Previous Article | Next Article 
Journal of Virology, September 2000, p. 7936-7942, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Existence of Distinct GB Virus C/Hepatitis G Virus
Variants with Different Tropism
Marta
Fogeda,
Juan Manuel
López-Alcorocho,
Javier
Bartolomé,
Carlos
Arocena,
Maria Angeles
Martín, and
Vicente
Carreño*
Department of Hepatology, Fundación
Jiménez Díaz, and Fundación para el Estudio de
las Hepatitis Virales, Madrid, Spain
Received 10 January 2000/Accepted 9 June 2000
 |
ABSTRACT |
To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV)
variants with different tropism, we have analyzed the heterogeneity and
quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and
PBMC from two chronically infected patients. For this purpose, the
GBV-C/HGV 5' noncoding region (5'NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5'NCR sequences isolated from the
in vitro-infected PBMC and from the PBMC isolated from the patient
whose serum was used as the inoculum were identical but different from
that of the inoculum. Furthermore, phylogenetic analysis revealed that
all PBMC sequences grouped together into a branch which was separate
from those of the inoculum. For one of the two chronically infected
patients, all the sequences from the PBMC and one from the liver
clustered into a single branch while the sequences from the serum and
all the other liver sequences grouped together in the other branch. For
the other patient, the sequences from the serum and PBMC and three
sequences from the liver grouped together into one branch, while the
remaining five sequences from the liver were separated in a different
cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.
| |
INTRODUCTION |
The GB virus C/hepatitis G virus
(GBV-C/HGV) is a positive-sense, single-stranded RNA (9.4 kb in length)
virus whose genetic structure resembles the hepatitis C virus (HCV) and
which is considered belong to the Flaviviridae family of
animal viruses (17, 18, 31, 32). Although GBV-C/HGV was
discovered as a putative agent of non-A-E hepatitis (2, 7)
and GBV-C/HGV RNA has been detected in the sera of patients with
various liver diseases including fulminant hepatitis (39),
chronic hepatitis C (1, 4, 34, 38), and cirrhosis with or
without hepatocellular carcinoma (13, 14), recent works have
shown that GBV-C/HGV may play a minor role in causing liver disease
(2, 3, 10, 11, 22).
The replication of GBV-C/HGV presumably occurs via a negative-strand
RNA intermediate. However, its replication site is still unknown, since
no conclusive evidence regarding the GBV-C/HGV cell tropism has been
reported. Thus, it remains unclear whether the liver is the main target
for GBV-C/HGV infection and replication, because, although several
authors have reported the detection of negative-polarity viral RNA in
the liver (16, 19, 29, 30), others have been unable to
detect this putative replicative intermediate (6, 15, 25,
28). Similarly, the results have been contradictory when
peripheral blood mononuclear cells (PBMC) from GBV-C/HGV-infected
patients have been examined for the presence of GBV-C/HGV-RNA of both
positive and negative polarity (19, 21, 27, 29). On the
other hand, in vitro studies have provided evidence that GBV-C/HGV is
able to replicate in established cell lines of hematopoietic origin
(MT-2C, a human T-cell leukemia virus type 1-infected human T-cell
line) and in immortalized hepatocytes (PH5CH, a non-neoplastic human
hepatocyte cell line immortalized with simian virus 40 large T antigen)
(12). Furthermore, we have recently demonstrated that
GBV-C/HGV can infect and replicate in PBMC from healthy donors after
incubation of these cells with GBV-C/HGV-RNA-positive serum
(8). In that work, in which a relatively small number of
clones were analyzed, we also demonstrated that only a fraction of the
GBV-C/HGV variants present in serum are able to infect and
replicate in PBMC in vitro. This finding suggests the existence
of lymphotropic variants of this virus. However there are no data on
the existence of these lymphotropic variants in vivo. Furthermore,
whether the GBV-C/HGV variants that infect PBMC in vivo are the
same as those that infect the liver and circulate in serum is not known.
In the present study, we have analyzed the genomic heterogeneity and
quasispecies composition of the GBV-C/HGV 5' noncoding region (5'NCR)
recovered from the PBMC of four healthy donors infected in vitro with a
GBV-C/HGV RNA-positive serum. We have also studied the in vivo
quasispecies composition in the 5'NCR from the GBV-C/HGV isolated from
the sera, livers, and PBMC of two chronically infected patients, and
our results clearly demonstrated that different GBV-C/HGV strains have
a different tropism.
| |
MATERIALS AND METHODS |
In vitro infection of PBMC.
