HCC tissues and normal
liver tissues (Fig. 4). No WHV CID mutant was identified in HCC tissues
from two other chronically WHV-infected woodchucks. Thus, WHV CID
mutants appeared to occur frequently in woodchucks with HCC.
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FIG. 4.
Detection of WHV CID mutants in HCC tissues from seven
chronically WHV-infected woodchucks. WHV core-specific PCR with DNA
extracted from HCC tissues from seven woodchucks is shown.
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Replication of WHV wild type and CID mutants in primary
hepatocytes.
So far, CID mutants were studied in biopsies or in
transfected cell lines. It is not possible yet to study the replication of CID mutants dynamically. We took advantage of studying replication of WHV CID mutants in primary hepatocytes. Primary hepatocytes were
prepared from perfused liver of WH69 and cultured for 7 days. Total WHV
replicative intermediates, encapsidated WHV DNA, and WHV cccDNA were
extracted from primary hepatocytes at days 1, 3, 5, and 7. WHV
replication in primary hepatocytes was examined by detection of WHV DNA
in extracted fractions by Southern blot hybridization with a
WHV-specific probe.
WHV replicative intermediates in primary hepatocytes changed only
slightly during the period of culturing (Fig.
5). The ratio between WHV wild-type and
CID mutant genomes was assessed by WHV core-specific PCR using total
DNA fractions, encapsidated WHV DNA, and WHV cccDNA-enriched fractions.
WHV CID mutants remained continuously predominant in total DNA
fractions and in fractions of encapsidated WHV DNA. However, the
wild-type WHV genome represented the predominant part in
cccDNA-enriched fractions. In addition, the ratio of WHV CID mutants to
wild type in cccDNA-enriched fractions decreased gradually during the
culturing (Fig. 5C). Thus, WHV CID mutants did not accumulate at the
level of cccDNA but as encapsidated WHV DNA and WHV genomes in
circulating viral particles.
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FIG. 5.
Detection of WHV CID mutants in a fraction of WHV
replication intermediates and cccDNA in woodchuck primary hepatocytes.
(A) WHV replication intermediates detected by Southern blot
hybridization. WHV core-specific PCRs were performed with total DNA
(B), with encapsidated WHV DNA (C), and with cccDNA-enriched
fractions (D) extracted from woodchuck primary hepatocytes.
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To examine whether cccDNAs of WHV CID mutants accumulated in liver
tissues of other chronically WHV-infected woodchucks, total DNA and WHV
cccDNA-enriched fractions from liver tissues of two woodchucks, DW3 and
DW15-8, were extracted and subjected to core-specific PCR. These
woodchucks were found to carry WHV CID mutants as predominant species
in serum in a previous study (4), with the same ratios of
wild type to CID mutants in total DNA fractions from liver by
core-specific PCRs (Fig. 6). CID mutants
were present as small fractions in cccDNA-enriched fractions from WH3
and WH15-8.
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FIG. 6.
Detection of WHV CID mutants in total DNA and
cccDNA-enriched fractions from normal liver tissues from woodchucks DW3
and DW15-8. WHV replication intermediates (A) and cccDNAs (B) were
detected by Southern blot hybridization. (C) The presence of WHV CID
mutants in the extracted DNA fractions was analyzed by WHV
core-specific PCR.
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Experimental infection of naive woodchucks with WHV stocks from
WH69 and WH70.
CID mutations lead to the loss of important
immunological determinants on WHcAg, e.g., a T-cell epitope which is
recognized by a large number of outbred woodchucks (19).
