Jpn. J. Infect. Dis., 59, 25-30, 2006

Original Article Prevalence and Clinical Significance of HGV/GBV-C in Patients with Chronic B or C Jeng-Fu Yang1,2, Chia-Yen Dai2,3, Wan-Long Chuang2, Wen-Yi Lin1,2, Zu-Yau Lin2, Shinn-Cherng Chen2, Ming-Yuh Hsieh2, Liang-Yen Wang2, Jung-Fa Tsai2, Wen-Yu Chang2 and Ming-Lung Yu1,2* 1Department of Preventive Medicine and 2Department of Medicine, Kaohsiung Medical University Hospital, and 3Department of Occupational Medicine, Kaohsiung Municipal HsiaoKang Hospital, Kaohsiung, Taiwan (Received July 5, 2005. Accepted November 11, 2005)

SUMMARY: Hepatitis G /GB virus-C (HGV/GBV-C) is a newly identified Flavivirus. Its clinical signifi- cance in chronic and C remains controversial. Infection with HGV/GBV-C was surveyed in 500 blood donors, 130 patients with chronic hepatitis B and 173 with , with chronic liver disease, cirrhosis, and/or (HCC). HGV/GBV-C RNA was detected by reverse transcription-polymerase chain reaction. An antibody to HGV/GBV-C’s second envelope protein (anti-E2 Ab) was detected using an enzyme immunoassay. The prevalence of HGV/GBV-C RNA was 3.4% and the exposure rate 10.2% in blood donors. The prevalence of HGV/GBV-C RNA in patients with chronic hepatitis B and hepatitis C was 7.7 and 17.3%, respectively (P = 0.002). The prevalence of the HGV/GBV-C infection in hepatitis B carriers increased with the severity of chronic liver disease and risk of HCC. The age and duration of infection were the more important contributing factors. Clinical and virological characteristics were comparable between those with and without of HGV/GBV-C and hepatitis C. The seroconversion rate was high. Coinfection of HGV/GBV-C with hepatitis B or C does not affect disease severity, but accelerates the progression of chronic liver disease and the development of HCC.

HGV/GBV-C infection has a high rate of spontaneous INTRODUCTION remission which involves the disappearance of HGV/GBV- A flavi-like virus designated the hepatitis G virus (HGV) or C RNA and the production of antibody to HGV/GBV-C’s GB virus-C (GBV-C) has been identified in two independent second envelope protein (anti-E2 Ab) (16-22). Most patients laboratories (1,2). Deinhardt et al. reported sera from a sur- positive for anti-E2 Ab were negative for HGV/GBV-C RNA, geon (GB) who induced acute hepatitis and hepatocellular and vice versa, indicating an inverse correlation of these two injury in primates (3). GB’s serum propagated in , viral markers. Anti-E2 Ab can be detected in the beginning and the viral agents were cloned and characterized as GB of HGV/GBV-C infection recovery and coexists with HGV/ A and B. Simons et al. detected and partially sequenced GBV-C RNA. There may be a window period between the a virus with a sequence similar to that of GB viruses A disappearance of HGV/GBV-C RNA and the appearance of and B, and named it GB virus C. Linnen et al. reported two anti-E2 Ab. isolates of the HGV, GBV-C and HGV, that shared 96-97% Taiwan is an endemic area of hepatitis B and C. Both of the amino acid sequence and 86% of the nucleotide (2). hepatitis B virus (HBV) and HCV may cause chronic liver Therefore, GBV-C and HGV could be two different genotypes disease and hepatocellular carcinoma (HCC). A high preva- of the same virus. lence of HGV/GBV-C infection in Taiwan has been reported The genomic organization of HGV/GBV-C is similar to (6,18). Although the damage induced by HGV/GBV-C infec- that of the family. Even though HGV/GBV-C tion to the