PBMC from four healthy blood
donors (who were not infected by GBV-C/HGV, HCV, hepatitis B virus,
human immunodeficiency virus, Epstein-Barr virus, or cytomegalovirus)
were isolated as previously described (8). One million
viable cells from each individual donor and a cell pool consisting of
equal parts of PBMC from each donor at a final density of
106 cells were incubated with 10 µl of a GBV-C/HGV
RNA-positive serum (inoculum). The culture method and the
characteristics of the inoculum have been described previously
(8). After the 30-day culture period, the cells were washed
five times with phosphate-buffered saline (PBS), and the last wash was
saved to be used as a negative control for GBV-C/HGV RNA detection.
Total RNAs from each cell culture, from the serum used as the inoculum,
and from the PBMC isolated from the patient whose serum was used as the
inoculum were extracted by a modification of the
guanidinium-phenol-chloroform method described by Chomczynski and
Sacchi (5).
In vivo study of GBV-C/HGV quasispecies.
Genetic
heterogeneity in the 5'NCR region of the GBV-C/HGV genome was analyzed
in paired serum (stored at 
20°C), liver, and PBMC (stored in liquid
N2) specimens from two patients coinfected with HCV. The
clinical features of these patients are summarized in Table
1. The patients presented anti-HCV
antibodies as detected by enzyme-linked immunosorbent assay III (Ortho
Diagnostic Systems, Raritan, N.J.) and confirmed by RIBA III (Ortho
Diagnostic Systems). These two patients were selected according to the
following criteria: (i) no previous antiviral or immunomodulatory
treatment and (ii) presence of GBV-C/HGV RNA by reverse
transcription-PCR (RT-PCR) in serum, liver, and PBMC samples obtained
at the same time. Total RNA was extracted from 200 µl of serum, 100 mg of liver tissue, and 106 PBMC as mentioned above
(5). Total RNAs from PBMC and liver tissue were quantitated,
and 1.5 µg of cell-derived RNA or the whole-serum-derived RNA was
used for GBV-C/HGV amplification. To prevent any possible contamination
of the liver and PBMC samples by the serum GBV-C/HGV particles, the
samples were washed five times with PBS before RNA extraction, and the
last wash of each sample was stored for further analysis by RT-PCR as a
negative control.
Amplification of the GBV-C/HGV 5'NCR.
GBV-C/HGV RNA from the
inoculum, the PBMC corresponding to the inoculum, the 30-day-cultured
cells, and the serum, liver, and PBMC specimens from the two patients
studied were amplified by RT-nested PCR with primers derived from the
5'NCR of the GBV-C/HGV genome, as previously described (8).
The amplified fragments covered nucleotides 36 to 356 of the 5'NCR
(17).
Cloning and sequencing.
The amplified GBV-C/HGV RNA 5'NCR
products were cloned into the pCR II-TOPO vector (TOPO TA Cloning kit;
Invitrogen, San Diego, Calif.). An average of 20 clones (range, 11 to
50 clones) from each PCR product were automatically sequenced in both
directions using the ALF Express DNA Sequencing System. In order to
exclude the possibility that the changes observed in the sequences of the samples were not due to nucleotide misincorporation induced by the
Taq polymerase, an in vitro-transcribed RNA from plasmid pCR-GNC, containing a cDNA corresponding to a GBV-C/HGV 5'NCR (nucleotides 10 to 387) whose nucleotide sequence was previously known,
was included in each PCR assay. Subsequently, the amplification products of this synthetic RNA were cloned and sequenced at the same
time as the in vitro and in vivo samples.
Analysis of the 5'NCR nucleotide sequences.
The sequences
were aligned and edited using the Clustal X (35) and GeneDoc
(version 2.5.000; K. B. Nicholas and H. B. Nicholas, Jr.,
1997) programs, respectively. Phylogenetic analysis was performed using
the DNADIST, NEIGHBOR, SEQBOOT, and CONSENSE programs from the
Phylogeny Inference Package (PHYLIP, version 3.5c; J. Felsenstein, Department of Genetics, University of Washington,
Seattle, 1993). The evolutionary distances were estimated by the Kimura
two-parameter method, and the unrooted phylogenetic trees were
constructed by the neighbor-joining method. The final outputs of the
trees were obtained with TreeView, version 1.5.2 (24).
