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High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors
Journal of Hepatology 1999; 31: 221-221 Printed in Denmark All rights reserved Munksgaard Copenhagen
Copyright 0 European Association for the Study of the Liver 1999
Journal of Hepatology ISSN 0168-8278
High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors Takanobu Kate’, Masashi Mizokami’, Etsuro Orito ’ , Tatsunori Nakano’, Yasuhito Tanaka’, Ryuzo Ueda’, Noboru Hirashima 2, Yoshihiko Iijima3, Tetsuo Kato3, Fuminaka Sugauchi4, Motokazu Mukaide’, Kazuhide Shimamatsu6, Masayoshi Kage6 and Masamichi Kojiro6 ‘Second Department of Medicine. Nagoya City University Medical Nagoya; ‘Section of Gastroenterology, Nagoya Daini Red Cross Hamamatsu; 5Center for Molecular Biology and Cytogenetics, SRL, Medicine,
School, Nagoya; 2Department of Gastroenterology, Chyukyo Hospital, Hospital, Nagoya: 4Department of Medicine. Enshu General Hospital, Inc., Tokyo, and 6Department of Pathology, Kurume University School of Kurume, Japan
Background/Aims: Although a novel DNA virus, TT virus (TTV), has been isolated from a patient with cryptogenic post-transfusion hepatitis, its pathogenic role remains unclear. To elucidate its prevalence and clinical impact in patients with liver diseases, the presence of TTV DNA was assessed in patients with liver diseases and blood donors (BDs) in Japan using two primer sets, one conventional and the other new and highly sensitive. Methods: We studied 261 samples, 72 with chronic hepatitis associated hepatitis C virus (HCV-CH), 57 with hepatocellular carcinoma associated HCV (HCV-HCC), 12 with HCC without either HCV or hepatitis B virus (NBNC-HCC), and 120 of BDs. Results: Using two primer sets, TTV DNA was detected in 68 (94.4%), 53 (93.0%), 12 (lOO%), and 98 (81.7%) HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The prevalence was not significantly different between HCV-CH and HCV-HCC, or be-
tween HCV-HCC and NBNC-HCC. Comparison between patients with and without TTV revealed no significant differences in backgrounds or biochemical findings. Histopathological findings in patients with HCV-CH, and number, maximum diameter, and histological differentiation of HCC also did not demonstrate any relation to TTV infection. TTV strains can be divided into five groups using phylogenetic analysis, but no disease-specific group appears to exist. Conclusions: Our data suggest that: 1) TTV is very prevalent among patients with liver diseases and even among BDs in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development or progression of HCC.
I
ence analysis from an acute phase serum of a patient with post-transfusion hepatitis of unknown etiology (1). The virus could be isolated from three of five Japanese patients with post-transfusion non A to G hepatitis, and has come to be regarded as a “transfusion-transmitted virus”. Using the polymerase chain reaction (PCR) with specific primers, the prevalence of TTV infection in Japanese patients with fulminant hepatitis and chronic liver diseases of unknown etiology has been reported (2). The virus has a singlestranded DNA genome and two overlapped open reading frames, suggesting that it belongs to the Circoviridue rather than the Parvoviridue (3). Since the virus was found more often in patients with various liver dis-
N 1997, a novel DNA virus, designated as the TT virus (TTV), was cloned by representational differ-
Received 10 November 1998; revised 15 February: accepted24
February 1999
Correspondence: Masashi Mizokami, Second Department of Medicine, Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya 4677860 1, Japan. Tel: 81 52 853 8216. Fax: 81 52 852 0849. e-mail: [email protected]
The nucleotide sequences in this paper will appear in the DDBJ/EMBL/GenBank with the following accession numbers, ABOl8850-AB018887 (JaCHC-), AB018888AB018918 (JaHCC-), ABO18919-AB018960 (JaBD-) and ABO18961-AB018965 (JaNBNC-).
Key words: Chronic hepatitis; Hepatocellular carcinoma; TTX
Genotype;
HCV,
221
T. Kato et al.
eases than in volunteer blood donors, some pathogenic role is conceivable. Recently, a new primer set for TTV detection, lo-100 times more sensitive than the conventional primer set, was generated, and a very high prevalence (92%) of TTV infection in the Japanese general population, especially in the older generation, was clarified (4). In this report, we document the prevalence of TTV infection using both primer sets in patients with chronic hepatitis and with hepatocellular carcinoma, and discuss the clinical impact of TTV infection on liver disease.
