- Email: [email protected]
TT virus infection among blood donors and patients with non-B, non-C liver diseases in Korea
1
Journal of Hepatology 1999; 30:389-393 Printed in Denmark • All rights reserved Munksgaard. Copenhagen
Copyright © European Association for the Study of the Liver 1999 Journal of Hepatoiogy ISSN 0168-8278
T r virus infection among blood donors and patients with non-B, non-C liver diseases in Korea Tatsunori Nakano 1, Young-Min Park 2, Masashi Mizokami 1, Jong-Young Choi 2, Etsuro Orito l, Tomoyoshi Ohno 1, Takanobu Kato 1, Yutaka Kondo 1, Yasuhito Tanaka a, Hideaki Kato 1, Tetsuo Kato 1 and Boo-Sung Kim 2 t Second Department of Medicine, Nagoya City University Medical School, Nagoya, Japan and 2Department of Internal Medicine, Kangnam St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Background~Aims: A novel virus, designated the TT virus (TTV), was isolated from the serum of a patient with posttransfusion hepatitis of unknown etiology, in Japan. Subsequently, TTV was suggested to be a causative agent in a proportion of cases with cryptogenic hepatitis in Japan. This study aimed to elucidate the significance of TTV infection in cases with cryptogenic liver disease in Korea, a neighbor of Japan. Methods: The prevalence of TTV infection was studied in 120 patients with liver diseases, including 85 patients diagnosed as having non-B, non-C liver diseases. As controls, 220 blood donors were also examined. TTV D N A was detected by polymerase chain reaction, and the sequence was analyzed by phylogenetic analysis. Results: Fourteen (14.0%) of 100 accepted blood donors, 23 (19.2%) of 120 rejected blood donors, and 15 (17.6%) of 85 patients with non-B, non-C liver dis-
eases were positive for TTV DNA. The prevalences of TTV infection among these groups were not significantly different. Phylogenetic analysis suggested the existence of four major genotypes of TTV. The proportions of each genotype among patients with non-B, non-C liver diseases were not different from those among accepted blood donors. Conclusions: TTV exists in Korea, but the prevalence among patients with non-B, non-C liver diseases was almost the same as that among blood donors. TTV may not be the main causative agent of cryptogenic liver disease in Korea. The relationship between nonB, non-C liver diseases and TTV genotype remains unclear, although TTV can be classified into four genotypes.
N 1995 and 1996, GB virus C and hepatitis G virus (GBV-C/HGV) (1,2), were identified as causative agents of non A - E hepatitis. However, it has become increasingly evident that GBV-C/HGV infection cannot account for a significant proportion of patients with non A - E hepatitis (3). Since GBV-C/HGV R N A was identified in only 7.7% of patients with non-B, non-C liver diseases in Korea (4), viral agents other than GBV-C/HGV must be responsible for inducing non A-E hepatitis in the country. Last year, a novel
D N A virus, designated the TT virus (TTV), was isolated in Japan, from the serum of a patient with posttransfusion hepatitis of unknown etiology (5). Subsequently, the prevalences of TTV infection among Japanese blood donors and Japanese patients with cryptogenic hepatitis were reported to be 12% and 46%, respectively, and TTV was suggested to be responsible for a proportion of cases with acute and chronic liver disease of unknown etiology in Japan (6). An epidemiologic aspect of chronic liver diseases in Korea is quite different from that in Japan, although Korea is a neighbor of Japan. Namely, the main cause of chronic liver diseases in Korea is hepatitis B virus (HBV) (7,8), whereas that in Japan is hepatitis C virus (HCV) (9,10). Therefore, the prevalence and significance of TTV infection in relation to liver diseases are also of interest in Korea, and we have investigated the
I
Received 27 July; rev&ed5 October; accepted 19 October 1998
Correspondence: Masashi Mizokami, Second Department of Medicine, Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya 467-8601, Japan. Tel: 81 52 853 8216. Fax: 81 52 852 0849. e-mail: [email protected]
Key words: Blood donor; Genotype; Korea; Non-B, non-C liver disease; TT virus (TTV).
389
T. N a k a n o et al.
presence of TTV DNA in the sera of blood donors and patients with liver diseases in Korea. TTV was classified into two genotypes and four subtypes by Okamoto et al. (6), and divided into three genotypes by Simmonds et al. (11), using phylogenetic analysis. Phylogenetic analysis is useful for identifying the origin of a virus (12), and tracing its routes of transmission (13). The genotyping of a virus may also be beneficial for the evaluation of liver disease progression or response to interferon, as has been observed for hepatitis C virus (HCV) (14,15). We therefore analyzed the heterogeneity of the detected TTV in a Korean population using phylogenetic analysis, and we discuss the association of liver diseases with genotypes of TTV.
The mean ages were 26.6 (range: 16-53), 24.7 (1848), 42.8 (2075), and 42.2 (20-67) years in the accepted blood donor group, rejected blood donor group, patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively (Table 1). The mean age of each group with liver diseases was significantly higher than that of the accepted blood donor group, whereas the mean age of the rejected blood donors was almost the same as that of the accepted blood donor group. Mean ALT levels were 18.6 (range: 337), 109.4 (66-333), 167.6 (10-1466), and 248.6 (32-1829) IU/I in the accepted blood donor group, rejected blood donor group, patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively (Table 1). Mean ALT levels in both the rejected blood donor group and each of the groups with liver diseases were significantly higher than the level in the accepted blood donor group.