Bootstrap analyses were determined on 1,000 resamplings of the data
sets. Bootstrap values of 
70% were considered statistically
significant for the observed grouping. In order to study the
quasispecies composition of each sample type, the complexity
coefficient (CC) was defined as the number of different clones from
each sample divided by the total number of clones analyzed
(23).
Nucleotide sequence accession numbers.
The GenBank accession
numbers of the nucleotide sequences presented in this paper are
AF125468 to AF12505 and AF197346 to AF197447.
| |
RESULTS |
Specificity of GBV-C/HGV detection.
A PCR fragment of 320 bp,
corresponding to the GBV-C/HGV 5'NCR amplification product, was
detected in the inoculum, the PBMC corresponding to the inoculum, the
30-day-cultured PBMC of the healthy donors, and the cell pool after
experimental infection, as well as in the serum, liver, and PBMC
samples of the two infected patients. In contrast, the last PBMC and
liver PBS washes and all the negative controls included in each PCR
assay were always GBV-C/HGV negative. On the other hand, sequencing of
the amplification products from the RNA transcribed in vitro from
plasmid pCR-GNC always gave the expected nucleotide sequence previously
known, confirming that no nucleotide misincorporation due to
Taq polymerase activity occurred during the PCR procedures.
Analysis of the GBV-C/HGV 5'NCR sequence of in vitro-infected
PBMC.
To study the sequence heterogeneity of PBMC after the
experimental infection, the PCR products from the inoculum and the
different cell cultures were cloned. A total of 50 clones from the
inoculum and 29 clones from the PBMC corresponding to the inoculum were sequenced. With respect to the in vitro-infected PBMC and cell pool, an
average of 22 clones (range, 19 to 27) were sequenced.
As shown in Table
2, CCs of 0.50 and 0.34 were obtained for the sequences isolated from the inoculum and the PBMC
corresponding
to the inoculum, respectively. On the other hand, CCs
ranged from
0.33 to 0.55 for the 5'NCR sequences of the PBMC infected
in vitro,
and the CC for the sequences of the GBV-C/HGV isolates
recovered
from the cell pool was 0.29. When the master sequences (the
master
sequence for each sample type is the sequence which occurs with
the highest frequency) were aligned and compared, we found that
those
recovered from the PBMC corresponding to the inoculum and
the in
vitro-infected PBMC and cell pool were all identical, but
different
from the master sequence recovered from the inoculum
(Fig.
1). None of the GBV-C/HGV 5'NCR sequences
derived from the
serum used as the inoculum were identical to the
master sequences
of the PBMC corresponding to the inoculum or the in
vitro-infected
PBMC and cell pool, and the mean genetic distance
between the
sequences was 0.0508 ± 0.0045 (range, 0.0406 to
0.0619).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 1.
Nucleotide alignment of the master sequences recovered
from the inoculum (I), the PBMC of the patient whose serum was used as
the inoculum (IP), the PBMC from the four healthy donors infected in
vitro with GBV-C/HGV (D1 through D4), and the pool of cells from the
four donors infected in vitro with GBV-C/HGV (PP).
|
|
The unrooted phylogenetic tree constructed with all these GBV-C/HGV
sequences revealed that the sequences derived from the
serum used as
the inoculum were grouped together in a cluster,
while the sequences
from the PBMC corresponding to the inoculum
and the in vitro-infected
PBMC and cell pool clustered together
into a separate branch, with a
bootstrap value of 96% (Fig.
2).
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 2.
Unrooted neighbor-joining tree constructed
with the 5'NCR nucleotide sequences from the in vitro-infected PBMC.
The isolates are designated I (inoculum), IP (PBMC of the patient whose
serum was used as the inoculum), D1 through D4 (PBMC from the four
healthy donors infected in vitro with GBV-C/HGV), or PP (pool of cells
from the four donors infected in vitro), followed by the clone number
and, in parentheses, the number of clones bearing the same sequence.
Bootstrap values are shown in the nodes of the tree.
|
|
Analysis of the 5'NCR sequence from serum, liver, and PBMC samples
of the GBV-C/HGV-infected patients.