Patients and Methods Patienfs A total of 141 patients (male:female=96:49, age=56.5?12.8 years) with chronic liver diseases were studied using serum samples. Totals of 72 patients with chronic hepatitis (CH) and 57 with hepatocellular carcinoma (HCC) with anti-hepatitis C virus (HCV) but without HBsAg, who consecutively visited our Institution from 1996 to 1998, were followed up for at least 6 months and diagnosed with biochemical liver function tests, ultrasonography, computerized tomography. and histology of biopsy specimens. Twelve consecutive patients with HCC without either HCV or hepatitis B virus (HBV) (NBNC-HCC), who visited our Institution from 1988 to 1998, were also studied. They were diagnosed as having HCC by histology and confirmed to be seronegative for anti-HCV, HCV RNA, HBsAg. anti-HBs, anti-HBc and HBV DNA (Table 1). As controls, 120 consecutive blood samples from voluntary blood donors (BDs) supplied from the Japanese Red Cross Blood Center, which were negative for HIV HBV and HCV markers, were also collected at the Department of Blood Transfusion, Nagoya City University as the attached tube for cross-match test. All samples were stored at -80°C until use. Anti-HCV was examined with a second-generation enzyme immunoassay kit (Ortho Diagnostics, Tokyo, Japan), and HCV-RNA was detected by reverse transcription polymerase chain reaction with 5’-UTR primers (5). HBsAg, anti-HBs and anti-HBc were examined by the PA method (Fujirebio, Tokyo, Japan). HBV-DNA was also detected by PCR with primers in the S region (6).
TABLE
1
Backgrounds
and characteristics
No. Sex (M:F) Mean age?SD (year) Family history of hepatitis History of BTF Alcohol abuse” Anti-HCV HBsAg ALT (mean?SD) (U/l) ALP (meant SD) (U/l) 7 GTP (meaniSD) (U/l) Platelet (mean%SD) (/pl)
of patients
CH HCV
HCC
72 44:28 48.5t12.0 8 (11.1%) 21 (29.2%) 15 (20.8%) 72 (100%) 0 98.3294.9 173.8C50.0 63.4k50.3 18.46-~5.20
57 12 41:16 7:5 65.026.7 64.327.9 3 (5.3%) 0 25 (43.9%) 1 (8.3%) 16 (28.1%) 2 (16.7%) 57 (100%) Ob 0 Ob 75.3545.5 47.0235.1 284.0t88.7 315.3’200.2 88.0257.8 82.8k48.7 9.2lk5.23 13.23+7.30
HCV
I
ORFl
588
l-l
2896
0RF2
c
1
107
1
712
c
1090
2ooo
I
3900
3739
Primerqlle” e-25
tK3$9lqcgl~ 1s*Cd
159bp
1900-1923
1915.1938
2161-2185
222bp
Fig. I. The two primer sets used in this study. Numbering is according to the sequence for TA278 (accession number; AB008394).
Detection of TTV Serum DNA was extracted from 200 ~1 samples using a High Pure Viral Nucleic Acid Kit (Boehringer Mannheim, Mannheim, Germany). TTV DNA was detected by PCR using two sets of primers, set A (NG059, NGO61 and NGO63) (2) and B (T801 and T935) (4) (Fig. 1). Thermal cycling conditions for primer set A, consisted of a preheat step for 9 min at 95°C. denaturation for 1 min at 95°C. annealing for 1 min at 60°C. and extension for 1 min at 72°C for 35 cycles as the first round of PCR with NGOS9 and NG063 using Ampli-Taq Gold (Perkin Elmer Applied Biosystems. Warrington, UK) on a GeneAmp PCR System 9600. The second round of PCR was performed under the same conditions for 25 cycles with NG061 and NG063. For primer set B, thermal cycling consisted of a preheat step for 9 min at 95°C denaturation for 1 min at 95”C, annealing for 1 min at 60°C and extension for 1 min at 72°C for 55 cycles. The expected sizes of products were confirmed by electrophoresis on 3% agarose gels. Phylogmetic analysis All products amplified with primer set A and some generated with primer set B were sequenced directly by the dideoxy chain termination method with a 373A DNA Sequencer (Applied Biosystems. Foster City, CA, USA). To analyze the relationship between the isolates in this study and previously reported strains. the genetic distances were calculated by the 6pdrameter method (7). Based on these estimates, a phylogenetic tree was constructed using the neighbor-joining method with the computer program ODEN version 1.1.1 (8,9). To confirm the reliability of the tree. bootstrap resampling tests were performed 1000 times ( 10).
NBNC
CH, patients with chronic hepatitis; HCC, patients with hepatocellular carcinoma; HCV, patients with HCV; NBNB, patients without either HCV or HBV; BTF, blood transfusion. a Alcohol abuse; ~30 mg/dayx 10 years. b All patients were negative for anti-HCV, HCV RNA. HBsAg, antiHBs, anti-HBc, and HBV DNA.