Materials and Methods Blood donors and patients" with liver diseases A total of 340 samples were tested in this study (Table 1). The sera of 220 blood donors, who were negative for hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus antibody (anti-HCV), were collected from the Korean Red Cross Blood Bank. One hundred of these samples were from accepted blood donors with normal alanine aminotransferase (ALT) levels (less than 40 IU/1), and 120 samples were from blood donors rejected because of elevated ALT levels (more than 40 IU/I). Patients were diagnosed by interview to obtain the history, serological tests, biochemical liver function tests, ultrasonography, computerized tomography, angiography, and/or liver biopsy at the Department of Internal Medicine, Kangnam St. Mary's Hospital, Catholic University Medical College, Seoul, Korea, from 1993 to 1996. Eighty-five patients were diagnosed as having non-B, non-C liver diseases by their negative results for both HBsAg and anti-HCV. The 85 patients did not have a nonviral etiology of liver injury such as alcohol addiction, drug administration, and metabolic diseases. Thirty-five were diagnosed as type B liver diseases by their positive results for HBsAg and negative results for anti-HC~ Detection of HBV DNA or HCV RNA, using polymerase chain reaction (PCR), could not be performed in all patients sera because of the small volume of some sera. Informed consent was obtained from all subjects who participated in this study.
Serological tests All serum samples were tested for HBsAg by radioimmunoassay and for anti-HCV by second-generation enzyme immunoassay (Abbott, North Chicago, IL, USA). All serum samples were tested for ALT levels. Detection o f T T V D N A Serum samples from all patients were stored at -80°C until assays. DNA was extracted from 100/A serum with proteinase K and sodium dodecyl sulfate, as reported previously (5). TTV DNA was amplified by AmpliTaq Gold DNA Polymerase (Roche Molecular Systems, Inc. Branchburg, NJ, USA) with semi-nested primers derived from the sequence of ORF 1. as reported previously (6). The specific primers used for PCR were as follows.
NG059: 5'-ACA GAC AGA GGA GAA GGC AAC ATG-3' [nucleotide position: 1900-1923 in TA278 (accession number: AB008394) (6)]. NG061: 5'-GGC AAC ATG YTR TGG ATA GAC TGG [Y=T or C, R = A or G, nucleotide position: 1915 1938]. NG063: 5'-CTG GCA TTT TAC CAT TTC CAA AGT T-3' [nucleotide position: 2161--2185]. In brief, the first round of PCR was performed with the sense primer NG059 and the anti-sense primer NG063 for 9 rain at 96°C, followed by 35 cycles, consisting of denaturation for 30 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C, using a 96well thermal cycler (GeneAmp 9600, Perkin-Elmer Cetus, Norwalk, USA). The second round of PCR was performed with the sense primer NG061 and the anti-sense primer NG063 for 25 cycles, under the same conditions as those used for the first round of PCR. The
TABLE 1 Subjects evaluated for positivity of TTV DNA in Korea Subjects
n
Blood donors Accepted blood donors
100
Rejected blood donors
120
Sex (M:F) (77:23) (118:2)
Age (year) mean-+SD (range)
ALT (IU/I) mean_+SD (range)
26.6_+8.6(16-53)
18.6-+8.3 (3 37)
/
24.7-+6.7(18-48) l
85 71 10 4
(54:31) (44:27) (6:4) (4:0)
42.8-+ 13.9 (20-75) ~ 40.4_ + 13.1 (20-75) 55.2-+8.2(41-66) 61.8-+6.7(56 71)
Type B liver diseases (CH:23, LC:12)
35
(21:14)
42.2-+12.1(20-67)
*p
390
340
(270:70)
Anti-HCV positive
0
0
109.4-+47.9 (66-333)
0
0
167.6-+237.2 (10 1466) 193.1-+260.1 (10-1466) 45.4-+34.7 (11-120) 57.5-+20.7 (31-81)
0
0
248.6-+342.7 (32-1829)
35
/
Non-B, non-C liver diseases Chronic hepatitis (CH) Liver cirrhosis (LC) Hepatocelular carcinoma
Total
*
HBsAg positive
31.6-+ 12.9 (16-75)
*
35
T T V infection in Korea amplified products were analyzed by electrophoresis on 3% agarose gels, stained with ethidium bromide, and observed under ultraviolet light.
Determination of nucleotide sequence of T T V The specificity of amplification was confirmed by direct sequencing of the amplified products with a 373A DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences of TTV DNA determined by us in this study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB017821 to AB017872. Molecular evolutionary analysis To investigate the relationship between the sequences obtained in this study and those of the previously reported strains (6,11), a phylogenetic tree was constructed by the neighbor-joining method (16) using genetic distances calculated by the 6-parameter method in the computer program ODEN ver. 1.1.1 (17,18). To confirm the reliability of the tree, we performed a bootstrap resampling test'1000 times (19). Statistics Data were analyzed by the Fisher exact test, Mann-Whitney U test, or chi-square test. A p-value less than 0.05 was regarded as statistically significant.