To study the complexity of the
GBV-C/HGV quasispecies in the 5'NCR infecting the serum, liver, and
PBMC in vivo, the PCR products from each compartment corresponding to
two patients with chronic GBV-C/HGV infection were cloned and
sequenced. A total of 41 clones from patient 1 (13 from the serum, 11 from the liver, and 17 from PBMC) and 45 clones from patient 2 (16 from
the serum, 15 from the liver, and 14 from PBMC) were analyzed.
In patient 1, the CCs in the serum and liver were similar (0.61 and
0.64, respectively) and higher than that found in the
isolates
recovered from the PBMC (0.35). In contrast, in patient
2, the CCs in
the three compartments studied were similar (0.81
in the serum, 0.80 in
the liver, and 0.86 in
PBMC).
When the frequency with which the evolutionary distances occurred in
the GBV-C/HGV isolates from the three compartments was
analyzed, the
distances in serum and PBMC samples were distributed
around a single
peak in both patients (Fig.
3). In
contrast, the
distribution of the distances among the GBV-C/HGV
isolates from
the liver showed two independent peaks in both cases
(Fig.
3).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Histograms of the frequency of each evolutionary
distance in the GBV-C/HGV 5'NCR sequences from serum, liver, and PBMC
specimens of the two patients with GBV-C/HGV infection.
|
|
With respect to the master sequences in the three compartments studied,
in patient 1, the master sequences from the serum
and the liver were
identical and represented 30.8 and 36.4% of
the total sequences from
the serum and liver, respectively; however,
this master sequence was
different from that of the PBMC, which
represented 70.% of total
sequences from this compartment. In
contrast, in patient 2, the master
sequences from the serum and
PBMC, which represented 18.7 and 21%,
respectively, of total sequences,
were identical, but different from
the predominant sequence from
the liver, which represented 20% of all
liver
sequences.
The neighbor-joining phylogenetic trees constructed with the GBV-C/HGV
5'NCR nucleotide sequence of the isolates from both
patients are shown
in Fig.
4. In patient 1, all the isolates
from
the PBMC samples and one isolate from the liver are separated
into
a single branch, while the remaining sequences from the serum
and liver
are clustered in another branch (Fig.
4a). In the other
patient, all
the sequences from the serum and PBMC samples and
seven sequences from
the liver are grouped into a single branch,
while the remaining five
different sequences from the liver biopsy
specimen are separated into a
different branch (Fig.
4b).
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 4.
Unrooted neighbor-joining trees constructed with the
5'NCR nucleotide sequences of GBV-C/HGV isolated from serum, liver, and
PBMC samples of patients 1 (a) and 2 (b). The clones are designated P1
or P2 (indicating those isolated from patient 1 or 2, respectively),
plus S, L, or P (indicating the source as serum, liver, or PBMC,
respectively), followed by the clone number and, in parentheses, the
number of clones with identical sequences in each compartments. The
bootstrap values are given in the nodes of the trees.
|
|
| |
DISCUSSION |
The tissue tropism of the recently described GBV-C/HGV is unknown.
In vitro studies have provided evidence which indicates that this virus
is able to infect and replicate in lymphoid cells and in immortalized
hepatocytes (8, 30). In contrast, contradictory results have
been reported with respect to the detection of GBV-C/HGV RNA of
negative polarity (the putative replicative intermediate) in liver and
PBMC samples from chronically infected patients (6, 15, 25).
These discrepancies could be partially explained if there are GBV-C/HGV
variants with different cell tropism.
In a previous study on in vitro infection of PBMC, we found that only a
minor proportion of the GBV-C/HGV variants present in serum are able to
infect and replicate in PBMC, which suggests the existence of
lymphotropic variants (8). However, there are no data on the
existence of these variants in vivo. Furthermore, it is not known
whether there are any variants with tropism for the liver cells and
whether all these variants are present in serum. Consequently we have
studied the genomic heterogeneity and quasispecies composition of the
5'NCR of viral genomes recovered from in vitro-infected PBMC and from
serum, liver, and PBMC samples from two chronically infected patients.