In addition, WHV CID mutants show a higher replication activity than
does the wild-type virus. Thus, the presence of WHV CID mutants in
inocula may have an influence on the anti-WHcAg antibody response,
WHcAg-specific lymphoproliferative responses, and virus clearance. To
clarify this possibility, stocks from WH60 and WH70 were used for
inoculation of naive woodchucks. Two naive woodchucks, WH10491 and
WH10840, were inoculated by intravenous injection of 100 µl of serum
of woodchuck WH69, corresponding to about 107 to
108 WHV genome equivalents. Both woodchucks developed acute
self-limiting WHV infection (Fig. 7). In
week 4 postinfection (p.i.), anti-WHcAg antibody was detectable in
WH10491 (Fig. 7A). Anti-WHsAg antibody appeared in week 6 p.i.,
which indicated a self-limiting course of WHV infection. WHV DNA was
detectable in PCR in week 3 p.i. and persisted to week 6 p.i.
Despite the predominance of WHV CID mutants over wild type in the
inoculum derived from WH69, WHV CID mutants were not observed in
WH10491. WH10491 was sacrificed in week 6, and liver tissues were
examined for the presence of WHV CID mutants. Total DNA was extracted
from liver tissues from different parts of the liver and was subjected
to WHV core gene-specific PCR (Fig. 7B). Only WHV wild type was found
in consistence with PCR results gained with serum samples. Similar
results were obtained from the infection of WH10840 (Fig. 7C). After
carryover anti-WHc antibodies decreased in WH10840, the anti-WHcAg
antibody titer raised from week 5 p.i. WHV DNA was detectable only
in weeks 3 and 4 p.i., with the appearance of anti-WHsAg at week
5 p.i. The proliferative response to WHcAg was assessed in both
woodchucks during the course of infection by in vitro stimulation of
woodchuck peripheral blood mononuclear cells (PBMCs) with recombinant
WHcAg (rWHcAg) (Fig. 7). PBMC from WH10491 and WH10840 showed
proliferative response to rWHcAg at weeks 3 and 4 p.i., as in
woodchucks infected by wild-type virus alone (19).
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FIG. 7.
Infection of naive woodchucks WH10491 (A) and WH10840
(C) with sera from WH69. OD 490, optical density at 490 nm. The course
of WHV infection was monitored by determining anti-WHcAg (anti-WHc
titer) and anti-WHsAg (anti-WHs OD 490) antibody levels in woodchuck
sera. For the detection of WHV CID, WHV core-specific PCR was performed
with DNA extracted from woodchuck sera. The specific
lymphoproliferative response of PBMCs to rWHcAg was indicated by a plus
if the stimulation index was higher than 3 (19). n.d., not
determined. (B) WHV core-specific PCR with DNA extracted from WHV
inoculum (WH69) and from liver tissues from woodchuck WH10491 is
shown.
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Two woodchucks, WH10838 and WH10839, were inoculated with 100 µl of
serum from woodchuck WH70. Similar to WH10491 and WH10840, both
woodchucks developed acute self-limiting infection. WHV appeared at
very low titers at week 3 p.i. and was cleared in week 4 p.i. (data not shown). No deletion mutants in these woodchucks were found by
PCR with WHV DNA extracted from sera.
| |
DISCUSSION |
WHV CID mutants appeared frequently in chronically WHV-infected
woodchucks. In this study, we confirmed results of a previous retrospective study on WHV CID mutants (4). WHV CID
mutations from WH69 and WH70 had different sizes and were located in
the center of the core gene, consistent with previous results. It is
not understood yet how heterogeneous WHV CID mutants emerge and coexist
in naturally WHV-infected woodchucks. It is likely that different WHV
CID mutants accumulate and replicate at low levels in chronically
infected animals. Under some as-yet-unknown conditions, WHV CID mutants
replicated rapidly and became predominant in a WHV population.
HBV CID mutants occur in chronically infected patients. HCC was found
in some patients harboring HBV CID mutants (16, 29). WHV
CID mutants appeared to occur often in woodchucks with HCC. However,
the association of the appearance of WHV CID mutants and HCC was not
close, as a number of woodchucks with WHV deletion mutants had no HCC.
Our results also clearly showed that WHV CID mutants replicated in
normal liver tissues and in primary woodchuck hepatocytes. In the liver
tumor of WH69, the replication of WHV CID mutants was rather limited.
