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TT virus infection in an area of high-endemicity for hepatitis C
Hepatology Research 13 (1999) 212 – 220
TT virus infection in an area of high-endemicity for hepatitis C Takeji Umemura a, Eiji Tanaka a,*, Masao Ota b, Koji Orii a, Kaname Yoshizawa a, Haruhiko Imai a, Takeshi Sodeyama a, Kendo Kiyosawa a a
Second Department of Internal Medicine, Shinshu Uni6ersity School of Medicine, 3 -1 -1 Asahi, Matsumoto 390 -8621, Japan b Department of Legal Medicine, Shinshu Uni6ersity School of Medicine, 3 -1 -1 Asahi, Matsumoto 390 -8621, Japan
Received 29 July 1998; received in revised form 8 September 1998; accepted 17 September 1998
Abstract TT virus (TTV) was recently identified as a candidate for a new hepatitis virus. In the present study, the clinical features and transmission routes of TTV infection were analyzed in an area highly endemic for hepatitis C virus (HCV) infection, and compared to those in an area not endemic. A total of 400 inhabitants 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 control area. TTV DNA in serum was tested by a semi-nested polymerase chain reaction. The prevalence of TTV DNA was 58% (116/200) in the high-endemicity area, and was significantly (P= 0.0083) higher than that in the control area (43%, 85/200). Age specific prevalence of TTV DNA was significantly (P=0.0007) higher in individuals over 60 years old (68.8%) than in those under 60 (45.1%) in the high-endemicity area, but was similar among the age groups in the control area. TTV infection was not associated with neither HCV and hepatitis G virus (HGV) infections nor histories of surgical operation, blood transfusion and folk remedies in both areas. In the high-endemicity area, the prevalence of TTV DNA was significantly (P =0.0311) lower in individuals with any HCV markers (25%) than in those without it (39%). The elevation of liver enzymes in serum
* Corresponding author. Tel.: +81 263 372634; fax: + 81 263 329412; e-mail: [email protected]. shinshu-u.ac.jp 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00090-4
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was dependent on HCV viremia but not on TTV viremia. In conclusion, the prevalence of TTV infection was as high as 58% in the high-endemicity area for HCV infection. The infection pattern for TTV was different from that of HCV and HGV. TTV infection showed a reciprocal association with HCV infection, and had limited pathogenetic effect on hepatitis. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Folk remedies; Hepatitis C virus RNA; TT virus DNA
1. Introduction Recently, a new virus, designated as the TT virus (TTV), has been identified from the serum of a patient with post-transfusion hepatitis of unknown etiology [1]. Okamoto et al. have reported that TTV is an unenveloped, single-stranded DNA virus. Further, they have reported that the amount of TTV DNA in liver tissue is equal or 10 – 100 times higher in corresponding sera, indicating a replication of TTV in the liver [2]. TTV infection has been supposed to be responsible for some cases of acute and chronic liver disease of unknown etiology. However, the epidemiology, clinical significance and transmission patterns of TTV infection still remain unclear. We have reported on an area of highly endemic for the hepatitis C virus (HCV) infection in which over 30% of the inhabitants are positive for the HCV antibody [3]. Folk remedies such as acupuncture and cutting of the skin using nonsterilized knives have been considered to be possible routes for HCV transmission in this area. In 1995, GB virus-C/hepatitis G virus (GBV-C/HGV) was, like TTV, identified as a candidate for a new hepatitis virus [4,5]. An analysis of GBV-C/HGV infection in the high endemic area clarified that the infection was also highly prevalent in the high-endemic area and was closely associated with HCV infection [6,7]. In the present study, we analyzed the prevalence, transmission routes, and clinical significance of TTV infection in the area of high hepatitis C endemicity compared with those in an area of no hepatitis C endemicity.
2. Materials and methods
2.1. Subjects A total of 420 individuals over 18 years old (62% of total inhabitants with corresponding ages) in an area in which HCV was highly endemic were medically screened for liver diseases in July 1993. Of those, 200 individuals whose serum samples were available were selected randomly for evaluation in the present study. Those subjects included 82 males and 118 females with a mean age of 56.29 17.6 years (range, 12 – 84 years). Medical screening was also conducted in an area in which HCV was not endemic and which is located near the high-endemic area. Out of 482 individuals (65% of total inhabitants with corresponding ages) who under-
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went medical screening in the non-endemicity area, 200 individuals were selected randomly for evaluation in the same manner as in the high-endemic area. These control subjects included 49 males and 151 females with a mean age of 57.1 9 13.5 years (range, 26 – 89 years). The medical screenings performed in the high- and non-endemicity areas were the same as the screenings reported previously, but the selected patients were different from those in the previous study [7]. The medical screenings and blood sample collections were carried out in the same manner as that reported previously. Informed consent was obtained from each subject. Serum samples were stored at − 70°C until assayed.