Results Positivities for T T V DNA among blood donors and patients with liver diseases in Korea TTV DNA was detected in 57 (16.8%) of 340 subjects (Table 2). Fourteen (14.0%) of the 100 accepted blood donors and 23 (19.2%) of the 120 rejected blood donors were positive for serum TTV DNA. Of the 85 patients with non-B, non-C liver diseases, 15 (17.6%) were positive for TTV DNA: 13 (18.3%) of 71 with chronic hepatitis, one (10.0%) of ten with liver cirrhosis and one (25%) of four with hepatocellular carcinoma.
TABLE 2 TTV DNA among blood donors and patients with liver diseases in Korea Subjects
Positivity of TTV* n (%)
Genotype of TTV t Gla:Glb:G2:G3:G4
Blood donors Accepted blood donors Rejected blood donors
14/100 (14.0°,4) 0:3:9:0:0 23/120 (19.2°,4) 11:7:5:0:0
Non-B, non-C liver diseases Chronic hepatitis (CH) Liver cirrhosis (LC) Hepatocellular carcinoma
15/85 (17.6%) 13/71 (18.3%) 1/10 (10.0%) 1/4 (25%)
2:5:4:0:1 1:4:4:0:1 1:0:0:0:0 0:1:0:0:0
5/35 (14.3%)
3:2:0:0:0
Type B liver diseases (CH:23, LC:12 Total
57/340 (16.8%)
16:17:18:0:1 ~
* No significant difference was observed in the positivities of TTV DNA among all groups. * The proportions of each genotype, which was determined using phylogenetic analysis, are shown. No significant difference was observed in the proportion of each genotype among all groups. Fifty-two nucleotide sequences of 57 TTV DNA could be determined by direct sequencing.
Five (14.3%) of the 35 patients with type B liver diseases were positive for TTV DNA. No significant difference was observed in the prevalence of TTV D N A between accepted blood donors, and rejected donors or patients with non-B, non-C liver diseases. Furthermore, the prevalences among patients with non-B, nonC liver diseases were not higher than that among patients with type B liver diseases. When the 340 subjects were divided into two groups (those with and those without TTV DNA), no significant difference in ALT levels was noted between these two groups (data not shown). There was also no significant difference between the two groups, with respect to age, sex, and positivity for HBsAg.
Sequence heterogeneities of T T V DNA in Korea Fifty-two nucleotide sequences of 57 TTV DNA-positive amplicons could be determined by direct sequencing. To analyze the heterogeneity of the sequences obtained in this study, we used reference sequences, for example, N22 and TA278, TX011, TS003, and NA004 obtained from Japanese subjects, as markers for the two major types and four serial subtypes of TTV, namely, genotype la (Gla), genotype lb (Glb), genotype 2a (G2a), and genotype 2b (G2b) (6). We also included sequences of TTV DNA reported recently by Simmonds et al. (11), with this analysis. We aligned the nucleotide sequences obtained in this study with the corresponding region of the reference sequences (222 bp, nucleotide position: 1939-2160) and estimated the genetic distances between them. Based on these estimates, a phylogenetic tree was constructed. These sequences were classified into four major genotypes (Fig. 1). The sequences, which were reported as genotype 3 (G3) (11), clustered to a branch distinct from those of G1 and G2. Among Korean clones, 33 isolates belonged to G1, 18 to G2, 0 to G3, and one, obtained from a patient with non-B, non-C chronic hepatitis, was assigned to a novel genotype, tentatively named genotype 4 (G4). On bootstrap analysis for evaluation of the statistical reliability of the tree, the clusters of G1, G2 and G3 exhibited 99.5, 94.5, and 99.5% reliability, respectively. Sixteen belonged to Gla, 17 to Glb, 2 to G2a, 9 to G2b, and 7 belonged to G2 but not to G2a or G2b. The clones isolated from accepted blood donors or patients with type B liver diseases and patients with non-B, non-C liver diseases or rejected blood donors clustered to G1 as well as G2. The proportions of each genotype among patients with non-B, non-C liver diseases or rejected blood donors were not significantly different from those among accepted blood donors or patients with type B liver diseases (Table 2). 391
T. Nakano et al.
I--'--
. . . . J~l~2 18
I
I i
99.5%
II
,..,£..~mz7+i*
V_A L
94.5% G2 81.2% 99.5% 99.5% G3
AFO~. NBCI52CH
G4 0.'2oo
0.i00
6
Fig. 1. A phylogenetic tree of T T virus isolated fi'om a Korean population based on the partial nucleotide sequences of open reading frame 1 (nucleotide length." 222 bp) (6). This tree was constructed by the neighbor-joining method with the reference sequences, N22 and TA278 (Gla), TX011 (Glb), TSO03 (G2a), and NAO04 (G2b) reported previously (6). Sequences reported by' Simmonds et al. were also included in this' analysis" (AF072738, AF072739, AFO72741-AF072749, AFO79537-AF079543) (11). The previously reported sequences are shown by asterisks (*). There are 4 major clusters, tentatively named genotype 1 (G1), genotype 2 (G2) genotype 3 (G3), and genotype 4 (G4). On bootstrap analysis for evaluation of the statistical reliability of the tree, G1, G2, and G3 exhibited 99.5, 94.5, and 99.5% reliability, respectively. The horizontal bar indicates the number of nucleotide substitutions' per site. The prefixes ABD, RBD, NBC, and B indicate that the clones were isolated fi'om accepted blood donors, rejected blood donors', patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively. The suffixes CH, LC, and HCC indicate that the clones were isolated from patients with chronic hepatitis, liver cirrhosis, and hepatoeellalar carcinoma, respectively.
