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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3986-3989
TRANSFUSION MEDICINE
Detection of active hepatitis C virus and hepatitis G virus/GB
virus C replication in bone marrow in human subjects
Marek Radkowski,
Joanna Kubicka,
Elzbieta Kisiel,
Janusz Cianciara,
Marek Nowicki,
Jorge Rakela, and
Tomasz Laskus
From the Institute of Infectious Diseases, Warsaw Medical Academy;
Municipal Hospital, Kielce, Poland; Maternal-Child Virology Research
Laboratory, University of Southern California, Los Angeles, CA; and
Division of Transplantation Medicine, Mayo Clinic Scottsdale, AZ.
 |
Abstract |
We have analyzed the presence of hepatitis C virus (HCV) and
hepatitis G virus (HGV) sequences in bone marrow and serum samples from
48 patients of a hematologic outpatient clinic. HCV RNA was detected in
18 (38%) and 15 (31%) and HGV RNA was detected in 6 (13%) and 9 (19%) of serum and bone marrow samples, respectively. In 3 patients,
HGV RNA was detectable in bone marrow but not in the serum; 2 of these
patients were negative for the presence of specific antibodies. Using a
highly strand-specific Tth-based reverse transcriptase-polymerase chain
reaction (RT-PCR), the presence of HCV RNA and HGV RNA negative strand
was demonstrated in 4 and 5 bone marrow samples, respectively. Our
study shows that HCV and HGV can replicate in bone marrow; in the case
of HGV, analysis of serum may underestimate the true prevalence of infection.
(Blood. 2000;95:3986-3989)
© 2000 by The American Society of Hematology.
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Introduction |
Hepatitis C virus (HCV) and the recently described
hepatitis G virus (HGV), known also as hepatitis GB virus C, are
similarly organized positive-strand enveloped viruses. As the
nomenclature of the latter agent has not been decided yet, for the
purpose of this article it will be referred to as "HGV." Genomes
of both viruses contain 5' and 3' untranslated regions,
flanking a single open reading frame encoding structural and
nonstructural proteins at its 5' and 3' ends,
respectively.1,2 The translated single long polyprotein is
subsequently cleaved by viral and host enzymes into a number of
structural and nonstructural proteins. It is assumed that both HCV and
HGV replicate through the RNA negative strand, the presence of which
could be regarded as a direct evidence of viral replication.
Whereas there is little doubt that HCV replicates primarily in the
liver, the presence of extrahepatic replication sites remains controversial. In support of such a possibility come studies reporting the relatively common detection of HCV RNA negative strand in peripheral blood mononuclear cells (PBMCs).3,4 This
evidence, however, has been questioned, as commonly used techniques are limited in their ability to discriminate between positive and negative
RNA strands.5 Importantly, in several studies that used
assays carefully optimized for strand specificity, HCV RNA negative
strand was not detected in PBMCs from infected patients.6-8 Similarly, although the presence of active replication in bone marrow
(BM) was suggested by in situ detection of viral RNA and viral
antigens,9 it was not confirmed by an investigation using strand-specific assay.10 However, the latter study was
relatively small, as it included only 6 patients.
HGV does not seem to be a primary hepatotropic virus. Although
originally associated with hepatitis, it is currently clear that, in
the absence of concomitant infection with another hepatotropic virus,
there is usually no liver injury and viral replicative forms were not
detected in the liver.8,11 The major replication sites of
HGV are not well defined; however, we have recently found viral
replicative forms in BM, spleen, and lymph nodes.12,13 In
support of the likely role of hematopoietic cells in supporting HGV
replication comes a recent report on successful infection of human
PBMCs in vitro.14
Nevertheless, strand-specific assays were not applied previously to the
study of HCV and HGV replication in BM in a larger group of subjects,
and only those found positive for markers of infection in serum were
included in past studies. Here we report on the results of our study
analyzing the presence of HGV and HCV viral replicative intermediaries
in BM from 48 patients of a hematologic outpatient clinic. The viral
negative strand RNAs were detected using highly strand-specific
Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR).
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Patients, materials, and methods |
The subjects were selected from consecutive patients seen between
May and July 1998 by the same consulting hematologist (E.K.) at an
outpatient hematology clinic in a large industrial city in central
Poland. The inclusion criteria were as follows: availability of BM
sample (BM aspiration biopsy was performed as part of clinical investigation), age older than or equal to 18 years, no serologic evidence of HIV-1 infection, and no antiviral therapy at the time of
the study. Forty-eight patients (28 men, 20 women, mean age 56.8 ± 11.7 years) satisfied the above criteria and were included in the study. Although many subjects have had chronic liver disease (Table 1), they were not preselected with
regard to markers of hepatitis. The high representation of the latter
patients among studied patients was due to the fact the hematology
clinic was serving a large liver disease clinic. The study protocol
adhered to local Institutional Review Board (IRB) requirements.