2.2. Laboratory tests Second-generation HCV antibody, hepatitis B surface (HBs) antigen, HBs antibody, and hepatitis B core (HBc) antibody were measured using commercially available enzyme-linked immunosorbent assay kits (International Reagents, Kobe, Japan). Aspartate aminotransferase (AST) (normal, 12–37 IU/l), alanine aminotranseferase (ALT) (normal, 7 – 45 IU/l), and zinc turbidity tests (ZTT) (normal, 4–12 Kunkel Unit) were carried out using a multichannel autoanalyzer. The ZTT test reflects the g-globulin level, and its value is usually elevated in patients with chronic liver diseases [3].
2.3. Detection of HCV RNA HCV RNA in serum was detected by a nested reverse transcription polymerase chain reaction (RT-PCR) using the two sets of primers synthesized from the 5% noncoding region of the HCV genome as reported previously [8]. In each PCR assay (including PCR for GBV-C/HGV RNA and TTV DNA), two negative controls and one positive control of estimated 10 copies/ml were tested in addition to the samples of interest. Standard precautions were taken to avoid contamination throughout the study by the application of appropriate measures [9].
2.4. Detection of GBV-C/HGV RNA GBV-C/HGV RNA in serum was detected by nested RT-PCR using primers in the 5% noncoding region of the GBV-C/HGV genome as described previously [10]. In each PCR assay, two negative controls and one positive control of 10 copies/ml were tested in addition to the samples of interest [11].
2.5. Detection of antibody to GBV-C/HGV en6elope-2 protein Antibody to the GBV-C/HGV envelope-2 protein (GBV-C/HGV-E2 antibody) was measured by an enzyme-linked immunosorbent assay in which recombinant E2 protein was bound to a microtiter plate [12,13]. After the addition of diluted serum samples to the plate, specifically bound antibodies against the E2 protein were detected with an anti-human immunoglobulin G conjugated with peroxidase. Positive or negative results were judged as reported previously [12,13].
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2.6. Detection of TTV DNA TTV DNA was detected by semi-nested PCR according to the report by Okamoto et al. [2]. Briefly, nucleic acids were extracted from 100 ml of serum and then resolved in 20 ml of Tris – EDTA buffer. After heating at 95°C for 15 min, resolved solution was chilled on ice. A half portion of the extract was subjected to semi-nested PCR. The first-round PCR was carried out for 35 cycles with the primers reported (NG059 and NG063). The second-round PCR was carried out for 25 cycles with primers NG061 and NG063 [2]. In each PCR assay, two negative controls and one positive control were tested in addition to the samples of interest.
2.7. Statistical analysis Statistical analyses were performed with the 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.
3. Results The prevalence of TTV DNA was significantly (P= 0.0083) higher in the high-endemicity area (58.0%) than in the non-endemicity area (42.5%) (Table 1). This tendency was also statistically significant when analyzed in male (62.2% in the Table 1 Comparison of clinical and virological characteristics between individuals in high- and non-endemicity areas Characteristics
High-endemicity (n = 200)
Non-endemicity (n = 200)
P
History of Surgical operation Blood transfusion Folk remedy
54 (27.0) 12 (6.0) 91 (47.2)
46 (23.0) 20 (10.0) 58 (29.0)
\0.2 0.1403 0.0006
HBs antigen HBs antibody HBc antibody Any HBV marker
3 50 52 62
4 42 43 46
\0.2 \0.2 \0.2 0.0715
HCV RNA HCV antibody Any HCV marker
49 (24.5) 62 (31.0) 62 (31.0)
1 (0.5) 2 (1.0) 2 (1.0)
B0.0001 B0.0001 B0.0001
HGV RNA HGV-E2 antibody Any HGV marker
9 (4.5) 58 (29.0) 63 (31.5)
1 (0.5) 8 (4.0) 9 (4.5)
0.0104 B0.0001 B0.0001
116 (58.0)
85 (42.5)
TTV DNA
(1.5) (25.0) (26.0) (31.0)
(2.0) (21.0) (21.5) (23.0)
0.0083
Data are expressed as positive no. (%). P values were obtained by the chi-square test in all characteristics except the HBs antigen (Fisher’s exact test).