392
Discussion
A high prevalence of TTV infection among patients with cryptogenic hepatitis has been reported in Japan (6). However, the significance of TTV as a causative agent of liver diseases remains unclear at present. We have investigated the prevalence of TTV DNA in serum by PCR with the primers reported by Okamoto et al. (6) among Korean patients with non-B, non-C liver diseases, and compared this prevalence with the prevalence among blood donors and patients with type B liver diseases, because hepatitis B virus infection is still the main cause of chronic liver diseases in Korea (7,8). Since none of the blood donors rejected due to elevated ALT levels had either HBsAg or anti-HCV, a certain proportion of these subjects were associated with non-B, non-C liver diseases. The prevalence of TTV infection not only among patients with non-B, non-C liver diseases, but also among rejected donors, whose age distribution was almost the same as that among accepted blood donors, was not high compared to that among accepted blood donors (Table 2). Furthermore, the prevalence among patients with non-B, non-C liver disease was not higher than that among patients with type B liver diseases (Table 2). When the subjects were divided into two groups (those with and those without TTV DNA), no significant difference in ALT levels was noted. These data indicate that TTV is not the main causative agent of cryptogenic liver disease in Korea. However, this virus has been reported to be a possible cause of posttransfusion hepatitis (5). At present, the genomic organization of this virus is still unknown and a more stable detection system for TTV infection is awaited. Further studies will be required for evaluation of the prevalence of TTV infection and its association with liver diseases. We analyzed the genotypes of TTV using the PCR amplicon used for the detection. A phylogenetic analysis suggested an additional TTV genotype distinct from G1, G2, and G3, and other subtypes of G2, distinct from G2a and G2b (Fig. 1). The high reliability of these results was confirmed by bootstrap analysis. The proportion of subjects with G2 in Korea was higher than that in Japan (6). The clones isolated from patients with non-B, non-C liver diseases or rejected blood donors clustered to three genotypes except G3. The proportions of each genotype among patients with non-B, non-C liver diseases or rejected blood donors were not different from those among accepted blood donors or patients with type B liver diseases (Table 2). These data indicate no association of non-B, non-C liver diseases with any specific genotype or subtype of
T T V infection in Korea
TTV. However, the significance of G3, G4, and the new subtypes of G2 and the association of liver disease progression with genotype could not be evaluated due to the small numbers of isolated clones. Further studies will be required on a larger scale. In conclusion, TTV exist in Korea, but the prevalence among patients with non-B, non-C liver diseases was almost the same as that among blood donors. TTV may not be the main causative agent of cryptogenic liver disease in Korea. The relationship between non-B, non-C liver diseases and TTV genotype remains unclear, although TTV can be classified into four major genotypes and G2 can be divided into more subtypes. Acknowledgements M. Mizokami was supported in part by a Grant-in-Aid for Scientific Research (06670571) from the Japanese Ministry of Education, Science, Sports, and Culture, and grants from the Japanese Ministry of Health and Welfare, Health Science Research Grants (Non-A, nonB Hepatitis Research Grants) and the Viral Hepatitis Research Foundation of Japan. Y.-M. Park was supported in part by a grant for health scientific research from the Korean Ministry of Health and Welfare (HMP-56-M-0037) and in part by grant for the Medical Science Research Foundation from the Catholic Medical Center of Korea.
References 1. Simons JN, Leary TP, Dawson GJ, Pilot MT, Muerhoff AS, Schlauder GG, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1995; 1: 564-9. 2. Linnen J, Wages J J, Zhang KZ, Fry KE, Krawczynski KZ, Alter H, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996; 271: 505-8. 3. Alter H J, Nakatsuji Y, Melpolder J, Wages J, Wesley R, Shih JW, et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 1997; 336: 747-54.
