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Journal of Clinical Microbiology, July 2000, p. 2584-2590, Vol. 38, No. 7
Department of Microbiology, University
Hospital of Trondheim,1 UNIGEN, Center
for Molecular Biology,2 and The
Foundation of Scientific and Industrial Research at the Norwegian
Institute of Technology,3 Trondheim, Norway
Received 29 November 1999/Returned for modification 22 February
2000/Accepted 24 April 2000
GB virus C (GBV-C), also called hepatitis G virus (HGV), occurs
worldwide, but the clinical significance of this virus is still
unclear. Plasma samples from 1,001 blood donors were tested by reverse
transcription PCR using primers from the NS5 region and by a commercial
enzyme-linked immunosorbent assay (ELISA) for the detection of
immunoglobulin G antibodies against the putative envelope of HGV
(anti-HGV E2). GBV-C/HGV RNA was present in the plasma from 2.5% of
the blood donors, and anti-HGV E2 antibodies could be detected in
10.5% of the samples. Only one of the blood donors with viremia had
elevated levels of alanine aminotransferase. Among ELISA-positive
donors, there was a significantly higher percentage (16.5%) of
individuals who had been treated by acupuncture than individuals who
had not been given this treatment (9.4%). No other variables showed
significant differences. Screening of medical records from 401 recipients of blood from PCR-positive donors revealed no association
with liver disease. Four of 12 partners (33%) were HGV RNA positive,
and sequence analyses of the strains showed that four of the couples
probably were infected with the same strains, while strains from
different couples were not identical. Anti-HGV E2 antibodies were
detected in serum samples from four other partners. The prevalence of
GBV-C/HGV among blood donors in our region is dramatically higher than
the prevalence of hepatitis C virus (0.03%).
The recently discovered GB virus C
(GBV-C), also called hepatitis G virus (HGV), belongs to the family
Flaviviridae, which also includes hepatitis C virus (HCV)
(17, 23, 33). The virus has a worldwide distribution with
various prevalences in different blood donor populations, ranging from
0.9% (25) to 14.6% (19). The clinical
significance of this agent is still uncertain, although the possibility
of it having a role in fulminant hepatitis (10, 12) and
aplastic anemia (26; J. J. Byrnes, A. T. Banks, M. Piatack, Jr., and J. P. Kim, Letter, Lancet
348:472, 1996) has been debated. GBV-C/HGV has been shown to
replicate in peripheral blood mononuclear cells both in vivo and in
vitro (9, 18). Several studies suggest that this virus does
not replicate in hepatocytes (5, 11, 15, 21), but
investigators also have reported replication in liver cells (30,
32).
GBV-C/HGV is parenterally transmissible (1), and
coinfections with hepatitis B virus and HCV are common (14).
The virus is more frequently transmitted to infants than human
immunodeficiency virus or HCV (35). Transmission of this
agent is associated with high-titer viremia and mode of delivery
(16). Sexual transmission has also been suggested
(22). However, little is known about other modes of
transmission that could explain the high prevalence and worldwide
distribution of this virus.
Our aims in this study were to determine the prevalence of GBV-C/HGV
markers in blood donors, explore risk factors, and investigate the
clinical consequences for patients who had received blood products from
carriers of this virus.
Blood donors and partners.
From March 1997 until July
1997 a total of 1,002 established blood donors attending the blood
bank at the University Hospital of Trondheim were consecutively
selected for screening of GBV-C/HGV markers and asked to participate.
Only 1 out of 1,002 responders refused to participate, and 1,001 blood
donors gave their written consent to take part in this study. The study
was approved by the Regional Committee of Medical Research Ethics,
Region IV, Norway.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Prevalence of GB Virus C (Also Called Hepatitis G
Virus) Markers in Norwegian Blood Donors
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Recipients. In the period from 1962 to 1997, the PCR-positive donors had given 697 donations of blood (2 to 77 donations per donor), and 536 of the recipients were identified. At our hospital, medical records from 401 (75%) of these recipients were traced and reviewed. All diagnoses made after the recipients had received the first transfusion from the PCR-positive donors were recorded. Many of the recipients were multitransfused, with the largest amount being 276 units. Twelve recipients had received blood products from two donors known to be GBV-C/HGV RNA positive, and one recipient had received blood from three positive donors.