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Table 2 Comparison of clinical and virological characteristics between TTV DNA positive and negative individuals Characteristics
TTV DNA positive
TTV DNA negative
P
High-endemicity area History of surgical operation History of blood transfusion History of folk remedy HBs antigen Any HBV marker HCV RNA Any HCV marker HGV RNA Any HGV marker
(n =116) 34 (29.3) 8 (6.9) 57 (49.1) 1 (0.9) 37 (31.1) 23 (19.8) 29 (25.0) 5 (4.3) 35 (30.2)
(n =84) 20 (23.8) 4 (4.8) 34 (40.5) 2 (2.4) 25 (29.8) 26 (31.0) 33 (39.3) 4 (5.0) 28 (33.3)
\0.2 \0.2 \0.2 \0.2 \0.2 0.0710 0.0311 \0.2 \0.2
Non-endemicity area History of surgical operation History of blood transfusion History of folk remedy HBs antigen Any HBV marker HCV RNA Any HCV marker HGV RNA Any HGV marker
(n =85) 18 (21.2) 8 (9.4) 24 (28.2) 2 (2.4) 20 (23.5) 0 (0) 0 (0) 0 (0) 5 (5.9)
(n =115) 28 (24.3) 12 (10.4) 34 (29.6) 2 (1.7) 26 (22.6) 1 (0.9) 2 (1.7) 1 (0.9) 4 (3.5)
\0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2
Data are expressed as positive no. (%). P values were obtained by the chi-square test for all characteristics except the HBs antigen (Fisher’s exact test). Any HBV marker:positive for HBs antigen, HBs antibody and/or HBc antibody. Any HCV marker:positive for HCV RNA and/or HCV antibody. Any HGV marker:positive for HGV RNA and/or HGV-E2 antibody.
high-endemicity area and 40.8% in the non-endemicity area, P= 0.0175) and female (similarly, 55.1% and 43.0%, P = 0.0499) individuals separately. Clinical and virological features of the 200 individuals in the high-endemicity area are compared to those of the 200 individuals in the non-endemicity area in Table 1. A history of folk remedy was significantly more prevalent in the high-endemicity area than in the non-endemicity area, but histories of surgical operation and blood transfusion were similarly distributed between the two areas. HCV and HGV infections were significantly more common in the high-endemicity area than in the non-endemicity area. These results are consistent with the results reported previously [7]. Clinical and virological features are compared between individuals with and without TTV DNA in Table 2. HBV and HGV infections were similarly prevalent in the two groups, but HCV infection was less prevalent in individuals with TTV viremia than in those without it in the high-endemicity area. In the non-endemicity area, prevalence of HBV, HCV, and HGV infections did not differ between the two groups. The clinical backgrounds were similar between the two groups in both highand non-endemicity areas.
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The age-specific prevalence of TTV, HCV, and HGV infections in the high-endemicity area is shown in Fig. 1. Patients who had a marker indicating the existence of viremia were defined as having ongoing or present infection, and those who had antibody in the absence of viremia were considered to have resolved or past infection. Present infection as well as total infection (past and present ones) with HCV was significantly (P B0.0001 for present infection and PB0.0001 for total infection) more prevalent in individuals with ages over 50 years (32.9% for present
Fig. 1. Age-specific prevalences of TTV, HCV, and HGV infections in the high-endemicity area. Prevalence of exposure is indicated by both filled and open bars and reflects a positive test for at least one viral marker (HCV RNA and/or HCV antibody for HCV; and HGV RNA and/or HGV-E2 antibody for HGV). Filled bars indicate a positive test for a marker of viremia (TTV DNA for TTV, HCV RNA for HCV, and HGV RNA for HGV).
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Table 3 Comparison of biochemical liver function tests according to the status of TTV DNA and HCV RNA Tests
TTV DNA alone (n=178)
HCV RNA alone (n = 27)
TTV DNA and HCV RNA (n = 23)
Neither TTV DNA nor HCV RNA (n =172)
ALT (IU/l) AST (IU/l) ZTT (KU)
17.19 11.5 21.39 8.4 8.4 9 3.5
43.4950.1a 37.19 21.7c 14.396.1e
34.3 9 24.5b 35.1 913.8d 14.2 9 6.7f
16.4 9 10.0 20.0 95.1 8.3 93.0
Data are expressed as mean 9 SD. Other comparisons among the four groups in each test are not statistically significant. P values were obtained by the Student’s t-test for all characteristics. a PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. b PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. c PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. d PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. e PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. f PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA.
infection and 40.7% for total infection) than in those under 50 (5.0% for present infection and 8.3% for total infection). Total infection of HGV was significantly more prevalent in individuals with ages over 50 years (40.7%) than in those under 50 (10.0%), but present infection with HGV was similar among the age groups. On the other hand, present infection with TTV was significantly (P= 0.0007) more prevalent in individuals aged over 60 years (68.8%) than in those under 60 (45.1%). In the non-endemicity area, the prevalence of TTV present infection was similar among the age groups; under 29 years; 0% (0/1), 30–39 years; 45.5% (10/22), 40–49 years; 29.7% (11/37), 50 – 59 years; 50.0% (20/40), 60–69 years; 41.3% (26/63), over 70 years; 48.6% (18/37). Mean levels of ALT, AST and ZTT in serum are compared according to the status of TTV DNA and HCV RNA in Table 3. The mean ALT level was significantly higher in individuals with TTV DNA and HCV RNA and in those with HCV RNA alone than in those with TTV DNA alone or in those with neither TTV DNA nor HCV RNA, respectively. Other comparisons among the four groups were not statistically significant, including the comparison between those with TTV DNA alone and those with neither TTV DNA nor HCV RNA. A similar tendency was observed for AST and ZTT tests.