BM samples were washed once in phosphate-buffered saline (PBS) to lower
contamination by serum and stored at  80°C until analysis. RNA was extracted from serum and BM samples by means of a modified guanidinium thiocyanate-phenol/chloroform technique using a
commercially available kit (RNAzol, Gibco/BRL, Rockville,
MD). One microgram of total RNA, (as determined by
spectrophotometry) was routinely used for RT-PCR. In the case of serum,
the amount of extracted RNA loaded into the reaction corresponded to 20 µL. However, all initially negative serum samples were retested using
RNA extracted from 100 µL of serum.
Strand-specific reverse transcriptase-polymerase chain reaction
Strand specificity of our RT-PCR for the detection of HCV and HGV
negative RNA strands was ascertained by conducting complementary DNA
(cDNA) synthesis at a high temperature using the thermostable enzyme,
Tth. The sensitivity and strand specificity of these reactions was
established using synthetic RNA as templates. A detailed description of
both strand-specific assays and sequence of used primers was published
previously.7,11 In brief, the cDNA was generated in 20 µL of reaction mixture containing 50 pmol/L of sense
primer, 1 × RT buffer (Perkin Elmer), 1 mmol/L
MnCl2, 200 µmol/L (each) dNTP, and 5 units Tth (Perkin
Elmer). After 20 minutes at 65°C, Mn2+ were chelated
with 8 µL of 10 × EGTA chelating buffer (Perkin Elmer), 50 pmol/L of antisense primer was added and the volume was adjusted to 100 µL, and the MgCl2 concentration was adjusted to 2.2 mmol/L. The amplification was performed in Perkin Elmer GenAmp PCR
System 9600 thermocycler as follows: initial denaturing for 1 minute at
94°C, 50 cycles of 94°C for 15 seconds, 58°C for 30 seconds
and 72°C for 30 seconds, followed by a final extension at 72°C
for 7 minutes. Twenty microliters of the final product were analyzed by
agarose gel electrophoresis and Southern hybridization with a
32P-labeled internal oligoprobe.
The strand-specific assays were capable of detecting approximately 100 genomic equivalent (eq) molecules of the correct strand, while
unspecifically detecting more than or equal to 107 to
108 genomic eq of the incorrect strand. The addition of 1 µg of total cellular RNA extracted from human tissues would lower the
the sensitivity of the reaction by no more than 1 log, whereas the specificity of the assays was not affected.7,11 Thus, both strand-specific assays were capable of detecting approximately 103 viral genomic eq in 1 µg of RNA. In case of serum,
the approximate detection limit was 103 eq per 1 mL.
Reverse transcriptase-polymerase chain reaction with Moloney murine
leukemia virus reverse transcriptase (MMLV-RT)
MMLV RT-based detection of HCV and HGV has been described in detail
elsewhere.7,11 These assays were capable of detecting approximately 10 genomic eq of the correct synthetic template but were
not strand specific. Similary to Tth-based assay, the addition of
cellular RNA would slightly lower the sensitivity by up to 1 log. The
established detection limit was approximately 100 genomic eq
per micrograms of total RNA or 1 mL of serum.
When deemed necessary, titers were determined by analyzing 10-fold
serial dilutions of the RNA template. Appropriate measures, described
elsewhere,11 were used to prevent and detect contamination. All RT-PCR runs included positive controls consisting of end-point dilutions of respective RNA strands and negative controls included normal BM, PBMCs, and normal sera. In addition, to account for RNA
extraction efficiency, synthetic template was routinely "spiked" into the negative control samples. The presence of anti-HCV and E2
antibody to the HGV envelope protein was determined by
commercially available tests (UBI HCV EIA, United Biochemical
and Quantinine Human GBV-C EIA, R&D Systems, Minneapolis, MN). The
infecting HCV genotypes were determined as described
previously.15
| |
Results and discussion |
The distribution of viral infection markers in different groups of
patients is shown in Table 1. When serum samples were analyzed, 18 (38%) patients were found to be HCV RNA positive and 6 (13%) were
found to be HGV RNA positive. Three patients were positive for both
viral sequences. Anti-HCV was demonstrated in 24 (50%) patients and
anti-HGV was found in 19 (40%) patients. However, although all
patients who were HCV RNA positive were also anti-HCV positive, only 1 patient who was HGV RNA positive had respective antibodies detectable
in serum. Thus, the overall prevalence of infection markers in serum
was 50% for both viruses.
When BM samples were analyzed, the presence of HCV RNA was detected in
15 (31%) patients, and the presence of HGV RNA was detected in 9 (19%) patients. The concordance between the presence of viral
sequences in serum and BM was not absolute. In 3 patients, HGV RNA was
detectable in the BM but not in the serum; 2 of these patients were
also anti-HGV negative and the presence of viral sequence in BM was the
only evidence of infection. In 3 patients, HCV sequences could be
demonstrated in serum but not in BM, and in 1 patient, HCV RNA was
detectable in BM but not in serum. However, the latter patient, in whom
the condition was diagnosed as chronic hepatitis C, was anti-HCV positive.