222
I
Histopathological analysis All liver tissue specimens were obtained by needle biopsy. Specimens were fixed in 10% neutral formalin, embedded in paraffin, cut in 4pm-thick sections and stained with hematoxylin-cosin. Each specimen was assessed independently by the three liver pathologists (M.K.. M.K., K.S.) by filling out the protocol of a scoring system which included a grading of necroinflammation and staging of fibrosis based on recommendations by Ichida et al (11) (Table 2). In the grading, inflammation in both the portal area and the lobular area was evaluated into 4 levels based on the degree of infiltration of lymphocytes and necrosis of hepatocytes. If the interpretations of the pathologists differed, the score was determined to be that on which two pathologists agreed. The histological grade of HCC was classified into three types according to the degree of differentiation: well, moderately and poorly differentiated. Statistical unalysis For statistical analysis, the Mann-Whitney and ANOVA were employed.
U-test, Kruskal-Wallis
test
TTY infection in Japan TABLE 2
HistopathologicalJindings
New Inuyama classification of chronic hepatitis
Histopathological examination was performed for all HCV-CH patients, and the results were expressed as fibrosis and activity scores according to the New Inuyama Classification (Table 2). Fibrosis was classified into five stages (FO-F4), and the mean score was 1.02820.872. Of 72 CH patients, 17 were classified into FO, 43 into Fl, 7 into F2, 3 into F3, and 2 into F4. Activity was classified into four grades (AO-A3), and the mean score was 1.33320.650. Of 72 CH patients, two were classified as AO, 49 as Al, 16 as A2, and 5 as A3. Comparison of CH patients with and without TTV DNA revealed no significant differences in histological mean scores, ratios of histological distribution and biochemical findings (Table 5).
Staging criteria
Grading criteria
FO: no fibrosis
AO: no necro-inflammatory reaction Al: Mild necro-intlammatory reaction A2: Moderate necro-inflammatory reaction
Fl: fibrous portal expansion F2: bridging fibrosis (portal-portal of portal-central linkage) F3: bridging fibrosis with lobular distortion (disorganization) F4: cirrhosis
A3: Severe necro-inflammatory reaction
Results Prevalence of TTV infection
Of the 261 samples, TTV DNA was detected in 116 (44.4%) and 228 (87.4%) with primer sets A and B, respectively. In total, it was found in 231 (88.5%) by combining results (primer sets A+B). Among patients with liver diseases, 38 (52.8%), 68 (94.4%) and 68 (94.4%) of 72 with HCV-associated chronic hepatitis (HCV-CH), 31 (54.4%), 52 (91.2%) and 53 (93.3%) of 57 with HCV-associated HCC (HCV-HCC), and 5 (41.7%), 12 (100%) and 12 (100%) of 12 with NBNCHCC were positive for TTV DNA using primer sets A, B, and A+B, respectively. The respective values for BDs were 42 (35.00/o), 96 (80.0%) and 98 (81.7%). TTV DNA was detected in 1331141 (94.3%) patients with liver diseases and 981120 (81.7%) BDs with primer sets A+B, the difference being significant ($0.05). However, the prevalence did not vary significantly between patients with HCV-CH and HCV-HCC, or between HCV-HCC and NBNC-HCC (Table 3). Comparison of patients with and without TTV revealed no significant differences for sex ratio, mean age, family history of hepatitis, history of blood transfusion, alcohol abuse, and serum ALT, ALP, yGTP, and platelet levels (Table 4).
for CH patients
Characteristics of HCC
All 69 patients with HCC were diagnosed histologically, and classified into well, moderately or poorly differentiated. The numbers and maximum diameters of tumors were measured by ultrasonography or computerized tomography. Of 69 patients with HCC, 39 had single and 30 had multiple lesions. The mean maximum diameter was 34.0225.2. Of 69 patients, 29 were classified as well, 36 as moderately, and 4 as poorly differentiated. Comparison of HCC patients with and without TTV DNA did not reveal any significant differences for the numbers of tumors, maximum diameters, tumor differentiations, or biochemical findings (Table 6). Phylogenetic analysis
A phylogenetic tree was constructed using alignment of sequences with primer set A. The TTV strains isolated in this study and previously reported are divided into at least five major genetic groups. Groups 1 and 2 were reported by Okamoto et al. (2), and group 3 by Simmonds et al. (12). The other two groups, 4 and 5, were observed among the Japanese population in this
TABLE 3 Prevalence of TTV DNA among patients and blood donors CH HCV No.