4. Park YM, Mizokami M, Nakano T, Choi JY, Cao K, Byun BH, et al. GB virus C/hepatitis G virus infection among Korean patients with liver diseases and general population. Virus Res 1997; 48: 185-92. 5. 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: 92-7. 6. Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H, et al. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res 1998; 10: 1-16. 7. Park BC, Han BH, Ahn SY, Lee SW, Lee DH, Lee YN, et al. Prevalence of hepatitis C antibody in patients with chronic liver disease and hepatocellular carcinoma in Korea. J Viral Hepat 1995; 2: 195-202. 8. Ahn YO. Strategy for vaccination against hepatitis B in areas with high endemicity: focus on Korea. Gut 1996; 38: $63-$66. 9. Nishioka K, Watanabe J, Furuta S, Tanaka E, Iino S, Suzuki H, et al. A high prevalence of antibody to the hepatitis C virus in patients with hepatocellular carcinoma in Japan. Cancer 1991; 67: 429-33. 10. Nishioka K. Hepatitis C virus infection in Japan. Gastroenterol Jpn 1991; 26: S152-S155. 11. Simmonds P, Davidson F, Lycett C, Prescott LE, MacDonald DM, Ellender J, et al. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet 1998; 352: 191-5. 12. Tanaka Y, Mizokami M, Orito E, Ohba K-I, Kato T, Kondo Y, et al. African origin of GB virus C/hepatitis G virus. FEBS Lett 1998; 423:143-48 13. Tahara T, Toyoda S, Mukaide M, Hikiji K, Ohba K, Mizokami M. Vertical transmission of hepatitis C through three generations. Lancet 1996; 347: 409. 14. Dusheiko G, Schmilovitz WH, Brown D, McOmish E Yap PL, Sherlock S, et al. Hepatitis C virus genotypes: an investigation of type-specific differences in geographic origin and disease. Hepatology 1994; 19: 13-8. 15. Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M, et aL Detection of hepatitis C virus by polymerase chain reaction and response to interferon-alpha therapy: relationship to genotypes of hepatitis C virus. Hepatology 1992; 16: 293-9. 16. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4: 40625. 17. Gojobori T, Ishii K, Nei M. Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide. J Mol Evol 1982; 18: 414-23. 18. Ina Y. ODEN: a program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Comput Appl Biosci 1994; 10:11-2. 19. Felsenstain J. Confidence limits on pylogenies. Evolution 1985; 39: 783-91.
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Journal of Hepatology 1999; 30:389-393 Printed in Denmark • All rights reserved Munksgaard. Copenhagen
Copyright © European Association for the Study of the Liver 1999 Journal of Hepatoiogy ISSN 0168-8278
T r virus infection among blood donors and patients with non-B, non-C liver diseases in Korea Tatsunori Nakano 1, Young-Min Park 2, Masashi Mizokami 1, Jong-Young Choi 2, Etsuro Orito l, Tomoyoshi Ohno 1, Takanobu Kato 1, Yutaka Kondo 1, Yasuhito Tanaka a, Hideaki Kato 1, Tetsuo Kato 1 and Boo-Sung Kim 2 t Second Department of Medicine, Nagoya City University Medical School, Nagoya, Japan and 2Department of Internal Medicine, Kangnam St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Background~Aims: A novel virus, designated the TT virus (TTV), was isolated from the serum of a patient with posttransfusion hepatitis of unknown etiology, in Japan. Subsequently, TTV was suggested to be a causative agent in a proportion of cases with cryptogenic hepatitis in Japan. This study aimed to elucidate the significance of TTV infection in cases with cryptogenic liver disease in Korea, a neighbor of Japan. Methods: The prevalence of TTV infection was studied in 120 patients with liver diseases, including 85 patients diagnosed as having non-B, non-C liver diseases. As controls, 220 blood donors were also examined. TTV D N A was detected by polymerase chain reaction, and the sequence was analyzed by phylogenetic analysis. Results: Fourteen (14.0%) of 100 accepted blood donors, 23 (19.2%) of 120 rejected blood donors, and 15 (17.6%) of 85 patients with non-B, non-C liver dis-
eases were positive for TTV DNA. The prevalences of TTV infection among these groups were not significantly different. Phylogenetic analysis suggested the existence of four major genotypes of TTV. The proportions of each genotype among patients with non-B, non-C liver diseases were not different from those among accepted blood donors. Conclusions: TTV exists in Korea, but the prevalence among patients with non-B, non-C liver diseases was almost the same as that among blood donors. TTV may not be the main causative agent of cryptogenic liver disease in Korea. The relationship between nonB, non-C liver diseases and TTV genotype remains unclear, although TTV can be classified into four genotypes.
N 1995 and 1996, GB virus C and hepatitis G virus (GBV-C/HGV) (1,2), were identified as causative agents of non A - E hepatitis. However, it has become increasingly evident that GBV-C/HGV infection cannot account for a significant proportion of patients with non A - E hepatitis (3). Since GBV-C/HGV R N A was identified in only 7.7% of patients with non-B, non-C liver diseases in Korea (4), viral agents other than GBV-C/HGV must be responsible for inducing non A-E hepatitis in the country. Last year, a novel
D N A virus, designated the TT virus (TTV), was isolated in Japan, from the serum of a patient with posttransfusion hepatitis of unknown etiology (5). Subsequently, the prevalences of TTV infection among Japanese blood donors and Japanese patients with cryptogenic hepatitis were reported to be 12% and 46%, respectively, and TTV was suggested to be responsible for a proportion of cases with acute and chronic liver disease of unknown etiology in Japan (6). An epidemiologic aspect of chronic liver diseases in Korea is quite different from that in Japan, although Korea is a neighbor of Japan. Namely, the main cause of chronic liver diseases in Korea is hepatitis B virus (HBV) (7,8), whereas that in Japan is hepatitis C virus (HCV) (9,10). Therefore, the prevalence and significance of TTV infection in relation to liver diseases are also of interest in Korea, and we have investigated the
I
Received 27 July; rev&ed5 October; accepted 19 October 1998
Correspondence: Masashi Mizokami, Second Department of Medicine, Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya 467-8601, Japan. Tel: 81 52 853 8216. Fax: 81 52 852 0849. e-mail: [email protected]
Key words: Blood donor; Genotype; Korea; Non-B, non-C liver disease; TT virus (TTV).