RNA extraction.
Total RNA was extracted from 250 µl of
EDTA-anticoagulated plasma or serum (stored samples) with TRIzol LS
Reagent (Life Technologies, Grand Island, N.Y.), followed by organic
extraction with chloroform, precipitation with isopropanol-ethanol, and
addition of an RNase inhibitor (RNasin; Promega, Madison, Wis.) (1 U/µl) and dithiothreitol (Life Technologies, Gaithersburg, Md.) (2 mM). The RNA extraction was performed within 6 h after the
donation. All specimens were kept frozen at 
70°C until tested.
GBV-C/HGV RT-PCR.
An in-house reverse transcriptase PCR
(RT-PCR) with primers and probe from the NS5b region (Table
1) was used to detect specific nucleotide
sequences of the GBV-C/HGV genome. Ten microliters of the RNA from each
specimen was used in a single-tube RT-PCR. Negative and positive
controls were included in each run. The reaction mixture consisted of
40 mM KCl, 16 mM Tris-HCl (pH 8.83), 1.2 mM MgCl2, 0.2%
NP-40, 0.5 mM dithiothreitol, 100 mM each deoxynucleoside triphosphate
(Life Technologies, Gaithersburg, Md.), 1× first strand buffer (Life
Technologies, Gaithersburg, Md.), 2.0 U of Moloney murine leukemia
virus RT (Life Technologies, Gaithersburg, Md.) per µl, 0.8 U of
RNasin (Promega) per µl, 0.6 pmol of each primer, and 1 U of AmpliTaq
Gold polymerase (Perkin-Elmer/Roche Molecular Systems Inc., Branchburg,
N.J.) in a final volume of 50 µl. Reverse transcription was completed
during the first step (37°C for 60 min) and was followed by enzyme
inactivation of RT and activation of AmpliTaq Gold polymerase at 94°C
for 15 min. Cycling conditions for amplification were 35 cycles of
94°C for 60 s, 55°C for 90 s, and 72°C for 120 s.
The amplicons (187 nucleotides) were detected by gel electrophoresis on
an ethidium bromide-stained 2% agarose gel (Sigma, St. Louis, Mo.)
under UV light. Twenty microliters of the amplified product was also
applied to a GeneScreen Plus nylon membrane (Dupont, Boston, Mass.) by
a slot blot procedure. The filter was prehybridized with 30 ml of
buffer containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate), 0.5% sodium dodecyl sulfate (SDS), 0.1% bovine serum
albumin (BSA) (Sigma), 0.1% Ficoll (Pharmacia, Uppsala, Sweden), and
0.1% polyvinylpyrrolidone (Sigma) for 1.5 h at 50°C and then
hybridized using the alkaline phosphatase-conjugated probe in fresh
prehybridization solution to a final concentration of 1.0 nM at 50°C
for 20 min. The filter was washed twice in washing buffer containing
1× SSC and 0.5% SDS for 5 min at 50°C and then twice in washing
buffer with 0.25× SSC and 0.5% SDS for 5 min at 50°C. After the
filter was rinsed in 2× SSC for 5 min at room temperature, CSPD
chemiluminiscence substrate (Tropix Inc., Bedford, Mass.) was added and
the results were visualized on films for autoradiography (X-OMAT;
Kodak, Rochester, N.Y.) after 90 min of incubation at room temperature.
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Sequence analysis.
The primers used for PCR and sequencing
are described in Table 2. The PCR
products were purified with QIAquick PCR purification kit (Qiagen) and
sequenced directly on an ABI 373 DNA sequencer using an ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems,
Warrington, Great Britain). Alignment analysis of the sequences was
performed using the program Sequence Navigator (PE Applied Biosystems).
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Molecular evolutionary genomic analysis. Twelve sequences from the NS5 region were manually aligned together with that of HGV isolate PNF2161 (23). Eight sequences were from PCR-positive couples, one was from a blood donor not encluded in the study (blood donor N in Fig. 2), and three were from patients who had received blood from this donor. The samples from the PCR-positive recipients were collected 185 to 425 days after the transfusion. A multiple-sequence file was made with the GeneDoc program, version 2.5. Using the DNADIST program, a distance matrix was calculated with the maximum-likelihood method. A tree file was produced with the Fitch program, which uses an algorithm based on the Fitch-Margoliash criterion (8). Both programs are from the PHYLIP program package, version 3.5c (6). The tree was rooted with isolate PNF2161, and a cladogram was produced with the TreView program, version 1.6.1 (27).