Primer set A Primer set B Total (primer sets A+B)”
12 38 (52.8%) 68 (94.4%) 68 (94.4%)
HCC HCV
NBNC
:: (54.4%) 52 (91.2%) 53 (93.0%)
12 5 (41.7%) 12 (100%) 12 (100%)
BDs
Total
120 42 (35.0%) 96 (80.0%) 98 (81.7%)
261 116 (44.4%) 228 (87.4%) 231 (88.5%)
CH, patients with chronic hepatitis; HCC, patients with hepatocellular carcinoma; HCV, patients with HCV, NBNB, patients without either HCV or HBV, BDs, blood donors. BCombined results using primer sets A and B.
223
T. Kato
et al.
TABLE
4
Comparison
of all patients
with and without
TTV DNA Total
No. Sex (M:F) Mean age_‘SD (year) Family history of hepatitis History of BTF Alcohol abuseb ALT (meantSD) (U/l) ALP (mean?SD) (U/l) yGTP (meantSD) (U/l) Platelet (mean?SD) (/pl)
141 96149 56.52 12.8 11 (7.8%) 47 (33.3%) 33 (23.4%) 84.6275.8 230.42 104.9 75.0254.3 14.2726.96
TTV DNA
p-value
Positivea
Negative
133 (94.3%) 85:48 56.52 12.9 10 (7.5%) 45 (33.8%) 29 (21.8%) 85.8277.7 230.9t 105.4 75.5’54.7 14.19k6.98
8 (5.7%) 7:l 56.9r 12.9 1 (lZ.S”/l,) 2 (25.0%) 4 (50.0%) 65.0121.0 220.95 102.7 67.1~~50.1 15.7026.82
NS NS NS NS NS
NS NS NS NS
BTF, blood transfusion a Combined results using primer sets A and B. b Alcohol abuse; >80 mg/dayx 10 years.
TABLE
5
Comparison
of CH patients
with and without
TTV DNA Total
Positive”
a Combined
IF-value
TTV DNA Negative
No. Histology Staging FO:Fl:F2:F3:F4 MeaniSD Grading AO:Al :A2:A3 Mean&SD
17143171312 1.028kO.872
17:39:7:3:2 1.029-tO.897
0:4:0:0:0 1.000r0
NS NS
2:49:16:5 1.333-tO.650
2~45~16~5 1.35320.664
0:4:0:0 1.000t0
NS NS
ALT (mean?SD) (U/l) ALP (mean?SD) (U/l) y GTP (mean&SD) (U/l) Platelet (meant SD) (/,ul)
98.3294.9 173.8~50.0 63.4k50.3 18.4625.20
100.3k97.1 174.6250.6 64.6251.4 18.33k5.20
62.3t28.7 160.0+42.2 42.52 12.5 20.7Oz5.51
NS NS NS NS
results using primer
72
68 (94.4%)
4 (5.6%)
sets A and B
study. On bootstrap analysis for evaluation of the statistical reliability of the tree, the clusters for groups l4 exhibited values of 97.9%, 96.1%, 97.9% and 96.1%, respectively (Fig. 2). The reliability of the cluster named group 5 could not be calculated, because only one strain included in this group was isolated in this study. Distribution of groups revealed that group 1 is
the predominant group in Japan, and that no diseasespecific group exists. The strains in group 3, reported by Simmonds et al. (12), were not observed in this study (Table 7). A phylogenetic tree using alignment sequences with primer set B did not allow division into these groups, because mean pairwise genetic distances between sequences were smaller than with primer set
Fig. 2. Phylogenetic tree drawn with an alignment of the sequences using primer set A with TTV strains divided into 5 major groups. This tree includes the 116 strains isolatedfrom Japanese in this study, the 5 strains reported by Okamoto et al., and the 23 strains reported by Simmonds et al. The names of the strains are indicuted on the right. N22 was reported by Nishizuwa et al. (I). TA278, TXOII, TSO03, and NAO04 were reported by Okamoto et al. (2). AF060545 - AF060549, AF072738 AF072749, AF079.537 - AF079543 were reported Simmonds et al. (12). Strains numbered with “JaCHC-“, “JaHCC-“, “JaNBNC-“, and “JaBD-” were isolated from patients with HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The percentages indicated in parentheses ure the results of bootstrap analysis. The root of the neighbor-joining tree was tentatively taken us the midpoint of the longest path. The length of the horizontal bars indicate the numbers qf nucleotide substitutions per site. 224
TTV infection in Japan
SW
Group 1 (97.9%)
W W
Group 4
?__JaBDSO
(Group 3
I
I
I_
-
JaHCC35 AR)79541 AFO7Q543
b 0.100
Group 5 JaNBNClO
225
T Kato et al. TABLE
6
Comparison
of HCC patients
with and without
TTV DNA Total
HCC
Positive”
Negative
HCV
NBNC
No.