4. Discussion Okamoto et al. have reported that the prevalence of TTV DNA is 12% in Japanese healthy blood donors [2]. Our preliminary data also shows that the prevalence is 12% (6/50) in blood donors (data not shown). Compared to those data, the prevalence of TTV DNA was about three times higher in the control area (43%) of the present study, in which the prevalence of other hepatitis viral markers
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was similar to that in other parts of Japan. The control area as well as the high-endemicity area is surrounded by mountains and is relatively isolated from other communities, but the lifestyle in this area does not seem to differ from that in other parts of Japan [3,6,7]. Thus, it is possible that the relatively isolated circumstances had contributed to the higher prevalence of TTV infection in the control area. Further studies are required to clarify whether our control area is extraordinary in terms of TTV infection, as sufficient epidemiological studies on TTV infection have not been carried out. Although the prevalence of TTV DNA was high in the control area, it was significantly higher in the high endemicity area (58%) than in the control area. The prevalence was similar (around 40%) in individuals under 60 years old between the two areas, but it increased to near 70% in individuals over 60 years old only in the high-endemicity area. HCV and HGV infections were much more common in individuals over 50 years old in the high-endemicity area, thus the age-specific prevalence of TTV infection differed from that of HCV or HGV infection. We have previously reported that HGV infection is closely associated with HCV infection, and have suggested that HGV spread in a similar manner to HCV in the high-endemicity area for hepatitis C [6,7]. On the contrary, TTV infection was not positively associated with either HCV infection or HGV infection. Furthermore, a history of folk remedies, which can be responsible for the spread of HCV and HGV, was similarly seen between individuals with and without TTV DNA. These results indicate that TTV is in a different manner than HCV and HGV. In support of the above results, the data from the present study suggest that histories of surgical operation and blood transfusion, both of which are potentially responsible for the transmission of blood-borne hepatitis viruses, were not associated with TTV infection either in the high-endemicity or the control areas. It is noteworthy that TTV infection showed a reciprocal association with HCV infection in the high-endemicity area, that is, the prevalence of any HCV marker was significantly lower in individuals with TTV DNA than in those without it. Furthermore, the two individuals with HCV infection in the control area were negative for TTV DNA. The following possibilities were considered as explanations. First, the main transmission routes are mutually exclusive between HCV and TTV. Second, a reciprocal interaction exists between TTV and HCV replications, as has been observed between HBV and HCV replications [14,15]. The elevation of transaminases and ZTT, which reflect the level of g-globulin, were dependent on HCV viremia but not on TTV viremia. A similar relationship has been reported by several investigators regarding HCV and HGV infections in which no or a limited pathogenecity of HGV as a hepatitis virus has been suggested [10,16 – 18]. Thus, a limited effect of TTV infection on the elevation of liver enzymes is suggested in the present study.
Acknowledgements This research was supported in part by a Grant-in-Aid from the Ministry of
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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 the members of the South Kiso study group for their assistance at the medical screenings, and Ms Yuki Sugiyama and Ms Kafumi Todoriki for their technical assistance. We also thank Professor Makoto Mayumi for providing us primers of TTV.
References [1] Nishizawa T, Okamoto H, Konishi K, Yoshizawa H, Miyakawa Y, Mayumi M. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun 1997;241:92 – 95. [2] Okamoto H, Nishizawa T, Kato N, et al. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res 1998;10:1 – 6. [3] Kiyosawa K, Tanaka E, Sodeyama T, et al. Transmission of hepatitis C in an isolated area in Japan: community acquired infection. Gastroenterology. 1994;106:1596 – 1602. [4] Simmons JN, Leary TP, Dawson GJ, et al. Isolation of novel virus-like sequences associated with human hepatitis virus. Nat Med 1995;1:564 – 569. [5] Linnen J, Wages Jr. J, Zhang-Keck ZY, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505 – 508. [6] Tanaka E, Nakatsuji Y, Kobayashi M, et al. Hepatitis G virus/GB virus C infection in an area of high endemic hepatitis C virus infecion. Hepatol Res 1997;7:130 – 135. [7] Tanaka E, Tacke M, Kobayashi M, et al. Past and present hepatitis G virus infections in an areas where hepatitis C is highly endemic and those where it is not endemic. J Clin Microbiol 1998;36:110–114. [8] Matsumoto A, Tanaka E, Suzuki T, et al. Viral and host factors that contribute to efficacy of interferon-a2a therapy in patients with chronic hepatitis C. Dig Dis Sci 1994;39:1273 – 1280. [9] Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237 – 238. [10] Tanaka E, Alter HJ, Nakatsuji Y, et al. Effect of hepatitis G virus infection co-infection on patients with chronic hepatitis C. Ann Intern Med 1996;125:740 – 743. [11] Nakatsuji Y, Shih JW, Tanaka E, et al. Prevalence and disease association of hepatitis G virus infection in Japan. J Viral Hepatitis 1996;3:307 – 316. [12] Tacke M, Kiyosawa K, Stark K, et al. Detection of antibodies to a putative hepatitis G virus envelope protein. Lancet 1997;349:318 – 320. [13] Tacke M, Schmolke S, Schlueter V, et al. Humoral immune response to the E2 protein of hepatitis G virus is associated with long-term recovery from infection and reveals a high frequency of HGV exposure among healthy blood donors. Hepatology 1997;26:1626 – 1633. [14] Sheen IS, Liaw YF, Chu CM, Pao CC. Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection. J Infect Dis 1992;165:831 – 834. [15] Pontisso P, Ruvoletto MG, Fattovich G, et al. Clinical and virological profies in patients with multiple hepatitis virus infections. Gastroenterology 1993;105:1529 – 1533. [16] Alter HJ, Nakatsuji Y, Melpolder J et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. New Engl J Med 1997;336:747 – 754. [17] Alter MJ, Gallagher M, Morris TT et al. Acute non A-E hepatitis in the United States and the role of hepatitis G virus infection. New Engl J Med 1997;336:741 – 746. [18] Kao JH, Chen PJ, Lai MY et al. GB virus-C/hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease, and liver cancer. Gastroenterology 1997;112:1265– 1270. .
TT virus infection in an area of high-endemicity for hepatitis C Takeji Umemura a, Eiji Tanaka a,*, Masao Ota b, Koji Orii a, Kaname Yoshizawa a, Haruhiko Imai a, Takeshi Sodeyama a, Kendo Kiyosawa a a
Second Department of Internal Medicine, Shinshu Uni6ersity School of Medicine, 3 -1 -1 Asahi, Matsumoto 390 -8621, Japan b Department of Legal Medicine, Shinshu Uni6ersity School of Medicine, 3 -1 -1 Asahi, Matsumoto 390 -8621, Japan
Received 29 July 1998; received in revised form 8 September 1998; accepted 17 September 1998
Abstract TT virus (TTV) was recently identified as a candidate for a new hepatitis virus. In the present study, the clinical features and transmission routes of TTV infection were analyzed in an area highly endemic for hepatitis C virus (HCV) infection, and compared to those in an area not endemic. A total of 400 inhabitants 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 control area. TTV DNA in serum was tested by a semi-nested polymerase chain reaction. The prevalence of TTV DNA was 58% (116/200) in the high-endemicity area, and was significantly (P= 0.0083) higher than that in the control area (43%, 85/200). Age specific prevalence of TTV DNA was significantly (P=0.0007) higher in individuals over 60 years old (68.8%) than in those under 60 (45.1%) in the high-endemicity area, but was similar among the age groups in the control area. TTV infection was not associated with neither HCV and hepatitis G virus (HGV) infections nor histories of surgical operation, blood transfusion and folk remedies in both areas. In the high-endemicity area, the prevalence of TTV DNA was significantly (P =0.0311) lower in individuals with any HCV markers (25%) than in those without it (39%). The elevation of liver enzymes in serum
* Corresponding author. Tel.: +81 263 372634; fax: + 81 263 329412; e-mail: [email protected]. shinshu-u.ac.jp 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00090-4
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was dependent on HCV viremia but not on TTV viremia. In conclusion, the prevalence of TTV infection was as high as 58% in the high-endemicity area for HCV infection. The infection pattern for TTV was different from that of HCV and HGV. TTV infection showed a reciprocal association with HCV infection, and had limited pathogenetic effect on hepatitis. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Folk remedies; Hepatitis C virus RNA; TT virus DNA
1. Introduction Recently, a new virus, designated as the TT virus (TTV), has been identified from the serum of a patient with post-transfusion hepatitis of unknown etiology [1]. Okamoto et al. have reported that TTV is an unenveloped, single-stranded DNA virus. Further, they have reported that the amount of TTV DNA in liver tissue is equal or 10 – 100 times higher in corresponding sera, indicating a replication of TTV in the liver [2]. TTV infection has been supposed to be responsible for some cases of acute and chronic liver disease of unknown etiology. However, the epidemiology, clinical significance and transmission patterns of TTV infection still remain unclear. We have reported on an area of highly endemic for the hepatitis C virus (HCV) infection in which over 30% of the inhabitants are positive for the HCV antibody [3]. Folk remedies such as acupuncture and cutting of the skin using nonsterilized knives have been considered to be possible routes for HCV transmission in this area. In 1995, GB virus-C/hepatitis G virus (GBV-C/HGV) was, like TTV, identified as a candidate for a new hepatitis virus [4,5]. An analysis of GBV-C/HGV infection in the high endemic area clarified that the infection was also highly prevalent in the high-endemic area and was closely associated with HCV infection [6,7]. In the present study, we analyzed the prevalence, transmission routes, and clinical significance of TTV infection in the area of high hepatitis C endemicity compared with those in an area of no hepatitis C endemicity.