389
T. N a k a n o et al.
presence of TTV DNA in the sera of blood donors and patients with liver diseases in Korea. TTV was classified into two genotypes and four subtypes by Okamoto et al. (6), and divided into three genotypes by Simmonds et al. (11), using phylogenetic analysis. Phylogenetic analysis is useful for identifying the origin of a virus (12), and tracing its routes of transmission (13). The genotyping of a virus may also be beneficial for the evaluation of liver disease progression or response to interferon, as has been observed for hepatitis C virus (HCV) (14,15). We therefore analyzed the heterogeneity of the detected TTV in a Korean population using phylogenetic analysis, and we discuss the association of liver diseases with genotypes of TTV.
The mean ages were 26.6 (range: 16-53), 24.7 (1848), 42.8 (2075), and 42.2 (20-67) years in the accepted blood donor group, rejected blood donor group, patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively (Table 1). The mean age of each group with liver diseases was significantly higher than that of the accepted blood donor group, whereas the mean age of the rejected blood donors was almost the same as that of the accepted blood donor group. Mean ALT levels were 18.6 (range: 337), 109.4 (66-333), 167.6 (10-1466), and 248.6 (32-1829) IU/I in the accepted blood donor group, rejected blood donor group, patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively (Table 1). Mean ALT levels in both the rejected blood donor group and each of the groups with liver diseases were significantly higher than the level in the accepted blood donor group.
Materials and Methods Blood donors and patients" with liver diseases A total of 340 samples were tested in this study (Table 1). The sera of 220 blood donors, who were negative for hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus antibody (anti-HCV), were collected from the Korean Red Cross Blood Bank. One hundred of these samples were from accepted blood donors with normal alanine aminotransferase (ALT) levels (less than 40 IU/1), and 120 samples were from blood donors rejected because of elevated ALT levels (more than 40 IU/I). Patients were diagnosed by interview to obtain the history, serological tests, biochemical liver function tests, ultrasonography, computerized tomography, angiography, and/or liver biopsy at the Department of Internal Medicine, Kangnam St. Mary's Hospital, Catholic University Medical College, Seoul, Korea, from 1993 to 1996. Eighty-five patients were diagnosed as having non-B, non-C liver diseases by their negative results for both HBsAg and anti-HCV. The 85 patients did not have a nonviral etiology of liver injury such as alcohol addiction, drug administration, and metabolic diseases. Thirty-five were diagnosed as type B liver diseases by their positive results for HBsAg and negative results for anti-HC~ Detection of HBV DNA or HCV RNA, using polymerase chain reaction (PCR), could not be performed in all patients sera because of the small volume of some sera. Informed consent was obtained from all subjects who participated in this study.
Serological tests All serum samples were tested for HBsAg by radioimmunoassay and for anti-HCV by second-generation enzyme immunoassay (Abbott, North Chicago, IL, USA). All serum samples were tested for ALT levels. Detection o f T T V D N A Serum samples from all patients were stored at -80°C until assays. DNA was extracted from 100/A serum with proteinase K and sodium dodecyl sulfate, as reported previously (5). TTV DNA was amplified by AmpliTaq Gold DNA Polymerase (Roche Molecular Systems, Inc. Branchburg, NJ, USA) with semi-nested primers derived from the sequence of ORF 1. as reported previously (6). The specific primers used for PCR were as follows.
NG059: 5'-ACA GAC AGA GGA GAA GGC AAC ATG-3' [nucleotide position: 1900-1923 in TA278 (accession number: AB008394) (6)]. NG061: 5'-GGC AAC ATG YTR TGG ATA GAC TGG [Y=T or C, R = A or G, nucleotide position: 1915 1938]. NG063: 5'-CTG GCA TTT TAC CAT TTC CAA AGT T-3' [nucleotide position: 2161--2185]. In brief, the first round of PCR was performed with the sense primer NG059 and the anti-sense primer NG063 for 9 rain at 96°C, followed by 35 cycles, consisting of denaturation for 30 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C, using a 96well thermal cycler (GeneAmp 9600, Perkin-Elmer Cetus, Norwalk, USA). The second round of PCR was performed with the sense primer NG061 and the anti-sense primer NG063 for 25 cycles, under the same conditions as those used for the first round of PCR. The
TABLE 1 Subjects evaluated for positivity of TTV DNA in Korea Subjects
n
Blood donors Accepted blood donors
100
Rejected blood donors
120
Sex (M:F) (77:23) (118:2)
Age (year) mean-+SD (range)
ALT (IU/I) mean_+SD (range)
26.6_+8.6(16-53)
18.6-+8.3 (3 37)
/
24.7-+6.7(18-48) l
85 71 10 4
(54:31) (44:27) (6:4) (4:0)
42.8-+ 13.9 (20-75) ~ 40.4_ + 13.1 (20-75) 55.2-+8.2(41-66) 61.8-+6.7(56 71)
Type B liver diseases (CH:23, LC:12)
35
(21:14)
42.2-+12.1(20-67)
*p
390
340
(270:70)
Anti-HCV positive
0
0
109.4-+47.9 (66-333)
0
0
167.6-+237.2 (10 1466) 193.1-+260.1 (10-1466) 45.4-+34.7 (11-120) 57.5-+20.7 (31-81)
0
0
248.6-+342.7 (32-1829)
35
/
Non-B, non-C liver diseases Chronic hepatitis (CH) Liver cirrhosis (LC) Hepatocelular carcinoma
Total
*
HBsAg positive
31.6-+ 12.9 (16-75)
*
35
T T V infection in Korea amplified products were analyzed by electrophoresis on 3% agarose gels, stained with ethidium bromide, and observed under ultraviolet light.