57
12
69
65 (94.2%)
Tumor number (single:multiple) Tumor diameter (mean?SD) (mm) Tumor histology (well:moderately:poorly)
29:28 33.2224.4 2712713
10:2 37.52 18.9 2:9: 1
39:30 34.0t25.2 29~3614
36~29 33.6225.9 27~3414
ALT (mean?SD) (U/l) ALP (mean-t-SD) (U/l) y GTP (mean?SD) (U/l) Platelet (mean_CSD) (/PI)
75.32455 284.0288.7 88.0t57.8 9.21T5.23
HCC, patients with hepatocellular carcinoma; HCV patients a Combined results using primer sets A and B.
TABLE
47.OL35.1 315.3k200.2 82.8248.7 13.23k7.30
patients
without
70.6e46.2 289.9-c 115.4 86.8-56.1 9.8625.92
4 (5.8%) 3:1 4O.O-c8.2 2:2:0
NS NS NS
66.82 14.0 281.8k113.9 91.8264.0 10.70t3.38
NS NS NS NS
either HCV or HBV
7
TTV genotypes
of patients
Group Group Group Group Group Total
and blood donors
1 2 3” 4 5
BDs
Total
CH HCV
HCC HCV
NBNC
27 (71.1%) 9 (23.7%) 0 2 (5.3%) 0
24 (77.4%) 7 (22.6%) 0 0 0
3 (60.0%) 1 (20.0%) 0 0 1 (20.0%)
28 (64.3%) .9 (21.4%) 0 5 (11.9%) 0
38
31
5
42
CH, patients with chronic hepatitis; HCC, patients or HBV; BDs, blood donors. a Group 3 was reported by Simmonds et al.
with hepatocellular
A, and the heterogeneity of the amplified sequences was too small for genotyping (data not shown).
Discussion Although the global distribution of TTV has been documented in previous reports, the incidence of TTV detection is not so frequently documented (2,12-14). TTV viremia was detected with a prevalence of 38.8 47.5% in Japan, 20.638.5% in the UK, and 4.&27.3% in North America among patients with chronic liver disease (2,13,14). Among blood donors, it was also detected at rates of 11.7% in Japan, 1.9% in the UK, and 1.O% in North America (2,12,14). According to our data, however, the prevalence of TTV seems to be significantly higher than previously reported. Our present findings suggest that: 1) TI’V is very prevalent among patients with liver diseases and even among blood donors in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development and progression of HCC. One possible explanation for the differences in
226
70.4245.0 289.5t 114.5 87.1256.1 9.9125.80
with HCV, NBNB,
o-value
T-IV DNA
carcinoma;
HCV; patients
81 (70.4%) 26 (22.6%) 0 7 (6.1%)
1 (0.9%) 115
with HCV, NBNB. patients
without
either HCV
prevalence of TTV in previous reports is the geographic variation in expansion of TTV The other possibility is the difference in the sensitivity of primers. Thus far, several primers for detection of TTV have been reported. The initial primer set, designed by Nishizawa et al. (I), detects TTV strains in genetic group 1, but not the other genetic groups. Although the primer set A used in this study, reported by Okamoto et al. (2), can detect TTV strains in different genetic groups, the sensitivity is relatively low. Simmonds et al. utilized another set of primers with a similar sensitivity to set A (11). In contrast, the primer set B used in our present study has high sensitivity, demonstrating most patients with liver diseases and BDs to be infected with TTV (4). Unfortunately, the region amplified with this primer set is not appropriate for genotyping because of the lower heterogeneity. The sequence in the targeted region may be essential for virus function, and therefore largely conserved. Using the region amplified by the primer set A, TTV strains could be divided into at least five major genetic groups. Two groups were reported by Okamoto et al., one group by Simmonds et
TTV infection in Japan
al., and the remaining two groups were newly discovered (2,12). An assessment of the association with disease indicated the absence of any genetic specificity. However, there are some TTV strains detected with primer set B but not A and not classified into groups, and they may be related to liver diseases. To evaluate the relationship between genetic groups and diseases conclusively, development of a detection system for TTV with higher sensitivity and availability for genotyping is needed. Among patients with HCV-CH, no association between TTV infection and histological score or serum ALT level could be found. This suggests that TTV infection cannot impact on liver damage in HCV infection. Among patients with HCC, the numbers and maximum diameters of tumors, and their histological differentiation also did not significantly differ with reference to TTV infection, suggesting no relation of TTV with progression of HCC. Moreover, the prevalence of TTV was not significantly different between HCV-CH and HCV-HCC, or between HCV-HCC and NBNCHCC. These findings indicate that the existence of TTV seems to affect the development of HCC in patients with HCV and that TTV is not the main causative agent of HCC among patients with NBNC-HCC. Thus, TTV seems not to be associated with HCC. Certainly, the prevalence of TTV in patients with liver diseases was significantly higher than that of BDs Q~0.05) as in a previous report (2), but the significance of this remains unclear. It may reflect only the risk of being exposed to TTV On the other hand, hepatotropism, with concentrations of TTV lO-loo-fold higher in liver tissue than in serum, and correlation between TTV titer and serum ALT level have been suggested (2). From our data, the association between TTV and hepatitis cannot be disproved completely. The relation between TTV titer and liver damage could not be estimated. A part of TTV infection with a high titer of TTV in liver tissue or in serum may relate to liver injury. To elucidate the association between TTV and liver damage, an accurate quantitative assay for TTV and further studies on a larger number of patients are required. In summary, the present study revealed a high prevalence of TTV infection in patients with liver diseases as well as in BDs. TTV does not seem to be associated with liver damage due to HCV and hepatocarcinogenesis.