2. Materials and methods
2.1. Subjects A total of 420 individuals over 18 years old (62% of total inhabitants with corresponding ages) in an area in which HCV was highly endemic were medically screened for liver diseases in July 1993. Of those, 200 individuals whose serum samples were available were selected randomly for evaluation in the present study. Those subjects included 82 males and 118 females with a mean age of 56.29 17.6 years (range, 12 – 84 years). Medical screening was also conducted in an area in which HCV was not endemic and which is located near the high-endemic area. Out of 482 individuals (65% of total inhabitants with corresponding ages) who under-
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went medical screening in the non-endemicity area, 200 individuals were selected randomly for evaluation in the same manner as in the high-endemic area. These control subjects included 49 males and 151 females with a mean age of 57.1 9 13.5 years (range, 26 – 89 years). The medical screenings performed in the high- and non-endemicity areas were the same as the screenings reported previously, but the selected patients were different from those in the previous study [7]. The medical screenings and blood sample collections were carried out in the same manner as that reported previously. Informed consent was obtained from each subject. Serum samples were stored at − 70°C until assayed.
2.2. Laboratory tests Second-generation HCV antibody, hepatitis B surface (HBs) antigen, HBs antibody, and hepatitis B core (HBc) antibody were measured using commercially available enzyme-linked immunosorbent assay kits (International Reagents, Kobe, Japan). Aspartate aminotransferase (AST) (normal, 12–37 IU/l), alanine aminotranseferase (ALT) (normal, 7 – 45 IU/l), and zinc turbidity tests (ZTT) (normal, 4–12 Kunkel Unit) were carried out using a multichannel autoanalyzer. The ZTT test reflects the g-globulin level, and its value is usually elevated in patients with chronic liver diseases [3].
2.3. Detection of HCV RNA HCV RNA in serum was detected by a nested reverse transcription polymerase chain reaction (RT-PCR) using the two sets of primers synthesized from the 5% noncoding region of the HCV genome as reported previously [8]. In each PCR assay (including PCR for GBV-C/HGV RNA and TTV DNA), two negative controls and one positive control of estimated 10 copies/ml were tested in addition to the samples of interest. Standard precautions were taken to avoid contamination throughout the study by the application of appropriate measures [9].
2.4. Detection of GBV-C/HGV RNA GBV-C/HGV RNA in serum was detected by nested RT-PCR using primers in the 5% noncoding region of the GBV-C/HGV genome as described previously [10]. In each PCR assay, two negative controls and one positive control of 10 copies/ml were tested in addition to the samples of interest [11].
2.5. Detection of antibody to GBV-C/HGV en6elope-2 protein Antibody to the GBV-C/HGV envelope-2 protein (GBV-C/HGV-E2 antibody) was measured by an enzyme-linked immunosorbent assay in which recombinant E2 protein was bound to a microtiter plate [12,13]. After the addition of diluted serum samples to the plate, specifically bound antibodies against the E2 protein were detected with an anti-human immunoglobulin G conjugated with peroxidase. Positive or negative results were judged as reported previously [12,13].
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2.6. Detection of TTV DNA TTV DNA was detected by semi-nested PCR according to the report by Okamoto et al. [2]. Briefly, nucleic acids were extracted from 100 ml of serum and then resolved in 20 ml of Tris – EDTA buffer. After heating at 95°C for 15 min, resolved solution was chilled on ice. A half portion of the extract was subjected to semi-nested PCR. The first-round PCR was carried out for 35 cycles with the primers reported (NG059 and NG063). The second-round PCR was carried out for 25 cycles with primers NG061 and NG063 [2]. In each PCR assay, two negative controls and one positive control were tested in addition to the samples of interest.
2.7. Statistical analysis Statistical analyses were performed with the 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.
3. Results The prevalence of TTV DNA was significantly (P= 0.0083) higher in the high-endemicity area (58.0%) than in the non-endemicity area (42.5%) (Table 1). This tendency was also statistically significant when analyzed in male (62.2% in the Table 1 Comparison of clinical and virological characteristics between individuals in high- and non-endemicity areas Characteristics
High-endemicity (n = 200)
Non-endemicity (n = 200)
P
History of Surgical operation Blood transfusion Folk remedy
54 (27.0) 12 (6.0) 91 (47.2)
46 (23.0) 20 (10.0) 58 (29.0)
\0.2 0.1403 0.0006
HBs antigen HBs antibody HBc antibody Any HBV marker
3 50 52 62
4 42 43 46
\0.2 \0.2 \0.2 0.0715
HCV RNA HCV antibody Any HCV marker
49 (24.5) 62 (31.0) 62 (31.0)
1 (0.5) 2 (1.0) 2 (1.0)
B0.0001 B0.0001 B0.0001
HGV RNA HGV-E2 antibody Any HGV marker
9 (4.5) 58 (29.0) 63 (31.5)
1 (0.5) 8 (4.0) 9 (4.5)
0.0104 B0.0001 B0.0001
116 (58.0)
85 (42.5)
TTV DNA
(1.5) (25.0) (26.0) (31.0)
(2.0) (21.0) (21.5) (23.0)
0.0083
Data are expressed as positive no. (%). P values were obtained by the chi-square test in all characteristics except the HBs antigen (Fisher’s exact test).
