Also Called Hepatitis G Virus - Journal of Clinical Microbiology

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Foundation of Scientific and Industrial Research at the Norwegian Institute of Technology,3 Trondheim, Norway. Received 29 ... PCR-positive donors revealed no association with liver disease. Four of 12 ..... The scale bar indicates the number ...
JOURNAL OF CLINICAL MICROBIOLOGY, July 2000, p. 2584–2590 0095-1137/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 38, No. 7

Prevalence of GB Virus C (Also Called Hepatitis G Virus) Markers in Norwegian Blood Donors SVEIN ARNE NORDBØ,1* SIDSEL KROKSTAD,1 PER WINGE,2 FINN EGIL SKJELDESTAD,3 1 AND ARE B. DALEN 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%). 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.

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

MATERIALS AND METHODS 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. The study population completed a questionnaire regarding marital status, number of children, surgery, reception of blood products, acupuncture, tattoos, earrings, and travels outside Europe. All blood donors had tested negative for markers of HIV (anti-HIV antibodies), hepatitis B virus (HBsAg and anti-HBc antibodies), and HCV (anti-HCV antibodies) using commercial enzyme immunoassays. Plasma samples from 1,001 donors were collected and tested for GBV-C/HGV markers. All blood donors who tested positive by PCR were examined and retested. Analyses for serum

TABLE 1. GBV-C/HGV RT-PCR with primers from the NS5b region (PCR product size, 187 nucleotides) Oligonucleotide

Sequence

Location

Sense primer Antisense primer Probea

5⬘-ATG GGT TAC ACT TAT GAG GAA-3⬘ 5⬘-AGA ACA CCT CCT TTT TCA CAG-3⬘ 5⬘-GGG CTG GGG ATC TAA GGT GTC GGT-3⬘

7629–7649 7795–7815 7682–7705

* 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 @medisin.ntnu.no. 2584

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TABLE 2. GBV-C/HGV RT-PCR used for sequencing of the NS5b region (PCR product size, 489 nucleotides) Oligonucleotide

Sequence

Location

Sense primer Antisense primer Sequencing primer

5⬘-AAC CAT ACA GCC TAT TGT GAC-3⬘ 5⬘-AGA ACA CCT CCT TTT TCA CAG-3⬘ 5⬘-TGG TGG GCA ATG AAC TTA CCT-3⬘

7269–7289 7795–7815 7327–7347

alanine aminotransferase (ALT), hemoglobin, C-reactive protein (CRP), and leukocyte counts were also performed. A third sample was collected from all patients who tested negative by PCR in the second sample. Two of the 25 GBV-C/HGV RNA-positive participants had no partner. Sera from 12 couples were obtained and tested for HGV markers. Samples from PCR-positive couples were sequenced and compared. 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 ␮l 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/␮l) 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 ␮l, 0.8 U of RNasin (Promega) per ␮l, 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 ␮l. 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 (XOMAT; Kodak, Rochester, N.Y.) after 90 min of incubation at room temperature. 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). Molecular evolutionary genomic analysis. Twelve sequences from the NS5 region were manually aligned together with that of HGV isolate PNF2161 (23).

TABLE 3. Laboratory results from 25 GBV-C/HGV RNA-positive blood donorsa Donor no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Gender

Female Female Male Female Male Male Male Male Female Male Male Male Female Male Male Male Male Female Male Male Female Female Male Female Male

Age (yr)

49 63 45 46 43 44 55 50 35 38 57 42 49 36 35 39 47 34 40 51 36 34 35 41 33

Hemoglobin (g/100 ml)

Leukocytes (109/liter)

HGV/GBV-C PCR

Anti-HGV E2 ELISA

ALT (IU/liter)

CRP (mg/liter)

1997

1998

1997

1998

1997

1998

1997

1998

1997

1998

1997

1998

9 24 21 15 16 30 29 20 58 36 27 12 21 24 31 38 17 12

1 20 21 37 26 28 20

⬍5 7.0 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5

18 ⬍5 ⬍5 5 ⬍5 ⬍5

12.5 13.5 14.4 12.1 15.2 16.4 13.8 13.1 12.6 15.1 13.9 12.8 13.4 16.8 14.2 14.1 14.1 12.8

13.9 14.3 14.7 13.2 15.9

8.0 4.6 7.0 6.2 4.3 4.9 5.9 5.7 5.7 4.0 4.7 3.6 8.8 5.2 6.5 5.2 4.0 4.2

8.8 5 6.9 10.8 4.9 5.8

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Pos Pos Pos Neg Neg Neg Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Pos Neg Neg