liver does not seem to be severe, there are some and (HCV) share strong sequence homolo- cases with prolonged (19,20). gies, there is too wide a divergence between the virus sequences To determine the contribution of HGV/GBV-C to chronic for HGV/GBV-C to be classified as genotypes of HCV. hepatitis pathogenesis and sequel, we investigated the pres- HGV/GBV-C is transfusion-transmissible and has a global ence of the HGV/GBV-C and anti-E2 Ab in different groups: distribution. It has high prevalence in high-risk groups (4-8), volunteer blood donors, and patients with chronic hepatitis such as patients receiving hemodialysis, hemophiliacs, and B and/or C who were diagnosed with chronic persistent hepa- intravenous drug users (9-12). HGV/GBV-C has been found titis, chronic active hepatitis, liver cirrhosis, or/and HCC. Our in some patients with chronic hepatitis or fulminant hepatitis goals were to examine the natural history of HGV/GBV-C without any evidence for known hepatitis virus infection (13). infection in those individuals in order to elucidate the epide- However, there are contradictory reports about the liver miology of HGV/GBV-C, determine the clinical significance tropicity of HGV/GBV-C (14,15). of HGV/GBV-C infection in chronic hepatitis, and determine the effect of coinfection with hepatitis viruses (B or C). *Corresponding author: Mailing address: Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical Univer- MATERIALS AND METHODS sity, No. 100, Tzyou 1st Rd, Kaohsiung 807, Taiwan. Tel: +886- 7-3121101 ext. 7475, Fax: +886-7-3123955, E-mail: fishya@ Patients: A total of 303 patients with chronic hepatitis ms14.hinet.net were divided into 2 groups according to disease etiology: 130

25 patients with chronic hepatitis B and 173 patients with chronic were retested for HCV with a recombinant immunoblot assay hepatitis C. Chronic hepatitis B was defined by a positive (Chiron*RIBA*HCV 3.0 Strip Immunoblot Assay; Bayer, reaction for HBsAg [HBsAg(+)], and HCV infection was Emeryville, Calif., USA). Alanine aminotransferase (ALT) defined by a positive second-generation anti-HCV assay (normal upper limit of serum ALT = 25 IU/L) was measured result [anti-HCV(+)]. Coinfection with HBV and HCV was on a multichannel autoanalyzer. defined as HBsAg(+) and anti-HCV(+), with or without HCV Detection of HCV RNA in serum and HCV genotyping: RNA(+). All chronic hepatitis patients received liver biopsy To detect serum HCV RNA, nested RT-PCR was performed examination and the biopsy tissue was sent to the Pathology using 5´UTR-specific primers as described previously. Department of Kaohsiung Medical University Hospital for Assignment to the HCV genotypes 1a, 1b, 2a, 2b, or 3a examination. HCC was diagnosed by the pathologist, mostly was determined by amplification of the core region using based on high α-fetoprotein levels and angiography studies. genotype-specific primers, as described by Okamoto et al. A total of 500 serum samples of healthy volunteer blood (23). donors were obtained from Kaohsiung Blood Center in 1996 Detection of HBV DNA: HBV DNA was detected and as controls. quantified in the serum with the Quantiplex HBV DNA kit Detection of HGV/GBV-C RNA in serum: HGV RNA (Bayer). in the serum was detected by nested reverse transcription- Statistical analyses: Statistical analysis was performed polymerase chain reaction (RT-PCR) using primers targeting using the SPSS computer software package (v10.0; SPSS, the 5´UTR. Briefly, the total RNA was