Our results revealed some interesting features of the replication of
WHV CID mutants. Though WHV CID mutants represented significant or even
predominant parts of serum viral populations in chronically WHV-infected woodchucks, just a small fraction of cccDNAs, if any,
harbored CID mutations. These results might be explained by the
instability or slower formation of mutant cccDNAs. The portion of
cccDNAs of WHV CID mutants decreased rapidly during the
culturing of woodchuck primary hepatocytes. Interestingly, HBV CID
mutants in patients disappeared quickly with treatments with
lamivudine, while HBV wild type persisted (18). The
vulnerability of HBV CID mutants to lamivudine might result from their
unstable cccDNA pools. By reduction of HBV replication activity, CID
mutants would not be able to maintain sufficient amounts of cccDNAs to persist.
It is still not clear how WHV CID mutants produced large amounts of
virions containing mutant genomes with such a small fraction of
cccDNAs. We demonstrated previously that CID mutations might lead to an
increased expression of WHV polymerase due to the deletion of some of
the 11 AUGs in the 5'-untranslated region preceding the WHV polymerase
start codon (4). An enhanced expression of WHV polymerase
may facilitate the packaging of mutant WHV pregenomic RNAs.
Alternatively, CID mutations resulted in a disregulation of
transcriptional control that led to an increased production of
pregenomic RNAs. Our preliminary data indicate that a large amount of
mRNA with CID mutations accumulated in liver tissues from WH69.
However, it could not be simply explained by an enhanced transcription
of mutant mRNAs. The subsequent steps, like the interaction with
polymerase and the packaging into virions, may greatly influence the
fate of pregenomic mRNAs. Further investigations should clarify whether
such mechanisms are responsible for the emergence of WHV CID mutants.
Yuan et al. (28, 29) found that HBV CID mutants have
properties of defective interfering particles. HBV CID mutants were
enriched if they were cotransfected with HBV wild-type DNA into a human
hepatoma cell line. Consistent with these results, Gunther et al.
demonstrated that HBV CID mutants show an enhanced replication
(15).
WHV CID mutants did not have an apparent influence on acute WHV
infections in naive woodchucks. It seems that WHV CID mutants did even
not have a chance to propagate in naive woodchucks after experimental
infection. WHV CID mutants need the trans-complementation of
wild-type viruses for their replication. Since only about
107 WHV genome equivalents were used for our infection
experiments, a simultaneous infection of single cells by WHV wild type
and mutants is supposed to be a very unlikely event. Thus, WHV CID mutants would disappear in the early phase of an acute infection due to
the lack of trans-complementation. The replication of CID mutants might be maintained in naive woodchucks if a sufficient part of
hepatocytes are coinfected with wild-type and mutant viruses by a
high-titer inoculum. An additional reason would be the lack of
appropriate selective conditions in naive woodchucks which favor the
replication of WHV CID mutants. Such selective conditions for CID
mutants are not defined so far. Our experiments demonstrated that naive
woodchucks developed immune responses to WHV proteins after infection
with WHV stocks containing WHV CID mutants and efficiently cleared WHV.
HBV CID mutants have consistently not been detected in acutely infected
patients so far or been found in an association with serious
consequences in acute infections. The role of HBV CID mutants for
pathogenesis in chronic HBV infection remains to be investigated.
| |
ACKNOWLEDGMENTS |
We are grateful to Hans Will for helpful discussions and critical
reading of the manuscript. We thank Ulrick Protzer and Ulla Schultz for
technical advice.
This work was supported by grants of German Bundesministerium für
Bildung und Forschung to M.R. and M.L. (BMBF, 01 KI 9862).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Virologie, Universitätsklinikum Essen, Hufelandstrasse
55, 45122 Essen, Germany. Phone: 49 201 7233530. Fax: 49 201 7235929. E-mail: mengji.lu{at}uni-essen.de.
| |
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Journal of Virology, April 2001, p. 3811-3818, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3811-3818.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
