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Journal of Clinical Microbiology, July 1998, p. 2084-2086, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Low-Level Viremia and Intracellular Expression of
Hepatitis B Surface Antigen (HBsAg) in HBsAg Carriers with
Concurrent Hepatitis C Virus Infection
Chia-Ming
Chu,*
Chau-Ting
Yeh, and
Yun-Fan
Liaw
Liver Research Unit, Chang Gung Memorial
Hospital and Medical College, Taipei, Taiwan
Received 14 January 1998/Returned for modification 3 March
1998/Accepted 30 March 1998
 |
ABSTRACT |
Assays of hepatitis B virus (HBV) replication and antigen
expression in HBV surface antigen (HBsAg) carriers with concurrent hepatitis C or D virus (HCV or HDV) infection revealed that HCV and HDV
can suppress HBV replication but that HCV also substantially suppresses
HBV surface protein expression. HBsAg carriers with concurrent HCV
infection thus have low-level viremia and intracellular HBsAg.
| |
TEXT |
In chronic hepatitis B virus (HBV)
infection, levels of HBV surface antigen (HBsAg) in serum and in the
liver bear an inverse relation to each other. During the highly
replicative phase, there are high serum titers of HBsAg but low tissue
levels of HBsAg. During the low-replicative phase, titers of HBsAg in
serum decrease and this decrease is accompanied by increased levels of
HBsAg in tissue (1, 2, 9, 13). Notably, patients with
fibrosing cholestatic hepatitis have high-level viremia, high titers of HBsAg in serum, and high-level intracellular HBsAg (10).
Immunosuppressive therapy probably plays an important role in the
evolution of fibrosing cholestatic hepatitis (11, 18).
Recently, we have noted that some HBsAg carriers had low-level viremia,
low titers of HBsAg in serum, and low-level intracellular HBsAg, and
many of them were found to have concurrent hepatitis C virus (HCV)
infection (6a). These preliminary observations prompted us
to study the effect of concurrent HCV infection on HBV
replication and antigen expression in HBsAg carriers.
Patients.
Between 1977 and 1993, 992 consecutive patients
with chronic hepatitis B were studied at our unit. Of these, 11%
were positive for the antibody to hepatitis D virus (HDV; anti-HDV) and
8% were positive for antibodies to HCV (anti-HCV)
(20). Three groups of patients, matched for age and sex, who
had available serum specimens drawn on the day of liver biopsy were
randomly selected for study. In one group, patients had concurrent HBV
and HCV infections (n = 29). All patients had been
HBsAg and anti-HCV positive for at least 12 months. All had HCV RNA
detectable by PCR. Genotyping of HCV revealed 1b in 19, 2a in 6, 2b in
3, and 1b plus 2a in 1. The duration and relative timing of the two
viral exposures were unknown. Seven patients had a history of blood
transfusion 3 to 23 years ago. In the second group, patients had
concurrent HBV and HDV infections (n = 35). All
patients had been HBsAg and anti-HDV positive for at least 12 months.
All had HDV antigen in liver tissue detectable by direct
immunofluorescence (3). In the third group, patients had
chronic HBV infection alone (n = 42). All patients were
anti-HCV and anti-HDV negative and had no detectable HCV RNA in serum
or HDV antigen in liver tissue. All patients denied any history of
homosexual behavior or intravenous drug abuse. None had ever received
antiviral or immunosuppressive therapy. Their clinical and laboratory
data are summarized in Table 1.
Laboratory methods.
HBsAg, HBV e antigen (HBeAg), anti-HBe,
and anti-HDV were assayed with radioimmunoassay kits (Abbott
Laboratories). Anti-HCV was assayed by second-generation enzyme
immunoassay (HCV-EIA; Upstate Biotechnology Inc.). HBV DNA
was assayed by spot hybridization with 32P-labeled
cloned HBV DNA. The detection sensitivity was 0.5 pg/50 µl. Levels of
HBV DNA in serum were semiquantitatively scored on a
1+-to-4+ scale corresponding to 
500, 501 to
1,000, 1,001 to 2,000, and >2,000 pg/ml, respectively (2).
Levels of HBV DNA in serum were also assayed by PCR (22)
when spot hybridization results were negative. Levels of HCV RNA were
assayed by reverse transcription-PCR (4). HCV genotypes were
analyzed by genotype-specific probe-based assay of the
5'-untranslated region (LiPA; Innogenetics, Ghent, Belgium).
Pre-S1, pre-S2, and HBsAg proteins were assayed by enzyme immunoassay
using pre-S1-, pre-S2-, and HBsAg-specific monoclonal antibodies
(Institute of Immunology, Tokyo, Japan) at serial 10-fold dilutions.
The highest dilution giving a positive result was designated the titer
of the appropriate antigen.
Cryostat sections of liver specimens were examined for HBV core antigen
(HBcAg), pre-S1, pre-S2, and HBsAg by indirect immunofluorescence,
as described previously (
1-3). Paraffin sections of liver
specimens
were also examined for HBcAg, pre-S1, pre-S2, and HBsAg by
the
avidin-biotin immunoperoxidase method, as reported before
(
5).
Statistical analyses were conducted by using the chi-square test with
Yates' correction, Student's
t test, or the Mann-Whitney
rank sum test where appropriate.
Results and discussion.