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Table 2 Comparison of clinical and virological characteristics between TTV DNA positive and negative individuals Characteristics
TTV DNA positive
TTV DNA negative
P
High-endemicity area History of surgical operation History of blood transfusion History of folk remedy HBs antigen Any HBV marker HCV RNA Any HCV marker HGV RNA Any HGV marker
(n =116) 34 (29.3) 8 (6.9) 57 (49.1) 1 (0.9) 37 (31.1) 23 (19.8) 29 (25.0) 5 (4.3) 35 (30.2)
(n =84) 20 (23.8) 4 (4.8) 34 (40.5) 2 (2.4) 25 (29.8) 26 (31.0) 33 (39.3) 4 (5.0) 28 (33.3)
\0.2 \0.2 \0.2 \0.2 \0.2 0.0710 0.0311 \0.2 \0.2
Non-endemicity area History of surgical operation History of blood transfusion History of folk remedy HBs antigen Any HBV marker HCV RNA Any HCV marker HGV RNA Any HGV marker
(n =85) 18 (21.2) 8 (9.4) 24 (28.2) 2 (2.4) 20 (23.5) 0 (0) 0 (0) 0 (0) 5 (5.9)
(n =115) 28 (24.3) 12 (10.4) 34 (29.6) 2 (1.7) 26 (22.6) 1 (0.9) 2 (1.7) 1 (0.9) 4 (3.5)
\0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2 \0.2
Data are expressed as positive no. (%). P values were obtained by the chi-square test for all characteristics except the HBs antigen (Fisher’s exact test). Any HBV marker:positive for HBs antigen, HBs antibody and/or HBc antibody. Any HCV marker:positive for HCV RNA and/or HCV antibody. Any HGV marker:positive for HGV RNA and/or HGV-E2 antibody.
high-endemicity area and 40.8% in the non-endemicity area, P= 0.0175) and female (similarly, 55.1% and 43.0%, P = 0.0499) individuals separately. Clinical and virological features of the 200 individuals in the high-endemicity area are compared to those of the 200 individuals in the non-endemicity area in Table 1. A history of folk remedy was significantly more prevalent in the high-endemicity area than in the non-endemicity area, but histories of surgical operation and blood transfusion were similarly distributed between the two areas. HCV and HGV infections were significantly more common in the high-endemicity area than in the non-endemicity area. These results are consistent with the results reported previously [7]. Clinical and virological features are compared between individuals with and without TTV DNA in Table 2. HBV and HGV infections were similarly prevalent in the two groups, but HCV infection was less prevalent in individuals with TTV viremia than in those without it in the high-endemicity area. In the non-endemicity area, prevalence of HBV, HCV, and HGV infections did not differ between the two groups. The clinical backgrounds were similar between the two groups in both highand non-endemicity areas.
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The age-specific prevalence of TTV, HCV, and HGV infections in the high-endemicity area is shown in Fig. 1. Patients who had a marker indicating the existence of viremia were defined as having ongoing or present infection, and those who had antibody in the absence of viremia were considered to have resolved or past infection. Present infection as well as total infection (past and present ones) with HCV was significantly (P B0.0001 for present infection and PB0.0001 for total infection) more prevalent in individuals with ages over 50 years (32.9% for present
Fig. 1. Age-specific prevalences of TTV, HCV, and HGV infections in the high-endemicity area. Prevalence of exposure is indicated by both filled and open bars and reflects a positive test for at least one viral marker (HCV RNA and/or HCV antibody for HCV; and HGV RNA and/or HGV-E2 antibody for HGV). Filled bars indicate a positive test for a marker of viremia (TTV DNA for TTV, HCV RNA for HCV, and HGV RNA for HGV).