Acknowledgements The authors are grateful to Dr. M. Mayumi, Jichi Medical School, for providing the primers NG059, NG061 and NG063, and to Dr. S. Mishiro, Toshiba General Hospital, for the primers T801 and T935. M. Mizokami was supported in part by a grant from the Japanese Ministry of Health and Welfare, Health Science Research Grants (Non-A, Non-B Hepatitis Research Grants), and by the Viral Hepatitis Research Foundation of Japan.
References 1. Nishizawa T, Okamoto H, Konisbi 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. 2. Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H, et al. Molecular cloning and characterization of a novel DNA virus (‘ITV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res 1998; 10: 1-16. 3. Takahashi K, Ohta Y, Mishiro S. Partial -2.4-kb sequences of TT virus (TTV) genome from eight Japanese isolates: diagnostic and phylogenetic implications. Hepatol Res 1998; 12: 11l-20. 4. Takahashi K, Hoshino H, Ohta Y, Yoshida N, Mishiro S. Very high prevalence of TT virus (TTV) infection in general population of Japan revealed by a new set of PCR primers. Hepatol Res 1998; 12: 233-9. 5. Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, et al. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the S-noncoding region. Jpn J Exp Med 1990; 60: 215-22. 6. Yokosuka 0, Omata M, Hosoda M, Tada T, Ehata T, Ohto M. Detection and direct sequencing of hepatitis B virus genome by DNA amplification method. Gastroenterology 1991; 100: 175-81. 7. Nei M, Gojobori T. Simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986; 3: 418-26. 8. Saito N, Nei M. The neighbor-joining method a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4: 40625. 9. Ina Y. ODEN: a program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Comput Appl Biosci 1994; 10: 1l-2. 10. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985; 39: 783-91. 11. Ichida F, Tsuji T, Omata M, Ichida T, Inoue K, Kamimura T, et al. New Inuyama Classification; new criteria for histological assessment of chronic hepatitis. Intern Hepatol Commun 1996; 6: 112-9. 12. Simmonds P Davidson F, Lysett C, Prescott L E, MacDonald D, M, Ellender J, et al. Detection of novel DNA virus (TTV) in blood donors and blood products. Lancet 1998; 352: 191-5. 13. Naoumov NV, Petrova EP Thomas MG, Williams R. Presence of a newly described human DNA virus (TTV) in patients with liver disease. Lancet 1998; 352: 195-7. 14. Charlton M, Adjei P, Poterucha J, Zein N, Moore B, Therneau T, et al. TTvirus infection in North American blood donors, patients with fulminant hepatic failure, and cryptogenic cirrhosis. Hepatology 1998; 28: 839-42.
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Copyright 0 European Association for the Study of the Liver 1999
Journal of Hepatology ISSN 0168-8278
High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors Takanobu Kate’, Masashi Mizokami’, Etsuro Orito ’ , Tatsunori Nakano’, Yasuhito Tanaka’, Ryuzo Ueda’, Noboru Hirashima 2, Yoshihiko Iijima3, Tetsuo Kato3, Fuminaka Sugauchi4, Motokazu Mukaide’, Kazuhide Shimamatsu6, Masayoshi Kage6 and Masamichi Kojiro6 ‘Second Department of Medicine. Nagoya City University Medical Nagoya; ‘Section of Gastroenterology, Nagoya Daini Red Cross Hamamatsu; 5Center for Molecular Biology and Cytogenetics, SRL, Medicine,
School, Nagoya; 2Department of Gastroenterology, Chyukyo Hospital, Hospital, Nagoya: 4Department of Medicine. Enshu General Hospital, Inc., Tokyo, and 6Department of Pathology, Kurume University School of Kurume, Japan
Background/Aims: Although a novel DNA virus, TT virus (TTV), has been isolated from a patient with cryptogenic post-transfusion hepatitis, its pathogenic role remains unclear. To elucidate its prevalence and clinical impact in patients with liver diseases, the presence of TTV DNA was assessed in patients with liver diseases and blood donors (BDs) in Japan using two primer sets, one conventional and the other new and highly sensitive. Methods: We studied 261 samples, 72 with chronic hepatitis associated hepatitis C virus (HCV-CH), 57 with hepatocellular carcinoma associated HCV (HCV-HCC), 12 with HCC without either HCV or hepatitis B virus (NBNC-HCC), and 120 of BDs. Results: Using two primer sets, TTV DNA was detected in 68 (94.4%), 53 (93.0%), 12 (lOO%), and 98 (81.7%) HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The prevalence was not significantly different between HCV-CH and HCV-HCC, or be-
tween HCV-HCC and NBNC-HCC. Comparison between patients with and without TTV revealed no significant differences in backgrounds or biochemical findings. Histopathological findings in patients with HCV-CH, and number, maximum diameter, and histological differentiation of HCC also did not demonstrate any relation to TTV infection. TTV strains can be divided into five groups using phylogenetic analysis, but no disease-specific group appears to exist. Conclusions: Our data suggest that: 1) TTV is very prevalent among patients with liver diseases and even among BDs in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development or progression of HCC.