Previous results have shown that HBeAg
and HBV DNA are significantly less prevalent in serum in HBsAg carriers
with concurrent HCV infection than in those without HCV infection
(7, 8, 14, 15, 19), suggesting that HCV, like HDV, might
suppress HBV replication. However, the possibility that the HBsAg
carriers with concurrent HCV or HDV infection might have been
previously anti-HBe-positive, asymptomatic HBsAg carriers with
low-level viremia cannot be excluded. All HBeAg-positive patients in
this series were found to be HBV DNA positive by PCR, but levels of HBV
DNA in serum were found to be significantly lower in those with
concurrent HCV (undetectable, 3 patients; 1+, 2 patients;
2+, 1 patient; P < 0.01) or HDV
infection (undetectable, 4 patients; 1+, 4 patients;
2+, 5 patients; 3+, 2 patients;
P < 0.001) than in those without concurrent HCV or HDV
infection (1+, 7 patients; 2+, 4 patients;
3+, 5 patients; 4+, 6 patients) by spot
hybridization. These data provide further evidence suggestive of the
suppressive effect of HCV and HDV on HBV replication. In keeping with
this postulation, the degree of intracellular expression of HBcAg was
significantly lower in HBeAg-positive carriers with concurrent HCV or
HDV infection (Fig. 1).
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FIG. 1.
The degree of intracellular nuclear and cytoplasmic
expression of HBcAg in the HBsAg- and HBeAg-positive patients with HBV
infection alone, concurrent HCV infection (HBV + HCV), and
concurrent HDV infection (HBV + HDV). The expression of HBcAg in
the liver was semiquantitatively scored on a 0-to-4 scale corresponding
to positivity in 0%, 1 to 10%, 11 to 25%, 26 to 50%, and >50% of
hepatocytes examined, respectively. The superscripts a and b indicate
that P < 0.05 and P < 0.01, respectively, versus HBV alone. SEM, standard error of the mean.
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Titers of pre-S1, pre-S2, and HBsAg in serum were significantly lower
in HBeAg-positive patients with concurrent HCV or HDV
infection (Table
2), most probably as a result of a decreased
level of viral
replication. Levels of HBV surface proteins in
tissue showed
little or no difference in HBeAg-positive patients
with HDV
infection but were modestly lower in those with HCV infection
compared
to those without HCV or HDV infection (Fig.
2). These
findings might suggest that in
the HBeAg-positive phase of chronic
HBV infection HCV not only
suppresses HBV replication but also
tends to suppress the expression of
HBV surface proteins.
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FIG. 2.
The degree of intracellular expression of pre-S1,
pre-S2, and HBsAg in HBsAg- and HBeAg-positive patients with HBV
infection alone, concurrent HCV infection (HBV + HCV), and
concurrent HDV infection (HBV + HDV). The expression of pre-S1,
pre-S2, and HBsAg in the liver was semiquantitatively scored on a
0-to-4 scale corresponding to positivity in 0%, 1 to 10%, 11 to 25%,
26 to 50%, and >50% of hepatocytes examined. For concurrent HBV and
HCV infections versus HBV infection, either alone or with concurrent
HDV infection, 0.05 < P < 0.1; for concurrent
HBV and HDV infections versus HBV infection alone, P > 0.3. SEM, standard error of the mean.
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All HBeAg-negative patients in this series were found to have no
detectable serum HBV DNA by spot hybridization or liver HBcAg
by
immunohistochemistry, though 80 to 90% of them were HBV DNA
positive
by PCR. However, levels of HBV surface proteins in tissue
were markedly
decreased (
P < 0.001) and titers of HBV surface
proteins in serum were modestly decreased (
P < 0.1) in
patients
with concurrent HCV infection (Table
2 and Fig.
3). These changes
are not observed in
patients with concurrent HDV infection and
thus do not appear to be
secondary to the increased inflammatory
activity in the liver. It has
been shown that acute HDV infection
can transiently suppress the
expression of HBV (
6,
12,
16).
The present findings that
chronic HDV infection had little or
no effect on the expression of HBV
surface proteins might be compatible
with the biologic characteristics
of HDV, which requires the helper
function of HBsAg (
17).
The current data thus demonstrate that
both HCV and HDV can suppress
the replication of HBV but that,
unlike HDV, HCV also can substantially
suppress the expression
of HBV surface proteins. These findings seem to
be in accordance
with the in vitro observation that HCV core protein
can suppress
HBV gene expression and replication (
21) as
well as the clinical
observation that concurrent HCV infection can
enhance the termination
of the HBsAg carrier state in chronic HBsAg
carriers (
20).
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TABLE 2.
Titers of pre-S1, pre-S2, and HBsAg in serum in HBsAg
carriers with HBV alone or with concurrent HCV or HDV infection
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FIG. 3.
The degree of intracellular expression of pre-S1,
pre-S2, and HBsAg in HBsAg-positive and HBeAg-negative patients
with HBV infection alone, concurrent HCV infection (HBV + HCV),
and concurrent HDV infection (HBV + HDV). The expression of
pre-S1, pre-S2, and HBsAg in the liver was semiquantitatively scored on
a 0-to-4 scale corresponding to positivity in 0%, 1 to 10%, 11 to
25%, 26 to 50%, and >50% of hepatocytes examined. For concurrent
HBV and HCV infections versus HBV infection, either alone or with
concurrent HDV infection, P < 0.001; for concurrent
HDV and HBV infections versus HBV infection alone, P > 0.5. SEM, standard error of the mean.
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ACKNOWLEDGMENTS |
This work was supported by a grant from the National Council of
Science, Republic of China (NSC 85-2331-B-182-024-MH).
The authors thank M. H. Tsai and S. C. Chen for preparation
of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Liver Research
Unit, Chang Gung Memorial Hospital, 199 Tung Hwa North Rd., Taipei, Taiwan 105. Phone: 886-3-3281200, ext. 8120. Fax: 886-3-3282824. E-mail: gi31208108{at}adm.cgmh.com.tw.
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Journal of Clinical Microbiology, July 1998, p. 2084-2086, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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