Determination of nucleotide sequence of T T V The specificity of amplification was confirmed by direct sequencing of the amplified products with a 373A DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences of TTV DNA determined by us in this study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB017821 to AB017872. Molecular evolutionary analysis To investigate the relationship between the sequences obtained in this study and those of the previously reported strains (6,11), a phylogenetic tree was constructed by the neighbor-joining method (16) using genetic distances calculated by the 6-parameter method in the computer program ODEN ver. 1.1.1 (17,18). To confirm the reliability of the tree, we performed a bootstrap resampling test'1000 times (19). Statistics Data were analyzed by the Fisher exact test, Mann-Whitney U test, or chi-square test. A p-value less than 0.05 was regarded as statistically significant.
Results Positivities for T T V DNA among blood donors and patients with liver diseases in Korea TTV DNA was detected in 57 (16.8%) of 340 subjects (Table 2). Fourteen (14.0%) of the 100 accepted blood donors and 23 (19.2%) of the 120 rejected blood donors were positive for serum TTV DNA. Of the 85 patients with non-B, non-C liver diseases, 15 (17.6%) were positive for TTV DNA: 13 (18.3%) of 71 with chronic hepatitis, one (10.0%) of ten with liver cirrhosis and one (25%) of four with hepatocellular carcinoma.
TABLE 2 TTV DNA among blood donors and patients with liver diseases in Korea Subjects
Positivity of TTV* n (%)
Genotype of TTV t Gla:Glb:G2:G3:G4
Blood donors Accepted blood donors Rejected blood donors
14/100 (14.0°,4) 0:3:9:0:0 23/120 (19.2°,4) 11:7:5:0:0
Non-B, non-C liver diseases Chronic hepatitis (CH) Liver cirrhosis (LC) Hepatocellular carcinoma
15/85 (17.6%) 13/71 (18.3%) 1/10 (10.0%) 1/4 (25%)
2:5:4:0:1 1:4:4:0:1 1:0:0:0:0 0:1:0:0:0
5/35 (14.3%)
3:2:0:0:0
Type B liver diseases (CH:23, LC:12 Total
57/340 (16.8%)
16:17:18:0:1 ~
* No significant difference was observed in the positivities of TTV DNA among all groups. * The proportions of each genotype, which was determined using phylogenetic analysis, are shown. No significant difference was observed in the proportion of each genotype among all groups. Fifty-two nucleotide sequences of 57 TTV DNA could be determined by direct sequencing.
Five (14.3%) of the 35 patients with type B liver diseases were positive for TTV DNA. No significant difference was observed in the prevalence of TTV D N A between accepted blood donors, and rejected donors or patients with non-B, non-C liver diseases. Furthermore, the prevalences among patients with non-B, nonC liver diseases were not higher than that among patients with type B liver diseases. When the 340 subjects were divided into two groups (those with and those without TTV DNA), no significant difference in ALT levels was noted between these two groups (data not shown). There was also no significant difference between the two groups, with respect to age, sex, and positivity for HBsAg.
Sequence heterogeneities of T T V DNA in Korea Fifty-two nucleotide sequences of 57 TTV DNA-positive amplicons could be determined by direct sequencing. To analyze the heterogeneity of the sequences obtained in this study, we used reference sequences, for example, N22 and TA278, TX011, TS003, and NA004 obtained from Japanese subjects, as markers for the two major types and four serial subtypes of TTV, namely, genotype la (Gla), genotype lb (Glb), genotype 2a (G2a), and genotype 2b (G2b) (6). We also included sequences of TTV DNA reported recently by Simmonds et al. (11), with this analysis. We aligned the nucleotide sequences obtained in this study with the corresponding region of the reference sequences (222 bp, nucleotide position: 1939-2160) and estimated the genetic distances between them. Based on these estimates, a phylogenetic tree was constructed. These sequences were classified into four major genotypes (Fig. 1). The sequences, which were reported as genotype 3 (G3) (11), clustered to a branch distinct from those of G1 and G2. Among Korean clones, 33 isolates belonged to G1, 18 to G2, 0 to G3, and one, obtained from a patient with non-B, non-C chronic hepatitis, was assigned to a novel genotype, tentatively named genotype 4 (G4). On bootstrap analysis for evaluation of the statistical reliability of the tree, the clusters of G1, G2 and G3 exhibited 99.5, 94.5, and 99.5% reliability, respectively. Sixteen belonged to Gla, 17 to Glb, 2 to G2a, 9 to G2b, and 7 belonged to G2 but not to G2a or G2b. The clones isolated from accepted blood donors or patients with type B liver diseases and patients with non-B, non-C liver diseases or rejected blood donors clustered to G1 as well as G2. The proportions of each genotype among patients with non-B, non-C liver diseases or rejected blood donors were not significantly different from those among accepted blood donors or patients with type B liver diseases (Table 2). 391