26 15 15 18 16 20

42 24 29 15 22 10 45 30 12 14 15 13 18 33

⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5 31

⬍5 ⬍5 ⬍5 ⬍5

⬍5 36 ⬍5 ⬍5 ⬍5 ⬍5 ⬍5

13.1 12.8 12.3 13.8 13.1 14.5

13.9 15.3 14.7 14.1

15.6 13.9 12.8

11.6 13.3 15

5.6 6.1 6.1 4.0 4.7 5.6

6.4 5.1 5.9 4.6 6.1 7.1 4.2 6.3

6.8 4.5 6.3

Pos Pos Pos Pos Pos Neg Pos Pos Pos Pos Pos Pos Neg Neg

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos

a Reference values: ALT, 0 to 35 IU/liter (females) and 0 to 50 IU/liter (males); CRP, 0 to 5 mg/liter; hemoglobin, 11.5 to 15.5 g/100 ml (females) and 13.5 to 17.4 g/100 ml (males); leukocytes, 3.7 ⫻ 109 to 10.0 0 ⫻ 109/liter (females) and 3.7 ⫻ 109 to 10.0 ⫻ 109/liter (males). Pos, positive; Neg, negative.

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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 multiplesequence 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.

RESULTS 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. One year later, 71% (15 of 21) of the GBV-C/HGV RNApositive donors were still PCR positive (four of the PCRpositive blood donors were lost for follow-up). Fifty percent (3 of 6) of the donors who had become PCR negative were antiHGV E2 positive. The distributions of ABO blood types and rhesus status are displayed in Table 4. There were no significant differences in the prevalence of these markers between the PCR-positive group and the ELISA-positive group. Nine hundred forty-two blood donors returned the questionnaire. A complete data set for 629 responders exists. The results are listed in Table 4. There was a significantly (P ⬍ 0.001) higher percentage (16.5%) of individuals who had been treated by acupuncture than individuals who had not been given this treatment (9.4%) among ELISA-positive donors. Regarding population characteristics and possible risk factors, no other significant difference was found between ELISA- or PCR-negative and -positive participants. Four out of 12 partners (33%) of PCR-positive blood donors were GBV-C/HGV RNA positive, and another four partners (33%) had antibodies against HGV E2 protein. The results of the nucleic acid sequencing of the GBV-C/HGV-RNA positive partners are shown in Fig. 1. The similarities between se-

J. CLIN. MICROBIOL. TABLE 4. Characteristics of blood donors (n ⫽ 1,001) tested for HGV markers Parameter

n

% PCR positive

ELISA positive

Age (yr) 19–35 23–44 45–51 52–70

236 297 237 231

2.5 3.0 3.0 1.3

6.4 11.8 12.7 10.8

Gender Male Female

547 454

2.9 2.0

9.9 11.2

Blood type O A B AB

519 369 84 29

2.3 2.7 1.2 6.9

11.4 8.4 11.9 17.2

Anti-D Negative Positive

395 606

2.5 2.5

8.9 11.6

Marital status Not married Married Unknown

328 606 67

4.3 1.8 0

13.4 8.9 10.4

Children None One or more Unknown

162 779 60

0.6 3.1 0

13.0 10.0 10.0

Hospitalization No Yes Unknown

315 613 73

1.9 3.1 0

7.6 11.9 11.0

Surgery No Yes Unknown

406 534 61

1.7 3.4 0

10.1 10.9 9.8

Blood transfusion No Yes Unknown

759 45 197

2.6 4.4 1.5

10.3 8.9 11.7

Anti-D immunization No Yes Unknown

733 93 175

2.9 2.2 1.1

10.9 7.5 10.3

Gamma globulin No Yes Unknown

693 189 119

2.7 2.6 0.8

10.1 11.6 10.9

Travel outside Europe No Yes Unknown

662 278 61

2.6 2.9 0

9.8 12.2 9.8

Acupuncture No Yes Unknown

794 139 68

2.4 2.2 4.4

9.4a 16.5a 10.3

Earring No Yes Unknown

519 414 68

2.5 2.2 4.4

10.2 10.9 10.3

Tattoo No Yes Unknown

897 36 68

2.5 0 4.4

10.1 19.4 10.3

a

Significant statistical differences (P ⬍ 0.05).