Altogether, the presence of HGV RNA and HCV RNA, either in serum or BM,
was demonstrated in 9 (19%) and 19 (40%) patients, respectively, and
at least 1 marker of infection was present in 26 (48%) and 24 (50%)
patients, respectively.
Using the Tth-based strand-specific assay, the presence of negative
strand HCV RNA and HGV RNA was detected in BM from 4 and 5 patients,
respectively, whereas all serum samples were negative (Table 1). These
results were confirmed in 2 independent experiments using 2 separate
extraction procedures. To exclude the possibility that these results
represent false-positives because of the presence of high titer
positive strand RNA, the viral titer was estimated by serial dilutions
and was found to range from 103 to 105 genomic
eq per microgram of total RNA, thus remaining well within the
specificity limits of our strand specific assay. The patients with
evidence of HCV and HGV replication in BM were not significantly different from the rest of the infected patients with respect to age,
gender, or history of blood transfusion; their infecting HCV genotypes
were either 1b or 1a, which was also not different from the rest of the
patients. Some clinical and virologic data on these patients are
presented in Table 2.
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Table 2.
Clinical and virologic data on 9 patients in whom HGV
RNA or HCV RNA negative strand sequences were detected in bone
marrow
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In this study, the presence of HCV RNA negative strand in BM was
demonstrated in 4 of 24 (17%) patients who were HCV positive and the
titer of positive strand was 1 to 2 logs higher than the titer of the
negative strands, which is a proportion similar to that expected for a
flavivirus at its replication site. However, the actual prevalence of
active infection could be higher, as the replication in other subjects
could have been below the sensitivity limit of our strand-specific
assay. Extrahepatic HCV replication is more likely to be detected in
the presence of immunosuppression: The presence of HCV RNA negative
strand was found in lymph nodes and BM from HIV-coinfected
subjects,15 as well as in hematopoietic cells derived from
patients who were HCV positive and transplanted into the severe
immunodeficiency mouse.16 However, even in these studies,
the presence of viral negative strand was by no means universal and the
titers were very low. This contrasts with the reported common detection
of HCV RNA and viral antigens in BM and PBMCs by in situ
techniques.9,17,18 Because the strand-specific assays are
relatively sensitive, our RT-PCR was capable of detecting approximately
103 viral genomic eq in 1 µg of total RNA, this suggests
a very low rate of replication or rapid degradation of negative strand
RNA. HCV invasion of hematopoietic cells may not be benign; HCV
infection was associated with such lymphoproliferative disorders as
non-Hodgkin's lymphoma and cryoglobulinemia.19 Although
the effect of HGV on BM is unclear, one study reported a possible link
between HGV infection and low-grade non-Hodgkin's
lymphoma.20
Interestingly, in our previous study, which used the same Tth-based
assay, we did not detect HCV replication in PBMCs from HIV-negative
subjects.7 Similarly, other studies6,8 failed to demonstrate the presence of HCV RNA negative strands in PBMCs when
using highly strand-specific RT-PCR assays. One possible explanation
for this discrepancy is that HCV may be particularly apt at infecting
CD34+ hematopoietic progenitor cells.18 It is
also possible that HCV replication becomes more efficient in
proliferating cells. In support of this concept, our recent
observations are that PBMCs from subjects with chronic hepatitis C
occasionally become positive for the presence of HCV RNA negative
strand after stimulation with phytohemagglutin (PHA)
mitogen.21
HCV circulates as a number of closely related but not identical
genomes, referred to as quasi-species, and the presence of this dynamic
mutant reservoir could facilitate viral adaptation to replication in
various secondary cells. HGV does not seem to be as
variable,13 and BM cells may represent the primary, not the
secondary, site of replication. BM replication may not be that unusual
for flaviviridae, for example, the hog cholera virus was found in
megakaryocytes,22 whereas dengue virus was shown to
replicate efficiently in BM progenitors and hematopoietic cell lines.23 Interestingly, we identified 3 patients in whom
HGV RNA was detectable in the BM but not in the serum; 2 of them were anti-HGV negative in serum. The latter 2 patients were probably in the
early phase of the infection when antibodies have not yet developed.24 Although it supports the notion of HGV
replication in BM, it also demonstrates that studying HGV RNA and
specific antibodies in serum may underestimate the true prevalence of
the infection. However, this may also be occasionally true for HCV infection as one of the patients studied was HCV RNA positive in BM but
not in serum. This could probably be explained by the fact that viral
levels in serum can fluctuate during chronic infection, becoming
occasionally undetectable by RT-PCR.25 Whether this was the
case is unclear, as follow-up serum samples were unavailable for analysis.
In summary, we have described the presence of active HCV and HGV
replication in BM from human subjects. For HGV, the presence of viral
RNA in BM was occasionally the only evidence of infection.
| |
Footnotes |
Submitted October 6, 1999; accepted February 10, 2000.
Reprints: T. Laskus, Division of Transplantation Medicine, SC
Johnson Bldg Sj3, Mayo Clinic Scottsdale, AZ 85259; email: laskus.tomasz{at}mayo.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction