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Journal of Clinical Microbiology, January 1998, p. 110-114, Vol. 36, No. 1
Second Department of Internal Medicine,
Shinshu University School of Medicine, Matsumoto 390, Japan1;
Boehringer Mannheim GmbH, 82377 Penzberg, Germany2; and
Department of
Transfusion Medicine, National Institutes of Health, Bethesda, Maryland
208923
Received 14 July 1997/Returned for modification 22 September
1997/Accepted 8 October 1997
We reported previously on an area in Japan where over 30% of the
inhabitants were positive for hepatitis C virus (HCV) antibody. In the
present study, clinical features of hepatitis G virus (HGV) infection
in this area of high endemicity were compared to those in an area where
HCV is not endemic. A total of 400 individuals were selected randomly
from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the
no-endemicity area. HGV RNA was measured by reverse transcription and
PCR with primers in the 5' noncoding region. Antibody to HGV envelope
protein E2 was measured by an enzyme-linked immunosorbent assay.
Prevalence of any HGV marker in the high-endemicity area (32%) was
significantly (P < 0.0001) higher than that in the
no-endemicity area (6%); similar differences, 32% versus 3%
(P < 0.0001), had been observed for HCV markers (HCV
RNA and HCV antibody). In areas of both high and no endemicity, HCV
markers were significantly more prevalent in individuals with any HGV
marker than in those without HGV markers, and age-specific prevalence
of HGV markers was distributed similarly to that of any HCV marker.
Among possible routes of HGV transmission that were analyzed, folk
medicine was significant in the high-endemicity area, but blood
transfusion was the major route in the no-endemicity area. The rate of
accompanying viremia in HGV infection (15%) was significantly lower
than that in HCV infection (78%) (P < 0.0001). In
conclusion, HGV infection was highly prevalent in the area of high HCV
endemicity and was closely associated with HCV infection. HGV seemed to
be transmitted via the practice of folk medicine as well as blood
transfusion. HGV resulted in a chronic carrier state less frequently
than did HCV.
The GB virus C and the hepatitis G
virus (HGV) were identified recently as possible causative agents of
human viral hepatitis (12, 17). Molecular characterization
of these two agents has shown them to be closely related strains of the
same virus, and they are supposed to represent a new genus in the
family Flaviviridae (3). As the nomenclature of
the new virus has not been settled, the term HGV is used in this paper.
HGV, like hepatitis C virus (HCV), is transmissible through blood
transfusion and is associated with acute and chronic infections
(4, 5, 15, 22, 24). Studies on HGV have depended on the
measurement of HGV RNA in serum, which reflects active HGV infection.
Recently, an assay for antibody to HGV envelope protein E2 (HGV-E2
antibody), which indicates recovery from HGV infection, has been
developed (6, 16, 18, 19). The combined use of these assays
has allowed for more comprehensive epidemiological studies of both past
and present HGV infection.
We previously reported on an area in which HCV is highly endemic, where
over 30% of the inhabitants were infected with HCV (10). In
that study, analyses of risk factors for HCV infection elucidated
inapparent modes of parenteral transmission, particularly folk medicine
procedures. In the present study, we determined the prevalence and
patterns of HGV infection in areas of high and low HCV endemicity to
compare the transmission patterns of these two common
Flaviviridae infections.
Patients.
A total of 420 individuals over 18 years old (62%
of total inhabitants with corresponding ages) in an area in which HCV
infection was endemic were medically screened for liver diseases in
July 1993. Of those, the first 200 individuals who prepared for
screening were selected randomly for evaluation in this study. Those
subjects included 79 males and 121 females aged 18 to 84 years
(mean ± standard deviation [SD], 56.3 ± 17.7 years).
Medical screening was also conducted in an area in which HCV was not
endemic and which is located near the high-endemicity area. Of 482 individuals (65% of total inhabitants with corresponding ages) who
underwent medical screening in the no-endemicity area, 200 individuals
were selected randomly for evaluation in the same manner as in the high-endemicity area. These control subjects included 48 males and 152 females aged 20 to 89 years (mean ± SD, 56.8 ± 13.4 years).
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Copyright © 1998, American Society for Microbiology. All rights reserved.
Past and Present Hepatitis G Virus Infections in
Areas Where Hepatitis C is Highly Endemic and Those Where It Is
Not Endemic
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until assayed.
Laboratory tests. Second-generation HCV antibody, hepatitis B surface (HBs) antigen, HBs antibody, and hepatitis B core (HBc) antibody were detected with commercially available enzyme-linked immunosorbent assay kits (International Reagents Co., Kobe, Japan).
Alanine aminotransferase (ALT) (normal range, 7 to 45 IU/liter) was measured on a multichannel autoanalyzer.Measurement of HCV RNA in serum. RNA extraction and reverse transcription (RT) were carried out in 100 µl of serum. The serum HCV RNA was measured by a nested RT-PCR with primers targeting the 5' noncoding region (14). Procedures to avoid contamination of samples were implemented throughout the study (11). In each PCR assay, two negative controls and one positive control of 10 copies/ml were tested in addition to the samples of interest.
Measurement of HGV RNA in serum. HGV RNA in serum was detected by nested RT-PCR using primers in the 5' noncoding region as described previously (21). Briefly, total RNA was extracted from 100-µl serum samples. After RT with Moloney murine leukemia virus reverse transcriptase, the first 30 cycles and then the second 30 cycles of PCR were performed (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min). PCR products were analyzed by gel electrophoresis with 3% agarose. In each PCR assay, two negative controls and one positive control of 10 copies/ml (15) were tested in addition to the samples of interest.
In the RT-PCR assays for HCV and HGV RNAs, all negative controls were negative and all positive controls were positive.Measurement of HGV-E2 antibody in serum. HGV-E2 antibody was measured by an enzyme-linked immunosorbent assay described previously (18, 19) in which recombinant E2 protein was bound to a microtiter plate. After addition of diluted serum samples, specifically bound antibodies against E2 protein were detected with an anti-human immunoglobulin G conjugated with peroxidase. Positive or negative results were judged as reported previously (18, 19).
Statistical analysis. Statistical analyses were performed with Student's t test, the chi-square test, and Fisher's exact test. A significance level was set at a P value of 0.05.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Backgrounds and viral markers in areas of endemicity versus areas of no endemicity. Clinical and virological features of the 200 individuals in the high-endemicity area were compared to those of the 200 individuals in the no-endemicity area (Table 1). A history of folk remedies was significantly more prevalent in the high-endemicity area than in the no-endemicity area, while histories of surgery and blood transfusion were similar in the two areas. Prevalence of HGV-related markers was significantly higher in the area of endemicity than in the no-endemicity area, as was observed for HCV-related markers. Prevalence of HBs antigen did not differ between the two areas, but that of any hepatitis B virus (HBV) marker was significantly higher in the area of endemicity. Of the 400 subjects, 75 (19%) were positive for HGV RNA and/or HGV-E2 antibody, 7 (9%) were positive for HGV RNA only, 4 (5%) were positive for both HGV RNA and HGV-E2 antibody, and 64 (86%) were positive for HGV-E2 antibody only.
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Age-specific prevalence. Age-specific prevalences of hepatitis viruses in the high-endemicity and no-endemicity areas are shown in Fig. 1. Individuals who had a marker indicating the existence of viremia were defined as having ongoing infection, the presence of HBs antigen was defined as indicating HBV infection, the presence of HCV RNA was defined as indicating HCV infection, and the presence of HGV RNA was defined as indicating HGV infection. On the other hand, individuals who had antibody in the absence of viremia were considered to have resolved or past infection. Age-specific prevalences of total infection (viremia plus antibody) were similar for HBV, HCV, and HGV in the high-endemicity area. The prevalence was around 10% in groups under 50 years old and around 40% in groups over 50 years old. This difference in distribution between groups under and over 50 was statistically significant (chi-square test) for each hepatitis virus: 10% versus 42% for HBV (P < 0.0001), 8% versus 42% for HCV (P < 0.0001), and 10% versus 41% for HGV (P < 0.0001). In the no-endemicity area, the prevalence did not differ between the two age groups for either HBV, HCV, or HGV.
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Current versus past infection. To analyze the proportion of present HGV infections to total HGV infections, cases in the high- and no-endemicity areas were combined, because the proportions were similar in each area for each hepatitis virus (6% versus 5% for HBV, 79% versus 60% for HCV, and 14% versus 17% for HGV, respectively). The overall percentage of current (to total) infections (15%, 11/75) was significantly higher for HGV than for HBV (6%, 6/106 [P = 0.04 by the chi-square test]) but significantly lower than for HCV (78%, 53/68 [P < 0.0001]).
HGV-infected versus noninfected groups. Clinical and virological features were compared between groups with and without HGV infection (including past and present infections) in the high- and low-endemicity areas (Table 2). A history of exposure to folk remedies was more frequent in HGV-positive subjects than in HGV-negative subjects in the high-endemicity area but not in the no-endemicity area. In contrast, a history of blood transfusion was significantly more common among HGV-positive subjects than among HGV-negative subjects in the no-endemicity area. The prevalence of HBV-related markers did not differ between the two groups, while that of HCV-related markers was significantly higher in the HGV-positive group.
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DISCUSSION |
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Introduction
Materials & Methods
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Discussion
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We previously reported that there was a small outbreak of community-acquired, non-A, non-B acute hepatitis among adults in the Arahiro area between 1981 and 1982. Subsequent study (10) showed that the outbreak was due to HCV infection spread mainly via folk remedies in which nonsterilized needles and knives were used. Age-specific prevalence of HCV antibody showed that inhabitants who were infected were predominantly over 40 years old when screened in 1986. By 1993 (present study), a high prevalence was found only in those over 50 years old, suggesting a cohort effect and indicating that the outbreak of HCV infection had already ceased in the Arahiro area following the adoption of sterilized tools in the practice of folk remedies.
HGV-E2 antibody has been reported as a marker of recovery from HGV infection, based on observations that HGV RNA and HGV-E2 antibody are generally mutually exclusive and that clearance of HGV RNA generally coincides with the appearance of HGV-E2 antibody (6, 16, 18). Our results showing that only 5% of individuals with any HGV marker were positive for both HGV RNA and HGV-E2 antibody further support the previous observations.
Tacke et al. (18) reported that 2.5% of healthy blood donors were positive for HGV RNA and that 9% were positive for HGV-E2 antibody. Similarly, Dille et al. (6) reported that 1% of donors were positive for HGV RNA and that 3% were positive for HGV-E2 antibody. Our data in the no-endemicity area were similar, showing a 1% prevalence of HGV RNA and a 5% prevalence of HGV-E2 antibody. Thus, in an area of low HCV endemicity in Japan, the rates of HGV infection are similar to those in Western nations.
When we previously compared HCV and HGV infections in the high-endemicity area by testing HCV and HGV RNAs (23), the prevalence of HGV infection (5%) appeared much lower than that of HCV infection (34%). However, with the advent of the HGV-E2 antibody assay, it became obvious that prevalence of both past and present HGV infection (32%) was as high as that of HCV (32%) or HBV (32%) infection in the high-endemicity area. The prevalence of total infection (past and present infections) for each virus was significantly higher in the high-endemicity area than in the no-endemicity area. However, the proportions of the infections that were active (viremic) were similar in the low- and high-endemicity areas for each virus. The overall proportions of subjects who were antigenic or viremic were 6% for HBV, 15% for HGV, and 78% for HCV. Seventy to 85% of patients with acute HCV infection become chronic HCV carriers (1, 2, 20) and usually maintain the carrier state for long periods afterwards (7, 9, 20). Although several reports have shown that HGV can cause a chronic carrier state (4, 5, 13, 15), the frequency with which it occurs and the rate by which it is maintained has not been clarified sufficiently. Our data suggest that the rate of persistence does not differ between areas with different prevalences of HGV infection, and it is higher than that of HBV but markedly lower than that of HCV.
HGV infection was closely associated with HCV infection both in areas of endemicity and in areas of no endemicity; individuals with total (past plus present) HGV infections had high prevalences of HCV markers and similar patterns of age-specific prevalence. Among possible routes of HGV transmission, folk remedies were significant in the area of endemicity, but blood transfusion was most significant in the no-endemicity area; similar trends were observed in our previous study of HCV transmission (10). Thus, our results indicate that HGV is transmissible not only by blood transfusion but also by folk remedies such as acupuncture and cutting of the skin with nonsterilized knives and that HGV infection had spread in parallel with HCV in the area of high HCV endemicity.
ALT levels of individuals with active HCV infection did not differ among those with and without concurrent HGV infection. Further, individuals with active HGV infection alone tended to exhibit normal or very-low-level elevations of ALT. These results are consistent with the findings of previous studies (4, 5, 8, 13, 21) that suggested a minimum-pathogenic-effect HGV.
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ACKNOWLEDGMENTS |
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This research was supported in part by a grant-in-aid from the Ministry of Health and Welfare in Japan and in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture (no. 09670529).
We thank members of the South Kiso hepatitis study group for assistance at the medical screenings performed in the Arahiro and Sakaue areas. We also thank Kafumi Todoriki for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Second Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan. Phone: 81-263-37-2634. Fax: 81-263-32-9412. E-mail: etanaka{at}gipac.shinshu-u.ac.jp.
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References
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Journal of Clinical Microbiology, January 1998, p. 110-114, Vol. 36, No. 1
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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