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Table 3 Comparison of biochemical liver function tests according to the status of TTV DNA and HCV RNA Tests
TTV DNA alone (n=178)
HCV RNA alone (n = 27)
TTV DNA and HCV RNA (n = 23)
Neither TTV DNA nor HCV RNA (n =172)
ALT (IU/l) AST (IU/l) ZTT (KU)
17.19 11.5 21.39 8.4 8.4 9 3.5
43.4950.1a 37.19 21.7c 14.396.1e
34.3 9 24.5b 35.1 913.8d 14.2 9 6.7f
16.4 9 10.0 20.0 95.1 8.3 93.0
Data are expressed as mean 9 SD. Other comparisons among the four groups in each test are not statistically significant. P values were obtained by the Student’s t-test for all characteristics. a PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. b PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. c PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. d PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. e PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA. f PB0.001 vs. TTV DNA alone and PB0.001 vs. neither TTV DNA nor HCV RNA.
infection and 40.7% for total infection) than in those under 50 (5.0% for present infection and 8.3% for total infection). Total infection of HGV was significantly more prevalent in individuals with ages over 50 years (40.7%) than in those under 50 (10.0%), but present infection with HGV was similar among the age groups. On the other hand, present infection with TTV was significantly (P= 0.0007) more prevalent in individuals aged over 60 years (68.8%) than in those under 60 (45.1%). In the non-endemicity area, the prevalence of TTV present infection was similar among the age groups; under 29 years; 0% (0/1), 30–39 years; 45.5% (10/22), 40–49 years; 29.7% (11/37), 50 – 59 years; 50.0% (20/40), 60–69 years; 41.3% (26/63), over 70 years; 48.6% (18/37). Mean levels of ALT, AST and ZTT in serum are compared according to the status of TTV DNA and HCV RNA in Table 3. The mean ALT level was significantly higher in individuals with TTV DNA and HCV RNA and in those with HCV RNA alone than in those with TTV DNA alone or in those with neither TTV DNA nor HCV RNA, respectively. Other comparisons among the four groups were not statistically significant, including the comparison between those with TTV DNA alone and those with neither TTV DNA nor HCV RNA. A similar tendency was observed for AST and ZTT tests.
4. Discussion Okamoto et al. have reported that the prevalence of TTV DNA is 12% in Japanese healthy blood donors [2]. Our preliminary data also shows that the prevalence is 12% (6/50) in blood donors (data not shown). Compared to those data, the prevalence of TTV DNA was about three times higher in the control area (43%) of the present study, in which the prevalence of other hepatitis viral markers
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was similar to that in other parts of Japan. The control area as well as the high-endemicity area is surrounded by mountains and is relatively isolated from other communities, but the lifestyle in this area does not seem to differ from that in other parts of Japan [3,6,7]. Thus, it is possible that the relatively isolated circumstances had contributed to the higher prevalence of TTV infection in the control area. Further studies are required to clarify whether our control area is extraordinary in terms of TTV infection, as sufficient epidemiological studies on TTV infection have not been carried out. Although the prevalence of TTV DNA was high in the control area, it was significantly higher in the high endemicity area (58%) than in the control area. The prevalence was similar (around 40%) in individuals under 60 years old between the two areas, but it increased to near 70% in individuals over 60 years old only in the high-endemicity area. HCV and HGV infections were much more common in individuals over 50 years old in the high-endemicity area, thus the age-specific prevalence of TTV infection differed from that of HCV or HGV infection. We have previously reported that HGV infection is closely associated with HCV infection, and have suggested that HGV spread in a similar manner to HCV in the high-endemicity area for hepatitis C [6,7]. On the contrary, TTV infection was not positively associated with either HCV infection or HGV infection. Furthermore, a history of folk remedies, which can be responsible for the spread of HCV and HGV, was similarly seen between individuals with and without TTV DNA. These results indicate that TTV is in a different manner than HCV and HGV. In support of the above results, the data from the present study suggest that histories of surgical operation and blood transfusion, both of which are potentially responsible for the transmission of blood-borne hepatitis viruses, were not associated with TTV infection either in the high-endemicity or the control areas. It is noteworthy that TTV infection showed a reciprocal association with HCV infection in the high-endemicity area, that is, the prevalence of any HCV marker was significantly lower in individuals with TTV DNA than in those without it. Furthermore, the two individuals with HCV infection in the control area were negative for TTV DNA. The following possibilities were considered as explanations. First, the main transmission routes are mutually exclusive between HCV and TTV. Second, a reciprocal interaction exists between TTV and HCV replications, as has been observed between HBV and HCV replications [14,15]. The elevation of transaminases and ZTT, which reflect the level of g-globulin, were dependent on HCV viremia but not on TTV viremia. A similar relationship has been reported by several investigators regarding HCV and HGV infections in which no or a limited pathogenecity of HGV as a hepatitis virus has been suggested [10,16 – 18]. Thus, a limited effect of TTV infection on the elevation of liver enzymes is suggested in the present study.
Acknowledgements This research was supported in part by a Grant-in-Aid from the Ministry of
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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 the members of the South Kiso study group for their assistance at the medical screenings, and Ms Yuki Sugiyama and Ms Kafumi Todoriki for their technical assistance. We also thank Professor Makoto Mayumi for providing us primers of TTV.
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