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Journal of Clinical Microbiology, January 1998, p. 255-257, Vol. 36, No. 1
Divisions of Clinical Virology,
Received 15 April 1997/Returned for modification 4 August
1997/Accepted 1 October 1997
We analyzed 224 and 163 serum samples from individuals in Sweden
and Honduras, respectively, for the presence of the hepatitis G virus
(HGV or GB virus-C) RNA. HGV infection in both Sweden and Honduras was
related to common risk factors for blood-borne infections, despite a
surprisingly high frequency in groups without known risk factors.
The recently identified hepatitis G
virus (HGV or GB virus-C [GBV-C]) (11, 19) has been found
worldwide by detection of HGV RNA by a reverse transcription-PCR
(RT-PCR) (3, 4, 9, 18). Similar to hepatitis B virus (HBV)
and hepatitis C virus (HCV), HGV seems to be transmitted by contact
with contaminated blood (11, 19) and vertically from mother
to child at partus (5). HGV RNA has been detected in serum,
saliva, and liver (2, 11, 13, 19). Interestingly, HGV
coinfections does not seem to enhance the pathology of chronic HCV
infection (1). A suggested relation between HGV and
fulminant hepatitis has been debated (6, 7, 10, 12, 17).
Thus, knowledge of HGV infection is still unclear with respect to both
transmission routes and any possible pathology.
We studied the prevalence and risk group correlations of HGV infection
in Sweden and Honduras using a total of 387 serum samples. Of 224 serum
samples from individuals residing in Sweden, 100 were from healthy
volunteers, 50 were from intravenous drug users (IVDUs), and 30 were
from homosexual men. Of 163 samples from individuals residing in
Honduras, 50 were from healthy university students, 10 were from female
sex workers, 18 were from homosexual men, 10 were from polytransfused
patients, and 17 were from hemophiliacs. Neither the Swedish nor the
Honduran healthy subjects had subjective or clinical signs of disease
at the time of sampling.
A total of 44 and 58 samples from patients with suspected viral
hepatitis of unknown origin were obtained from Sweden and Honduras,
respectively. The samples were all negative for immunoglobulin M (IgM)
antibodies to hepatitis A virus (Abbott Laboratories, Chicago, Ill.),
negative for HBV surface antigen (HBsAg; Abbott), and negative for
antibodies to HCV (Abbott). The Swedish samples were also serologically
negative for active cytomegalovirus and Epstein-Barr virus infections.
For all of these subjects, the clinical diagnosis excluded hepatitis of
other nonviral origin.
Total RNA was extracted from 100 µl of serum by guanidinium
extraction as described previously (2). cDNA synthesis was initiated by using reverse transcriptase (Boehringer Mannheim, Mannheim, Germany). HGV DNA was amplified by PCR of the 5' noncoding (5'-NC) region by using either of two different outer primer pairs followed by nesting with the same inner primer pair. The two outer pairs were as follows (locations are according to reference
11); sense HGV-1X (nucleotides [nts] 205 to 222;
5'-TTGTGCCTGCGGCGAGAC-3') and antisense HGV-2X (nts 533 to
516; 5'-AATGCCACCCGCCCTCAC-3') or sense HGV-1V (nts 147 to
166; 5'-ATTCCCGGTCACCTTGGTAGC-3') and antisense HGV-2V (nts
595 to 579; 5'-ATGGCCCCGGCCTCAAC-3'). The inner primer pair
has been described previously (2, 5). The nested PCR
amplifies a product of approximately 245 bp. All samples positive by
any of the 5'-NC region PCRs were subjected to a new RNA extraction and
were run with primers for the NS3 region for HGV as described
previously (2, 5, 22). The expected size of the product of
this PCR was 140 bp. For sequence analysis the product from the
first-round PCR (5 µl) was further amplified with biotinylated
primers from the NS3 region (22). The sequencing reaction
was carried out by using the Cy5 AutoRead Sequencing kit and an
ALFexpress DNA Sequencer (Pharmacia Biotech, Uppsala, Sweden).
Alignment and analysis of the NS3 region sequences was performed by
using the GeneWorks, version 2.3, software package (Intelligenetics,
Mountain View, Calif.), and simple dendrograms were constructed by the
unweighted pair group method with arithmetic averages (UPGMA)
(15) included in the GeneWorks software package.