HGV E2 antibody testing. All of the samples were tested with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Anti-HGenv EIA; Boehringer GmbH, Mannheim, Germany) for the detection of specific antibodies against HGV E2 protein (34). Reactive specimens were retested in duplicate with and without HGV E2 antigen added according to the instructions of the manufacturer.
Statistical analysis. Data were processed through the SPSS (Chicago, Ill.) data package. All analyses were done with the chi-square test or the Mann-Whitney test.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Donors and partners. The mean age of the study population was 43.3 years (547 men and 454 women; age range, 19 to 70 years). GBV-C/HGV RNA was detected in plasma samples from 25 blood donors with mean a age of 43.1 years (9 females and 16 males; age range, 33 to 63 years). Plasma samples from two of these donors were negative when retested by PCR. However, both donors tested positive again by PCR in the third sample.
All but one of the PCR-positive blood donors (no. 9 in Table 3) had normal ALT levels. However, this donor had only a slightly elevated ALT level (42 to 58 IU/liter), and there was no history of liver disease. Two of the blood donors (no. 2 and 25) had elevated CRP levels for unknown reasons, but they showed normal values (<5 mg/liter) 1 year later. None of the female donors were anemic, but three of the male donors (no. 8, 12, and 20) had subnormal hemoglobin values (12.8 to 13.1 g/100 ml). All PCR-positive participants tested had normal leukocyte counts. Plasma samples from 116 blood donors were initially positive by the anti-HGV E2 ELISA. All of these specimens were GBV-C/HGV PCR negative. By repeat testing, 105 of these specimens were confirmed positive. Fifty-one females and 54 males (mean age, 44.9 years; range, 22 to 66 years) were anti-HGV E2 ELISA positive.
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Recipients.
The groups of the most recently recorded main
diagnoses of the recipients (n = 401) are listed in
Table 5. The mean time between the
transfusion and the most recent recorded main diagnosis was 825 days
(median time, 181 days; range, 0 to 9,218 days).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, GBV-C/HGV was detected in plasma samples from 2.5% of Norwegian blood donors. This is in agreement with other studies of blood donors in Europe, with results ranging from 1.3 to 4.2% (3, 4, 7, 24, 29).
The prevalence of GBV-C/HGV viremia reported will vary not only due to the actual prevalence in the population examined but also due to the storage condition of the samples, the choice of RNA extraction method used, and the design of the RT-PCR. The 5' noncoding region of the GBV-C/HGV genome contains several well-conserved regions that should be ideal for GBV-C/HGV RNA detection (20). However, even if the sequence variabilities within the NS3 and NS5 regions are greater than the 5' noncoding region of the GBV-C/HGV genome, practical studies have shown that there are no obvious differences in efficacy between the amplification targets used (2, 28). In our hands, an in-house RT-PCR based on primers from the NS5b region yielded a higher detection rate than several other published primers from the 5' noncoding region (data not shown). To increase the sensitivity and specificity of the in-house RT-PCR, all amplicons were subjected to probe hybridization. In our study, 2 out of 25 GBV-C/HGV RNA-positive cases would have been lost if amplicon detection had been based on gel electrophoresis only. However, subsequently collected plasma samples from these donors and the two donors that tested negative by PCR in the second sample were positive by gel electrophoresis after amplification. These results may be due to fluctuations in the GBV-C/HGV RNA level in plasma over time, enzyme inhibitors in the samples, or loss of RNA during extraction. Since our group of blood donors was a highly selected group of individuals with a very low risk of being carriers of blood-borne agents, the overall carrier rate in the Norwegian population is probably higher than 2.5%.
Among the first 5,914 blood donors tested for anti-HCV in Trondheim, sera from 28 blood donors (0.47%) were repeatedly reactive in a first-generation anti-HCV ELISA. However, only two donors (0.03%) were confirmed positive by recombinant immunoblot assay (RIBA) and PCR. Hence, the vast difference between the rates of GBV-C/HGV and HCV carriage in our blood donor population (2.5 versus 0.03%) indicates that there are important routes of transmission of GBV-C/HGV other than parenteral.