I
ence analysis from an acute phase serum of a patient with post-transfusion hepatitis of unknown etiology (1). The virus could be isolated from three of five Japanese patients with post-transfusion non A to G hepatitis, and has come to be regarded as a “transfusion-transmitted virus”. Using the polymerase chain reaction (PCR) with specific primers, the prevalence of TTV infection in Japanese patients with fulminant hepatitis and chronic liver diseases of unknown etiology has been reported (2). The virus has a singlestranded DNA genome and two overlapped open reading frames, suggesting that it belongs to the Circoviridue rather than the Parvoviridue (3). Since the virus was found more often in patients with various liver dis-
N 1997, a novel DNA virus, designated as the TT virus (TTV), was cloned by representational differ-
Received 10 November 1998; revised 15 February: accepted24
February 1999
Correspondence: Masashi Mizokami, Second Department of Medicine, Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya 4677860 1, Japan. Tel: 81 52 853 8216. Fax: 81 52 852 0849. e-mail: [email protected]
The nucleotide sequences in this paper will appear in the DDBJ/EMBL/GenBank with the following accession numbers, ABOl8850-AB018887 (JaCHC-), AB018888AB018918 (JaHCC-), ABO18919-AB018960 (JaBD-) and ABO18961-AB018965 (JaNBNC-).
Key words: Chronic hepatitis; Hepatocellular carcinoma; TTX
Genotype;
HCV,
221
T. Kato et al.
eases than in volunteer blood donors, some pathogenic role is conceivable. Recently, a new primer set for TTV detection, lo-100 times more sensitive than the conventional primer set, was generated, and a very high prevalence (92%) of TTV infection in the Japanese general population, especially in the older generation, was clarified (4). In this report, we document the prevalence of TTV infection using both primer sets in patients with chronic hepatitis and with hepatocellular carcinoma, and discuss the clinical impact of TTV infection on liver disease.
Patients and Methods Patienfs A total of 141 patients (male:female=96:49, age=56.5?12.8 years) with chronic liver diseases were studied using serum samples. Totals of 72 patients with chronic hepatitis (CH) and 57 with hepatocellular carcinoma (HCC) with anti-hepatitis C virus (HCV) but without HBsAg, who consecutively visited our Institution from 1996 to 1998, were followed up for at least 6 months and diagnosed with biochemical liver function tests, ultrasonography, computerized tomography. and histology of biopsy specimens. Twelve consecutive patients with HCC without either HCV or hepatitis B virus (HBV) (NBNC-HCC), who visited our Institution from 1988 to 1998, were also studied. They were diagnosed as having HCC by histology and confirmed to be seronegative for anti-HCV, HCV RNA, HBsAg. anti-HBs, anti-HBc and HBV DNA (Table 1). As controls, 120 consecutive blood samples from voluntary blood donors (BDs) supplied from the Japanese Red Cross Blood Center, which were negative for HIV HBV and HCV markers, were also collected at the Department of Blood Transfusion, Nagoya City University as the attached tube for cross-match test. All samples were stored at -80°C until use. Anti-HCV was examined with a second-generation enzyme immunoassay kit (Ortho Diagnostics, Tokyo, Japan), and HCV-RNA was detected by reverse transcription polymerase chain reaction with 5’-UTR primers (5). HBsAg, anti-HBs and anti-HBc were examined by the PA method (Fujirebio, Tokyo, Japan). HBV-DNA was also detected by PCR with primers in the S region (6).