extracted from 140 μL Inc., Chicago, Ill., USA), with descriptive statistics, Student of serum sample. After the RT reaction (using the Moloney t test, chi-square test and Spearman’s correlation coefficient. murine leukemia virus reverse transcriptase), the first 30 Values were deemed statistically significant at P < 0.05. cycles of PCR were performed with primers 5 grl (5´-GCGAC GTGGGTACRTGGGCG-3´) and 5 grl (5´-GGTTGGTAGGT RESULTS CGTAAATCCCGGTCA-3´) (each cycle: 94°C for 1 min, 62°C for 45 sec, and 72°C for 1 min), and the next 35 cycles HGV/GBV-C in chronic hepatitis: HGV/GBV-C RNA were performed with primers 5gf2, (5´-TGGTAGCCACT was detected in 10 of 130 patients with chronic hepatitis B ATAGGTGGGT-3´) and 5grl (each cycle: 94°C for 1 min, (7.7%) and 30 of 173 patients with chronic hepatitis C 62°C for 45 sec, and 72°C for 1 min). The PCR products (17.3%). Anti-E2 Ab was found in 9 (6.9%) and 38 (22%) were analyzed by electrophoresis on a 3% agarose gel. In patients with hepatitis B and C, respectively. The exposure each PCR assay, two negative controls and one positive rate to HGV/GBV-C was significantly higher in patients with control were tested in addition to the samples of interest. hepatitis C than in patients with hepatitis B (37% versus Measurement of anti-E2 Ab in serum: Anti-E2 Ab titer 14.6%, P = 0.002). was measured by enzyme-linked immunosorbent assay Patients with HBV and HGV/GBV-C coinfection were (ELISA) from Boehringer Mannheim GmbH, Heidelberg, older than those infected by HBV alone (48.90 ± 6.89 versus Germany according to the manufacturer’s instructions. 40.11 ± 14.75, P = 0.003). The mean ALT value of patients HCV antibody and HBsAg detection: HCV antibody and with a simple HBV infection was higher than that of patients HBsAg were detected with commercially available ELISA coinfected with HBV and HGV/GBV-C (138.57 ± 205.77 kits (Abbott, Chicago, Ill., USA). Serum samples that were versus 67 ± 31.16, P = 0.001). Sex, age, history, blood positive for anti-HCV (ELISA) and negative for HCV RNA transfusion, HBV DNA level, and hepatitis B e antigen

Table 1. Demographic and clinical characteristics of HBV carriers with and without HGV/GBV-C infection HGV RNA(+) HGV RNA(–) HGV exp(+) HGV exp(–) No. (%) 10 (7.7%) 120 (92.3%) 19 (14.6%) 111 (84.5%) SEX M/F 8/2 98/22 14/5 92/19 Age (mean ± SD) 48.90 ± 6.891) 40.11 ± 14.75 50.21 ± 10.5162) 39.17 ± 14.47 BT HX (+) 2 (1.6%) 9 (7.1%) 3 (2.4%) 8 (6.3%) ALT (mean ± SD) 67 ± 31.163) 138.57 ± 205.77 73.68 ± 90.924) 143.32 ± 210.32 HBV DNA (pg/mL) 40.27 ± 645.35 54.51 ± 1276.70 856.3 ± 1883.34 612.34 ± 1218.05 HBe Ag(+) 4 (3.2%) 65 (52.0%) 9 (7.2%) 60 (48%) Chronic hepatitis CPH 1 (0.8%)5) 16 (12.3%) 2 (1.6%) 15 (11.5%) CAH 1 (0.8%) 52 (40.0%) 4 (3.1%) 49 (37.7%) Liver cirrhosis 1 (0.8%) 24 (18.5%) 3 (2.3%) 22 (16.9%) Hepatocellular carcinoma 7 (5.4%)6) 28 (21.5%) 10 (7.7%)7) 25 (19.2%) 1): P = 0.003, Significance 6): odds ratio=3 (95% CI) 2): P = 0.002, Significance 7): odds ratio=2.34 (95% CI) 3): P = 0.001, Significance 4): P = 0.19, No significance 5): P = 0.022, Significance (Spearman’s correlation) (+) HGV exp (+) =HGV/GBV-C exposure positive, ongoing infection plus past infection (with HGV/GBV-C RNA positive and/or anti-E2 Ab positive). HGV exp (–) =HGV/GBV-C exposure negative, without ongoing infection nor past infection (with HGV/ GBV-C RNA negative and anti-E2 Ab negative). BT HX, blood transfusion history; CPH, chronic persistent hepatitis; CAH, chronic active hepatitis.