As expected, the mean age of the anti-HGV E2-positive blood donors was higher than the mean age of the PCR-positive donors (44.9 versus 43.1 years), but the difference was not statistically significant.
The sensitivity and specificity of the anti-HGV E2 ELISA are unknown. There are no supplementary tests using different antigens that are commercially available. Taking into account that the vast majority of sera (26 out of 28) that tested positive by the first-generation anti-HCV ELISA were positive due to unspecific reactions, we have reason to believe that the use of the anti-HGV E2 ELISA without retesting the sera by a supplementary test will overestimate the number of individuals who have seroconverted, even if the sensitivity of the test is less than 100%.
In our study, six persons became PCR negative after 1 year. Three of them had seroconverted, and three of them were both PCR and ELISA negative. It may be that the last group did not react by ELISA due to complex formation between the virus and the antibodies. Another explanation may be that the viral RNA load was below the detection limit of the PCR or that the antibodies produced were not detected by the particular E2 antigen used in the ELISA. In our study, more males (2.9%) than females (2.0%) tested positive by PCR. On the other hand, relatively more females (11.2%) than males (9.9%) were anti-HGV E2 positive. However, these differences were not statistically significant.
GBV-C/HGV can be transmitted parenterally, and by using our primers for sequencing of the NS5b region, we have observed 100% sequence identity between strains from PCR-positive donors and recipients (Fig. 2).
It has been documented that HCV and GBV-C/HGV can be transmitted through contaminated anti-D batches (31). In our study, we have no indications that any of the PCR-positive blood donors have been infected this way.
In our study there was a significantly higher proportion of anti-HGV E2-positive blood donors who had been treated with acupuncture than in the group that had not received this treatment. No such differences were seen in the PCR-positive group. One can speculate that this treatment was given under poorer hygienic conditions than in the professional health care system and that exposure to very small amounts of virus reduced the overall time of seroconversion. The same tendency was also observed in the group with tattoos, but the difference was not statistically significant. However, since the specificity of the anti-HGV E2 test has not been proven, these data should be interpreted with great care.
Only one PCR-positive blood donor had a slightly elevated ALT level, but there was no history of liver disease. All donors tested had normal leukocyte counts, and only three male donors had subnormal hemoglobin values. No cases of severe anemia were found.
The prevalence of GBV-C/HGV markers among partners of the carriers (66.7%) was much higher than the prevalence among blood donors (13.0%), but the number was to small to be subjected to statistical analysis.
Strains from three of the four PCR-positive partners showed identity of between 99.2 and 100% of the sequence from the chosen part of the NS5b region of the GBV-C/HGV genome. Strains from the fourth couple showed less identity (93.1%), but according to the phylogenetic analysis (Fig. 2), each couple probably had strains of the same origin. Sequential serum samples from one of the partners over a period of 6 years showed 100% identity within the sequenced region. We therefore believe that this region of the genome is suitable for epidemiological investigations (13).
Among the 401 recipients of blood from PCR-positive donors, only 3 out of 11 cases of liver disorders were recorded after the transfusions. All three patients had diagnosed liver cirrhosis. One patient developed cirrhosis due to cardiac failure, and two other patients died within 2 years after the transfusions. It is therefore very unlikely that GBV-C/HGV infection could have caused any of these conditions. Only long-term prospective studies will give a final answer regarding the potential pathogenicity of this virus.
Parenteral and vertical transmission of GBV-C/HGV is well documented. Our study strongly supports the hypothesis that the sexual route may be a significant way of transmission. Other modes of transmission are possible but poorly understood.
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ACKNOWLEDGMENTS |
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This study was partly supported by grants from the Norwegian Board of Health.
We thank Ann-Charlotte Åström at the Department of Microbiology for technical assistance and Ellen Berg and the staff at the Department of Immunology and Blood Bank for their cooperation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University Hospital of Trondheim, N-7006 Trondheim, Norway. Phone: 47 73 86 74 70. Fax: 47 73 86 77 65. E-mail: Svein.A.Nordbo{at}medisin.ntnu.no.
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Abstract
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Materials and Methods
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Discussion
References
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Journal of Clinical Microbiology, July 2000, p. 2584-2590, Vol. 38, No. 7
0095-1137/00/$04.00+0
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