Regarding the in vitro-infected PBMC, we found that the predominant
GBV-C/HGV 5'NCR sequence was the same in all in vitro-infected PBMC and
in the PBMC isolated from the patient whose serum was used as the
inoculum. This sequence was different from the predominant sequence
found in the inoculum. In fact, the predominant sequence of the PBMC
was not detected among the 50 clones sequenced from the inoculum,
showing that this variant represents a minority of the circulating
GBV-C/HGV variants. Furthermore, the phylogenetic analysis of these
sequences shows that the sequences derived from the inoculum grouped
together in a cluster, while those of PBMC grouped in a separate branch
(bootstrap value, 96%).
Considered as a whole, all these findings show that there are GBV-C/HGV
lymphotropic variants and that in vitro infection of PBMC depends on
the presence of these variants in the inoculum but not on host factors,
as the PBMC of the four unrelated donors were infected with similar efficiencies.
In the naturally infected patients, we have found that GBV-C/HGV in the
liver and PBMC exists as a complex distribution of nonidentical but
closely related genomes (quasispecies), as has been reported previously
for serum (26, 36). This situation is similar to that of the
hepatitis C virus (20, 23). When the frequency at which the
evolutionary distances occurred in the GBV-C/HGV isolated from the
three compartments was analyzed, it was seen that while sequences
isolated from the serum and PBMC of both patients were distributed
around a single peak, the sequences isolated from the livers of the two
patients were distributed into two independent peaks. This finding
shows that the GBV-C/HGV quasispecies composition is more complex in
the liver than in the serum or PBMC.
When phylogenetic analysis of these sequences was performed, it was
found that the sequences isolated from the serum and liver in patient 1 grouped into a single branch, while the sequences from PBMC (all except
one) were grouped into a different branch. In contrast, for patient 2, all the sequences from the serum and PBMC and some of those from the
liver grouped within in one branch, while the remaining liver sequences
grouped into a different branch. Furthermore, for patient 1, the
predominant sequence in the serum (representing 30.8% of the
total serum sequences) and the predominant sequence in the liver
(36.4% of all liver sequences) were identical, and this sequence was
different from the predominant sequence found in PBMC (70.5% of the
total PBMC sequences). A different situation occurs in patient 2, for
whom the predominant sequence in the serum and the predominant
sequence in PBMC (representing 18.7 and 21% of all sequences
from the serum and PBMC, respectively) were identical to each other and
different from the predominant sequence from the liver (20% of all
liver sequences). All these results strongly support the existence of
GBV-C/HGV variants with different tropism for the liver and lymphoid cells.
Finally, the fact that for patient 1 the sequences from serum and liver
grouped together and that the predominant sequences were identical
could indicate that the liver was the main contributor to the
quasispecies composition, while in patient 2 the clustering of PBMC and
serum sequences and some of the liver sequences in a single branch
suggests that in this case both the liver and PBMC contribute to the
quasispecies composition in the serum. However, it should be noted that
both patients for whom serum, liver and PBMC samples were analyzed were
also coinfected by HCV. Since HCV infects and replicates in both the
liver and PBMC (9, 37), the possibility that HCV may
influence the tropism of the GBV-C/HGV variants by facilitating the
replication of some strains of these lines cannot be excluded. So, to
definitively conclude that there are GBV-C/HGV variants with different
tropism, further studies analyzing patients infected only by GBV-C/HGV
should be performed. Furthermore, the samples analyzed were obtained at a single time point from each patient; therefore, changes in GBV-C/HGV quasispecies composition in the serum, liver, and PBMC over time cannot
be excluded. However, the fact that GBV-C/HGV does not seem to have a
high mutation rate (33) argues against this hypothesis.
In conclusion, our results have demonstrated tissue
compartmentalization of GBV-C/HGV 5'NCR sequences from PBMC, liver, and serum samples obtained at the same time from chronic GBV-C/HGV carriers
coinfected by HCV, suggesting the existence of GBV-C/HGV variants with
different tropism. The question of the factors involved in this
different tropism, as well as its pathological implications, deserves
future research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fundación
para el Estudio de las Hepatitis Virales, C/ Guzmán el Bueno, 72 Semisótano, 28015 Madrid, Spain. Phone: 34 91 544 60 13. Fax: 34 91 544 92 28. E-mail: fehv{at}tdi.es.
| |
REFERENCES |
| 1.
|
Aikawa, T.,
Y. Sugay, and H. Okamoto.
1996.
Hepatitis G infection in drug abusers with chronic hepatitis C.