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FIG. 1. Alignment of nucleotide sequences from the NS5b regions of GBV-C/HGV strains from four couples compared to that of strain U44402 (from GenBank).

quences of a 320-nucleotide fragment from the NS5b region for each couple were 99.2, 99.7, 100, and 93.1%, respectively. Phylogenetic analysis (Fig. 2) showed that the four couples were infected with different strains but that each couple probably had identical strains. These strains were different from strain PNF2161 and the other isolates. Strains from the three recipients showed 100% identity with the isolate from blood donor N (Fig. 2). 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). Eleven cases of liver disorders were recorded, with six belonging to the group of other diseases, four in the group of gastrointestinal diseases, and one in the group of cardiovascular diseases. Nine of these cases were patients with liver cirrhosis, and there was one case of neonatal icterus and only one case of hepatitis of unknown origin. Five cases of liver cirrhosis were caused by long-term alcohol abuse, and two cases were

recorded as liver cirrhosis of unknown etiology. In addition, there was one case of primary biliary cirrhosis and one case of cirrhosis due to cardiac failure. In only three cases was the diagnosis of liver disease recorded after the first transfusion from PCR-positive donors. One patient had liver cirrhosis of unknown origin and died from myocardial infarction 2 years after the transfusion. Another patient with liver cirrhosis of unknown origin died from massive variceal hemorrhage due to portal hypertension only a few months after the transfusion. The third patient developed portal hypertension and cirrhosis due to cardiac failure caused by a growing myxoma in the right atrium and died from acute appendicitis with peritonitis 4 years after the transfusion. DISCUSSION 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).

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FIG. 2. Phylogenetic tree of 12 GBV-C/HGV sequences from the NS5 region aligned together with that of HGV isolate PNF2161. Strains from four PCRpositive couples, one blood donor not included in the study (donor N), and three recipients of blood from this donor were sequenced and analyzed. The scale bar indicates the number of nucleotide substitutions per position.

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

TABLE 5. Main groups of most recent recorded diagnoses of recipients (n ⫽ 401) of blood products from GBV-C/HGV RNA-positive blood donors Group of diseases

n

Cardiovascular diseases ..........................................................................95 Malignant tumors ....................................................................................69 Orthopedic conditions ............................................................................57 Lymphomas and leukemia .....................................................................36 Gastrointestinal diseases ........................................................................32 Infectious diseases...................................................................................15 Various blood disorders .........................................................................10 Diseases of the nervous system ............................................................. 8 Pulmonary diseases ................................................................................. 3 Other diseases..........................................................................................76

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 RNApositive 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 GBVC/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 PCRpositive 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 PCRpositive 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

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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. ACKNOWLEDGMENTS This study was partly supported by grants from the Norwegian Board of Health. We thank Ann-Charlotte Åstro ¨m 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. REFERENCES 1. Alter, H. J., Y. Nakatsuji, J. Melpolder, J. Wages, R. Wesley, J. W. 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. 2. Andonov, A., C. Sauder, H. Jacobsen, and R. Chaudhary. 1998. Comparison of six sets of PCR primers from two different genomic regions for amplification of GB virus C/hepatitis G virus RNA. J. Clin. Microbiol. 36:286–289. 3. Bjo ¨rkman, P., G. Sundstro¨m, and A. Widell. 1998. Hepatitis C virus and GB virus C/hepatitis G virus viremia in Swedish blood donors with different alanine aminotransferase levels. Transfusion 38:378–384. 4. Blair, C. S., F. Davidson, C. Lycett, D. M. McDonald, G. H. Haydon, P. L. Yap, P. C. Hayes, P. Simmonds, and J. Gillon. 1998. Prevalence, incidence, and clinical characteristics of hepatitis G virus/GB virus C infection in Scottish blood donors. J. Infect. Dis. 178:1779–1782. 5. Fan, X., Y. Xu, H. Solomon, S. Ramrakhiani, B. A. Neuschwander-Tetri, and A. M. Di Bisceglie. 1999. Is hepatitis G/GB virus-C virus hepatotropic? Detection of hepatitis G/GB virus-C viral RNA in liver and serum. J. Med. Virol. 58:160–164. 6. Felsenstein, J. 1984. Distance methods for inferring phylogenies: a justification. Evolution 38:16–24. 7. Feucht, H. H., B. Zollner, S. Polywka, B. Knodler, M. Schroter, H. Nolte, and R. Laufs. 1997. Distribution of hepatitis G viremia and antibody response to recombinant proteins with special regard to risk factors in 709 patients. Hepatology 26:491–494.

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