Two sets of outer PCR primers from the 5'-NC region of the HGV genome
were used to analyze the prevalence of HGV viremia in serum samples
from Sweden and Honduras. Of a total of 387 samples, 27 (7%) samples
were positive for HGV RNA. HGV RNA was identified in one of the
Honduran samples with the 5'-NC region HGV-1V-HGV-2V primer set but
not the 5'-NC region HGV-1X-HGV-2X or NS3 primer set. Of the 26 (96%)
samples in which HGV RNA was detected with the 5'-NC region primer set
HGV-1X-HGV-2X, HGV RNA was also identified in 20 (74%) samples with
the NS3 primer set, indicating a lower sensitivity of the NS3-based PCR
(8, 14, 23).
HGV RNA was detected in 3 (3%) of 100 healthy Swedish volunteers and 1 (2%) of 50 healthy Honduran university students (Table 1). These frequencies are similar to
those from other studies of healthy subjects (11, 19). The
frequency of samples with HGV RNA was higher among the Swedish
IVDUs than among the Swedish healthy volunteers
(P < 0.0001; Fisher's exact test). No significant differences in the frequencies of detection of HGV RNA were found between the healthy volunteers and the homosexual men or patients with
suspected cases of non-A, non-C hepatitis. The finding may be
influenced by the low number of samples analyzed. Overall, our findings
are in line with those from other reports from European countries
(4, 18) and support a relation between HGV infection and
known risks for blood-borne infections.
Among the Honduran samples no difference in the frequency of HGV
viremia was found among the different groups (Table 1). There was a
trend of an elevated frequency of HGV viremia among the polytransfused
patients compared to that among the healthy university students
(P = 0.0693; Fisher's exact test).
Among the 27 HGV-RNA positive samples (21 Swedish and 6 Honduran), 20 were partially sequenced for the NS3 region and a limited analysis
confirmed an HGV origin for all samples (Fig.
1). Almost all the Swedish HGV strains
grouped with the two HGV prototype strains which are distinct from the
GBV-C prototype strain (20). Interestingly, HGV from two
Swedish HGV-infected IVDUs had NS3 sequences that contained five of the
six nucleotides of the motif which was found in HGV from German
patients with fulminant hepatitis (6).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Hepatitis G Virus RNA in Persons with
and without Known Risk Factors for Blood-Borne Viral Infections in
Sweden and Honduras
ABSTRACT
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TABLE 1.
Prevalence of HGV RNA in serum in relation to those of
serological markers for human immunodeficiency virus, HBV, and HCV in
different Swedish and Honduran populations with and without known
risk factors for blood-borne infections
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FIG. 1.
Alignment and phylogenetic analysis, using the UPGMA
algorithm, of 70-bp NS3 region sequences obtained from the Swedish
healthy population (NORM), IVDUs (DRUG), homosexual men (HS), and
patients suspected of having non-A, non-C hepatitis (NANC). The numbers
used with these abbreviations indicate individual subjects. Also
included in the analysis are NS3 sequences from three full-length HGV
genomes (GBV-C, HGV44402, and HGV45966) and, as an outgroup, an NS3
sequence from a full-length HCV genome (HCVJTNS3).
In the Swedish material we observed an elevated prevalence of HGV RNA in blood from groups at risk for blood-borne infections, such as IVDUs. The high prevalence of HGV RNA in the healthy population and homosexual men emphasizes the question of whether chronic HGV infection may be transmitted by sexual contacts (16, 21). Thus, chronic HGV infection might also be transmitted by social contacts or other routes yet to be defined.
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ACKNOWLEDGMENTS |
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We thank Rene Stefan and Lilian Garcia for kindly providing samples.
The study was supported by grants from the Swedish Medical Research Council, the Cancer Foundation, and Karolinska Institute Research and Training Programme for Central American Countries.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Clinical Virology, F 68, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Phone: 46-8-58587939. Fax: 46-8-58581305. E-mail: masa{at}vird01.hs.sll.se.
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Journal of Clinical Microbiology, January 1998, p. 255-257, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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