N. Engl. J. Med.
334:195[Free Full Text].
|
| 2.
|
Alter, H. J.,
Y. Nakatsuyi,
J. Melpolder,
J. Wages,
R. Wesley,
J. W. K. Shih, and J. P. Kim.
1997.
The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease.
N. Engl. J. Med.
336:747-754[Abstract/Free Full Text].
|
| 3.
|
Alter, M. J.,
M. Gallagher,
T. T. Morris,
L. A. Moyer,
E. L. Meeks,
K. Krawczynski,
J. P. Kim, and H. S. Margolis.
1997.
Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection.
N. Engl. J. Med.
336:741-746[Abstract/Free Full Text].
|
| 4.
|
Berenguer, M.,
N. A. Terrault,
M. Piatak,
A. Yun,
J. P. Kim,
J. Y. N. Lau,
J. R. Lake,
J. R. Roberts,
N. Ascher,
L. Ferrel, and T. Wright.
1996.
Hepatitis G virus infection in patients with hepatitis C virus infection undergoing liver transplantation.
Gastroenterology
111:1569-1575[CrossRef][Medline].
|
| 5.
|
Chomczynski, P., and N. Sacchi.
1987.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:156-159[Medline].
|
| 6.
|
Fan, X.,
Y. Xu,
H. Solomon,
S. Ramrakhiani,
B. A. Neuschwander-Tetri, and A. M. Di Bisceglie.
1999.
Detection of hepatitis G/GB virus-C viral RNA in liver and serum.
J. Med. Virol.
58:160-164[CrossRef][Medline].
|
| 7.
|
Fiordalisi, G.,
I. Zanella,
G. Mantero,
A. Bettinardi,
R. Stellini,
G. Paraninfo,
G. Cadeo, and D. Primi.
1996.
High prevalence of GB virus C infection in a group of Italian patients with hepatitis of unknown etiology.
J. Infect. Dis.
174:181-183[Medline].
|
| 8.
|
Fogeda, M.,
S. Navas,
J. Martín,
M. Casqueiro,
E. Rodríguez,
C. Arocena, and V. Carreño.
1999.
In vitro infection of human peripheral blood mononuclear cells by GB virus C/hepatitis G virus.
J. Virol.
73:4052-4061[Abstract/Free Full Text].
|
| 9.
|
Fong, T. S.,
M. Shindo,
S. M. Feinstone,
J. H. Hoofnagle, and A. M. Di Bisceglie.
1991.
Detection of the replicative intermediates of hepatitis C viral RNA in liver and serum samples of patients with chronic hepatitis C.
J. Clin. Investig.
88:1058-1060.
|
| 10.
|
Fried, M. W.,
Y. E. Khudyakov,
G. A. Smallwood,
M.-E. Cong,
B. Nichols,
E. Diaz,
P. Siefert,
K. Gutekunst,
R. D. Gordon,
T. D. Boyer, and H. A. Fields.
1997.
Hepatitis G virus co-infection in liver transplant recipients with chronic hepatitis C and non-viral chronic liver disease.
Hepatology
25:1271-1275[CrossRef][Medline].
|
| 11.
|
Hadziyannis, S. J.
1997.
The "hepatitis" G virus: biology, epidemiology and search for disease.
Prog. Liver Dis.
14:219-245.
|
| 12.
|
Ikeda, M.,
K. Sugiyama,
T. Mizutani,
T. Tanaka,
K. Shimotohno, and N. Kato.
1997.
Hepatitis G virus replication in human cultured cells displaying susceptibility to hepatitis C virus infection.
Biochem. Biophys. Res. Commun.
235:505-408[CrossRef][Medline].
|
| 13.
|
Kanda, T.,
O. Yokosuka,
F. Imazeki,
M. Tagawa,
T. Ehata, and H. Saisho.
1997.
GB virus-C RNA in Japanese patients with hepatocellular carcinoma and cirrhosis.
J. Hepatol.
27:464-469[CrossRef][Medline].
|
| 14.
|
Kao, J. H.,
P. J. Chen,
M. Y. Lai,
W. Chen,
D. P. Liu,
J. T. Wang,
M. C. Shen, and D. S. Chen.
1997.
GB virus-C/hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease, and liver cancer.