26 (HBeAg) positivity were comparable between patients with 11.19 versus 29.08 ± 9.41 years, P = 0.02). In the blood chronic hepatitis B with and without HGV/GBV-C (Table 1). donors, the rate of seroconversion in HGV/GBV-C infection The existence of HGV/GBV-C RNA seemed to accelerate was 66.7%. the progression of chronic liver disease (P = 0.002, Spearman’s correlation) (Table 1). The risk of HCC development increased DISCUSSION with the existence of HGV/GBV-C RNA and the exposure to HGV/GBV-C infection, ongoing infection or past infection In the present study, HGV/GBV-C RNA was detected (with HGV/GBV-C RNA positive and/or anti-E2 Ab posi- in 3.4% of volunteer blood donors and the exposure rate tive) (Table 1). (ongoing infection plus past infection) was 10.1%. Thus, the Among the 173 patients with chronic hepatitis C, there were prevalence of HGV/GBV-C infection in southern Taiwan no significant differences in sex, age, blood transfusion, ALT (3.4%), where HBV and HCV infection are endemic, is higher value, and distribution of genotype between those with and than that reported for northern Taiwan (2.1%). This indicates without HGV/GBV-C infection. HGV/GBV-C infection. that HGV/GBV-C may share the same routes of infection HGV/GBV-C infection had no influence on the severity of with HBV and HCV. A high rate of exposure to HGV/GBV- chronic liver disease and the development of HCC (Table 2). C indicates that, besides parenteral , additional There was no relationship between HCV seroconversion (the route(s) of viral transmission may exist. The patients with disappearance of HCV RNA from the serum) and HGV/GBV- HGV/GBV-C infection were older than those without infec- C viremia or anti-E2 Ab (Table 2). The rate of seroconversion tion. There was also no difference in the mean ALT level in HGV/GBV-C infection was 47.4 and 53% in the HBV and between the patients with and without HGV/GBV-C infec- HCV carriers, respectively. tion, suggesting that HGV/GBV-C may not cause obvious The odds ratio for the presence of HGV/GBV-C RNA hepatocellular injury. was 3 (95% CI) in HBV carriers and 1.1 (95% CI) in HCV Coinfection with HGV/GBV-C in patients with chronic carriers. The odds ratio estimate for HGV/GBV-C exposure hepatitis B and C has frequently been observed. In this was 2.34 (95% CI) and 0.62 (95% CI) for HBV and HCV study, 18% patients with HGV/GBV-C infection were HBV carriers, respectively. carriers and 55% of the patients were coinfected with HCV, HGV/GBV-C infection in volunteer blood donors: HGV/ suggesting that HGV/GBV-C and HCV may share the same GBV-C RNA was detected in 17 of 500 blood donors (3.4%), transmission route. and anti-E2 Ab was detected in 34 donors (6.8%). The preva- Patients coinfected with HBV and HGV/GBV-C were older lence of HGV/GBV-C exposure, ongoing infection or past than those infected with HBV alone. However, there was no infection (with HGV/GBV-C RNA positive and/or anti-E2 difference in age between HCV carriers with and without Ab positive) was 10.2% (51 of 500 donors). The male/female HGV/GBV-C infection. Most HBV carriers in Taiwan be- ratio, mean age, and ALT value were comparable between came infected with HBV during the perinatal period of their the HGV/GBV-C RNA positive group and negative group. childhood, and superinfection with HGV/GBV-C occurred Donors who had HGV/GBV-C exposure were significantly thereafter. However, in Taiwan, the prevalence of HCV older than those without HGV/GBV-C exposure (34.47 ± infection increases with age. Thus, caution should be taken

Table 2. Demographic, clinical, and viral characteristics of patients with chronic hepatitis C, infected and not infected with HGV/GBV-C HGV RNA (+) HGV RNA (–) HGV exp (+) HGV exp (–) No. (%) 30 (17.3%) 143 (82.7%) 64 (37%) 109 (63%) SEX M/F 24/6 85/58 45/19 64/45 Age (mean ± SD) 46.37 ± 15.13 49.28 ± 13.07 47.47 ± 13.49 49.54 ± 13.43 BT HX(+) 6 (3.5%) 33 (19.1%) 13 (7.5%) 26 (15%) ALT value 81.17 ± 69.57 89.27 ± 98.97 97.23 ± 108.06 82.36 ± 85.44 HCV RNA (+) 22 102 48 76 RNA (–) 8 41 16 33 HCV genotype (%) I b 15 (50%) 70 (48.9%) 25 (39%) 50 (45.9%) II a 11 (36%) 63 (44%) 21 (32.8%) 40 (36.7%) II b 3 (10%) 5 (3.5%) 8 (12.5%) 5 (4.6%) I b + II a 0 0 3 (4.7%) 6 (5.5%) I b + II b 0 3 (2.1%) 2 (1.6%) 3 (2.8%) Unclassified 1 (3%) 2 (1.4%) 5 (7.8%) 5 (5.5%) Chronic hepatitis CPH 6 (3.5%) 26 (15.0%) 13 (8.1%) 19 (11.0%) CAH 13 (7.5%) 73 (42.2%) 33 (19.6%) 53 (30.6%) Liver cirrhosis 6 (3.5%) 19 (11.0%) 10 (7.0%) 15 (8.7%) Hepatocellular carcinoma 5 (2.9%)1) 25 (14.5%) 8 (4.6%)2) 22 (12.7%) No significance between infected and not infected groups in all parameters. 1): odds ratio=1.1 (95% CI) 2): odds ratio=0.62 (95% CI) HGV exp (+), HGV/GBV-C exposure positive; HGV exp (–), HGV/GBV-C exposure negative. Abbreviations are in Table 1.