T. Nakano et al.
I--'--
. . . . J~l~2 18
I
I i
99.5%
II
,..,£..~mz7+i*
V_A L
94.5% G2 81.2% 99.5% 99.5% G3
AFO~. NBCI52CH
G4 0.'2oo
0.i00
6
Fig. 1. A phylogenetic tree of T T virus isolated fi'om a Korean population based on the partial nucleotide sequences of open reading frame 1 (nucleotide length." 222 bp) (6). This tree was constructed by the neighbor-joining method with the reference sequences, N22 and TA278 (Gla), TX011 (Glb), TSO03 (G2a), and NAO04 (G2b) reported previously (6). Sequences reported by' Simmonds et al. were also included in this' analysis" (AF072738, AF072739, AFO72741-AF072749, AFO79537-AF079543) (11). The previously reported sequences are shown by asterisks (*). There are 4 major clusters, tentatively named genotype 1 (G1), genotype 2 (G2) genotype 3 (G3), and genotype 4 (G4). On bootstrap analysis for evaluation of the statistical reliability of the tree, G1, G2, and G3 exhibited 99.5, 94.5, and 99.5% reliability, respectively. The horizontal bar indicates the number of nucleotide substitutions' per site. The prefixes ABD, RBD, NBC, and B indicate that the clones were isolated fi'om accepted blood donors, rejected blood donors', patients with non-B, non-C liver diseases, and patients with type B liver diseases, respectively. The suffixes CH, LC, and HCC indicate that the clones were isolated from patients with chronic hepatitis, liver cirrhosis, and hepatoeellalar carcinoma, respectively.
392
Discussion
A high prevalence of TTV infection among patients with cryptogenic hepatitis has been reported in Japan (6). However, the significance of TTV as a causative agent of liver diseases remains unclear at present. We have investigated the prevalence of TTV DNA in serum by PCR with the primers reported by Okamoto et al. (6) among Korean patients with non-B, non-C liver diseases, and compared this prevalence with the prevalence among blood donors and patients with type B liver diseases, because hepatitis B virus infection is still the main cause of chronic liver diseases in Korea (7,8). Since none of the blood donors rejected due to elevated ALT levels had either HBsAg or anti-HCV, a certain proportion of these subjects were associated with non-B, non-C liver diseases. The prevalence of TTV infection not only among patients with non-B, non-C liver diseases, but also among rejected donors, whose age distribution was almost the same as that among accepted blood donors, was not high compared to that among accepted blood donors (Table 2). Furthermore, the prevalence among patients with non-B, non-C liver disease was not higher than that among patients with type B liver diseases (Table 2). When the subjects were divided into two groups (those with and those without TTV DNA), no significant difference in ALT levels was noted. These data indicate that TTV is not the main causative agent of cryptogenic liver disease in Korea. However, this virus has been reported to be a possible cause of posttransfusion hepatitis (5). At present, the genomic organization of this virus is still unknown and a more stable detection system for TTV infection is awaited. Further studies will be required for evaluation of the prevalence of TTV infection and its association with liver diseases. We analyzed the genotypes of TTV using the PCR amplicon used for the detection. A phylogenetic analysis suggested an additional TTV genotype distinct from G1, G2, and G3, and other subtypes of G2, distinct from G2a and G2b (Fig. 1). The high reliability of these results was confirmed by bootstrap analysis. The proportion of subjects with G2 in Korea was higher than that in Japan (6). The clones isolated from patients with non-B, non-C liver diseases or rejected blood donors clustered to three genotypes except G3. The proportions of each genotype among patients with non-B, non-C liver diseases or rejected blood donors were not different from those among accepted blood donors or patients with type B liver diseases (Table 2). These data indicate no association of non-B, non-C liver diseases with any specific genotype or subtype of
T T V infection in Korea
TTV. However, the significance of G3, G4, and the new subtypes of G2 and the association of liver disease progression with genotype could not be evaluated due to the small numbers of isolated clones. Further studies will be required on a larger scale. In conclusion, TTV exist in Korea, but the prevalence among patients with non-B, non-C liver diseases was almost the same as that among blood donors. TTV may not be the main causative agent of cryptogenic liver disease in Korea. The relationship between non-B, non-C liver diseases and TTV genotype remains unclear, although TTV can be classified into four major genotypes and G2 can be divided into more subtypes. Acknowledgements M. Mizokami was supported in part by a Grant-in-Aid for Scientific Research (06670571) from the Japanese Ministry of Education, Science, Sports, and Culture, and grants from the Japanese Ministry of Health and Welfare, Health Science Research Grants (Non-A, nonB Hepatitis Research Grants) and the Viral Hepatitis Research Foundation of Japan. Y.-M. Park was supported in part by a grant for health scientific research from the Korean Ministry of Health and Welfare (HMP-56-M-0037) and in part by grant for the Medical Science Research Foundation from the Catholic Medical Center of Korea.
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