Gastroenterology
112:1265-1270[CrossRef][Medline].
|
| 15.
|
Laskus, T.,
M. Radkowski,
L.-F. Wang,
H. Vargas, and J. Rakela.
1997.
Lack of evidence for hepatitis G virus replication in the livers of patients coinfected with hepatitis C and G viruses.
J. Virol.
71:7804-7806[Abstract].
|
| 16.
|
Laskus, T.,
M. Radkowski,
L.-F. Wang,
H. Vargas, and J. Rakela.
1998.
Detection of hepatitis G virus replication sites by using highly strand-specific Tth-based reverse transcriptase PCR.
J. Virol.
72:3072-3075[Abstract/Free Full Text].
|
| 17.
|
Leary, T. P.,
A. S. Muerhoff,
J. N. Simons,
T. J. Pilot-Matias,
J. C. Erker,
M. L. Chalmers,
G. G. Schlauder,
G. J. Dawson,
S. M. Desai, and I. K. Mushahwar.
1996.
Sequence and genomic organization of GBV-C: a novel member of the flaviviridae associated with human non-A-E hepatitis.
J. Med. Virol.
48:60-67[CrossRef][Medline].
|
| 18.
|
Linnen, J.,
J. Wages,
Z.-Y. Zhank-Kek,
K. E. Fry,
K. Z. Krawczynski,
H. Alter,
E. Koonin,
M. Gallagher,
M. Alter,
S. Hadziyannis,
P. Karayiannis,
K. Fung,
I. Nakatsuji,
J. W. K. Shih,
L. Young,
M. Piatak, Jr.,
C. Hoover,
J. Fernandez,
S. Cheng,
J.-C. Zou,
T. Morris,
K. C. Hyams,
S. Ismay,
J. D. Lifson,
G. Hess,
S. K. H. Foung,
H. Thomas,
D. Bradley,
H. Margolis, and J. P. Kim.
1996.
Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.
Science
271:505-508[Abstract].
|
| 19.
|
Madejón, A.,
M. Fogeda,
J. Bartolomé,
M. Pardo,
C. González,
T. Cotonat, and V. Carreño.
1997.
GB virus C RNA in serum, liver, and peripheral blood mononuclear cells from patients with chronic hepatitis B, C, and D.
Gastroenterology
113:573-578[CrossRef][Medline].
|
| 20.
|
Maggi, F.,
C. Fornai,
M. L. Vatteroni,
M. Giorgi,
A. Morrica,
M. Pistello,
G. Cammarota,
S. Marchi,
P. Ciccorossi,
A. Bionda, and M. Bendelli.
1997.
Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma.
J. Gen. Virol.
78:1521-1525[Abstract].
|
| 21.
|
Mellor, J.,
G. Haydon,
C. Blair,
W. Livingstone, and P. Simmonds.
1998.
Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.
J. Gen. Virol.
79:705-714[Abstract].
|
| 22.
|
Mushahwar, I. K., and J. N. Zuckerman.
1998.
Clinical implications of GB virus C.
J. Med. Virol.
56:1-3[CrossRef][Medline].
|
| 23.
|
Navas, S.,
J. Martín,
J. A. Quiroga,
I. Castillo, and V. Carreño.
1998.
Genetic diversity and tissue compartmentalization of the hepatitis C virus genome in blood mononuclear cells, liver, and serum from chronic hepatitis C patients.
J. Virol.
72:1640-1646[Abstract/Free Full Text].
|
| 24.
|
Page, R. D. M.
1996.
TreeView. An application to display phylogenetic trees on personal computers.
Comput. Appl. Biosci.
12:357-358[Free Full Text].
|
| 25.
|
Pessoa, M. G.,
N. A. Terrault,
J. Detmer,
J. Kolberg,
M. Collins,
H. M. Hassoba, and T. L. Wright.
1998.
Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic.
Hepatology
27:877-880[CrossRef][Medline].
|
| 26.
|
Pickering, J. M.,
H. C. Thomas, and P. Karayiannis.
1997.
Genetic diversity between hepatitis G virus isolates: analysis of nucleotide variation in the NS-3 and putative `core' peptide genes.
J. Gen. Virol.
78:53-60[Abstract].
|
| 27.
|
Radkowski, M.,
L.-F. Wang,
H. Vargas,
J. Rakela, and T. Laskus.
1998.