27 in interpreting the effects of HGV/GBV-C infection in chronic HGV/GBV-C coinfection was related to a particular HCV hepatitis B and C. HGV/GBV-C infection in the HCV/HBV genotype (45), our results did not reveal this relationship. endemic area is not associated with the presence of HBsAg. HGV/GBV-C and HCV, both Flaviviridae RNA viruses, did For HBV-HGV/GBV-C coinfection, discordant results were not seem to interact with each other. We also showed that found in the literature; HGV/GBV-C RNA prevalence in there was no significant difference in terms of HCV RNA hepatitis B chronic infection may be low: 0/48 in Wang and positivity, HBeAg positivity, and HBV DNA levels between Jin (survey in professional blood donors, Taiwan) (24) and 7/ patients with a single HBV or HCV infection and patients 220 (3.2%) Kao et al. (survey in patients with asymptomatic with HBV + HGV or HCV + HGV dual . This result hepatitis B carriage and with chronic liver disease, northern indicates that HGV/GBV-C coinfection has no suppressive Taiwan) (25), or high in other studies: 7/27(9.7%) (patients effect on HBV and HCV and does not aggravate the clinical with chronic , USA) for Linnen et al. (2), 32/ course of chronic hepatitis B or C. 100 (32%) (patients with acute viral hepatitis, USA) for Alter A higher prevalence of HGV/GBV-C infection was found et al. (26). A recent report showed that, in France, a high in patients with HCC (46). Another study supported the prevalence of HGV/GBV-C RNA correlated with chronic hypothesis of an association between HGV/GBV-C infection HBV carriers that were coinfected with the immuno- and HCC, but the causality of the association was unclear deficiency virus (27). This finding is consistent with the view (47). In contrast, studies by Kao et al. and Lightfoot et al. that the spread of HGV/GBV-C in HBV/HCV endemic areas showed that HGV/GBV-C infection did not increase the risk is different from the spread of HBV in Taiwan, which is of HCC development (25,48). predominantly transmitted vertically from carrier mother to In the present study, HGV/GBV-C superinfection seemed newborn or horizontally from child to child (28-30). The to increase the relative risk of HCC development in HBV difference in the HBV-HGV/GBV-C coinfection pattern carriers, but not in HCV carriers. The increasing prevalence among the Far East, USA and Western Europe may be attrib- of HGV/GBV-C RNA in HBV-infected patients is associated uted to differences in HBV transmission routes in those areas with aging in Taiwan, where HBV carriers acquire HBV infec- (30). tion in their perinatal or childhood period. Age itself may The fact that the blood transfusion history was similar in contribute to the increased risk of HCC development. Even both HGV/GBV-C infected and noninfected patients, and that though this cross-sectional study does not clearly reveal the HGV/GBV-C RNA was found in the absence of hepatitis B correlations between HGV/GBV-C and HCC, the role of and C infection indicates that the HGV/GBV-C virus is HGV/GBV-C in HCC etiology seems modest. Additional capable of independent transmission. Vertical transmission longitudinal studies that include more cases are needed to has been reported, and interspousal transmission was also resolve this issue. suggested (25,31-33), although it was not well characterized. In summary, we found that HGV/GBV-C infection is HGV/GBV-C viremia seemed to accelerate the progres- common in volunteer blood donors and more prevalent in sion of chronic liver disease in HBV carriers. However, the patients with chronic hepatitis B or C. Blood transfusion is patient’s age and the duration of HBV infection may be the an important route for transmission. HGV/GBV-C seemed to important contributing factors. The fact that the ALT value cause little hepatic injury and did not aggravate the clinical was similar in all chronic hepatitis patients regardless of HGV/ course of chronic hepatitis B and C in this cross-sectional GBV-C infection suggests that HGV/GBV-C cannot cause study. additive hepatic injury in patients with chronic hepatitis B or C. This result is comparable with that previously reported ACKNOWLEDGMENTS (34,35). In the present study, gender, age, ALT levels, and the patho- This work was supported by a grant NSC 87-2314-B037- logical distribution of chronic hepatitis were comparable 079 from the National Science Council, Taiwan. between HGV/GBV-C viremic and nonviremic patients with chronic hepatitis. Additionally, blood donors with HGV/GBV- REFERENCES C viremia exhibited normal ALT levels. 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