Lack of evidence for GB virus C/hepatitis G virus replication in peripheral mononuclear cells.
J. Hepatol.
28:179-183[Medline].
|
| 28.
|
Radkowski, M.,
L.-F. Wang,
J. Cianciara,
J. Rakela, and T. Laskus.
1999.
Analysis of hepatitis G virus/GB virus C quasispecies and replication sites in human subjects.
Biochem. Biophys. Res. Commun.
258:296-299[CrossRef][Medline].
|
| 29.
|
Saito, S.,
K. Tanaka,
M. Kondo,
K. Morita,
T. Kitamura,
T. Kiba,
K. Numata, and H. Sekihara.
1997.
Plus- and minus-stranded hepatitis G virus RNA in liver tissue and in peripheral blood mononuclear cells.
Biochem. Biophys. Res. Commun.
237:288-291[CrossRef][Medline].
|
| 30.
|
Seipp, S.,
M. Scheidel,
W. J. Hofmann,
U. Töx,
L. Theilmann,
T. Goeser, and B. Kallinowski.
1999.
Hepatotropism of GB virus C (GBV-C): GBV-C replication in human hepatocytes and cells of human hepatoma cell lines.
J. Hepatol.
30:570-579[CrossRef][Medline].
|
| 31.
|
Simons, J. N.,
T. P. Leary,
G. J. Dawson,
T. J. Pilot-Matias,
A. S. Muerhoff,
G. G. Schlauder,
S. M. Desai, and I. K. Mushahwar.
1995.
Isolation of novel virus-like sequences associated with human hepatitis.
Nat. Med.
1:564-569[CrossRef][Medline].
|
| 32.
|
Simons, J. N.,
T. J. Pilot-Matias,
T. P. Leary,
G. J. Dawson,
S. M. Desay,
G. G. Schlauder,
A. S. Muerhoff,
J. C. Erker,
S. L. Buijk,
M. L. Chalmers,
C. L. Van Sant, and I. K. Mushahwar.
1995.
Identification of two flavivirus-like genomes in the GB hepatitis agent.
Proc. Natl. Acad. Sci. USA
92:3401-3405[Abstract/Free Full Text].
|
| 33.
|
Suzuki, Y.,
K. Katayama,
S. Fukushi,
T. Kageyama,
A. Oya,
H. Okamura,
Y. Tanaka,
M. Mizokami, and T. Gojobori.
1999.
Slow evolutionary rate of GB virus C/hepatitis G virus.
J. Mol. Evol.
48:383-389[CrossRef][Medline].
|
| 34.
|
Tanaka, E.,
J. Alter,
Y. Nakatsuji,
W.-K. Shih,
J. P. Kim,
A. Matsumoto,
M. Kobayashi, and K. Kiyosawa.
1996.
Effect of hepatitis G virus infection on chronic hepatitis C.
Ann. Intern. Med.
125:772-773[Free Full Text].
|
| 35.
|
Thompson, J. D.,
T. J. Gibson,
F. Plewniak,
F. Jeanmougin, and D. G. Higgins.
1997.
The CLUSTAL-X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res.
25:4876-4882[Abstract/Free Full Text].
|
| 36.
|
Viazov, S.,
M. Riffelmann,
Y. Khoudyakov,
H. Fields,
C. Varenholz, and M. Roggendorf.
1997.
Genetic heterogeneity of hepatitis G virus isolates from different parts of the world.
J. Gen. Virol.
78:577-581[Abstract].
|
| 37.
|
Wang, J. T.,
J. C. Sheu,
J. T. Lin,
T. H. Wang, and D. S. Chen.
1992.
Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells.
J. Infect. Dis.
166:1167-1169[Medline].
|
| 38.
|
Wang, J. T.,
F. C. Tsai,
C.-Z. Lee,
P.-J. Chen,
J. C. Sheu,
T. H. Wang, and D. S. Chen.
1996.
A prospective study of transfusion-associated GB virus C infection: similar frequency but different clinical presentation compared with hepatitis C virus.
Blood
88:1881-1886[Abstract/Free Full Text].
|
| 39.
|
Yoshiba, M.,
H. Okamoto, and S. Mishiro.
1995.
Detection of the GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown aetiology.
Lancet
346:1131-1132[CrossRef][Medline].
|
Journal of Virology, September 2000, p. 7936-7942, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