TABLE
1
Backgrounds
and characteristics
No. Sex (M:F) Mean age?SD (year) Family history of hepatitis History of BTF Alcohol abuse” Anti-HCV HBsAg ALT (mean?SD) (U/l) ALP (meant SD) (U/l) 7 GTP (meaniSD) (U/l) Platelet (mean%SD) (/pl)
of patients
CH HCV
HCC
72 44:28 48.5t12.0 8 (11.1%) 21 (29.2%) 15 (20.8%) 72 (100%) 0 98.3294.9 173.8C50.0 63.4k50.3 18.46-~5.20
57 12 41:16 7:5 65.026.7 64.327.9 3 (5.3%) 0 25 (43.9%) 1 (8.3%) 16 (28.1%) 2 (16.7%) 57 (100%) Ob 0 Ob 75.3545.5 47.0235.1 284.0t88.7 315.3’200.2 88.0257.8 82.8k48.7 9.2lk5.23 13.23+7.30
HCV
I
ORFl
588
l-l
2896
0RF2
c
1
107
1
712
c
1090
2ooo
I
3900
3739
Primerqlle” e-25
tK3$9lqcgl~ 1s*Cd
159bp
1900-1923
1915.1938
2161-2185
222bp
Fig. I. The two primer sets used in this study. Numbering is according to the sequence for TA278 (accession number; AB008394).
Detection of TTV Serum DNA was extracted from 200 ~1 samples using a High Pure Viral Nucleic Acid Kit (Boehringer Mannheim, Mannheim, Germany). TTV DNA was detected by PCR using two sets of primers, set A (NG059, NGO61 and NGO63) (2) and B (T801 and T935) (4) (Fig. 1). Thermal cycling conditions for primer set A, consisted of a preheat step for 9 min at 95°C. denaturation for 1 min at 95°C. annealing for 1 min at 60°C. and extension for 1 min at 72°C for 35 cycles as the first round of PCR with NGOS9 and NG063 using Ampli-Taq Gold (Perkin Elmer Applied Biosystems. Warrington, UK) on a GeneAmp PCR System 9600. The second round of PCR was performed under the same conditions for 25 cycles with NG061 and NG063. For primer set B, thermal cycling consisted of a preheat step for 9 min at 95°C denaturation for 1 min at 95”C, annealing for 1 min at 60°C and extension for 1 min at 72°C for 55 cycles. The expected sizes of products were confirmed by electrophoresis on 3% agarose gels. Phylogmetic analysis All products amplified with primer set A and some generated with primer set B were sequenced directly by the dideoxy chain termination method with a 373A DNA Sequencer (Applied Biosystems. Foster City, CA, USA). To analyze the relationship between the isolates in this study and previously reported strains. the genetic distances were calculated by the 6pdrameter method (7). Based on these estimates, a phylogenetic tree was constructed using the neighbor-joining method with the computer program ODEN version 1.1.1 (8,9). To confirm the reliability of the tree. bootstrap resampling tests were performed 1000 times ( 10).
NBNC
CH, patients with chronic hepatitis; HCC, patients with hepatocellular carcinoma; HCV, patients with HCV; NBNB, patients without either HCV or HBV; BTF, blood transfusion. a Alcohol abuse; ~30 mg/dayx 10 years. b All patients were negative for anti-HCV, HCV RNA. HBsAg, antiHBs, anti-HBc, and HBV DNA.
222
I
Histopathological analysis All liver tissue specimens were obtained by needle biopsy. Specimens were fixed in 10% neutral formalin, embedded in paraffin, cut in 4pm-thick sections and stained with hematoxylin-cosin. Each specimen was assessed independently by the three liver pathologists (M.K.. M.K., K.S.) by filling out the protocol of a scoring system which included a grading of necroinflammation and staging of fibrosis based on recommendations by Ichida et al (11) (Table 2). In the grading, inflammation in both the portal area and the lobular area was evaluated into 4 levels based on the degree of infiltration of lymphocytes and necrosis of hepatocytes. If the interpretations of the pathologists differed, the score was determined to be that on which two pathologists agreed. The histological grade of HCC was classified into three types according to the degree of differentiation: well, moderately and poorly differentiated. Statistical unalysis For statistical analysis, the Mann-Whitney and ANOVA were employed.
U-test, Kruskal-Wallis
test
TTY infection in Japan TABLE 2
HistopathologicalJindings
New Inuyama classification of chronic hepatitis
Histopathological examination was performed for all HCV-CH patients, and the results were expressed as fibrosis and activity scores according to the New Inuyama Classification (Table 2). Fibrosis was classified into five stages (FO-F4), and the mean score was 1.02820.872. Of 72 CH patients, 17 were classified into FO, 43 into Fl, 7 into F2, 3 into F3, and 2 into F4. Activity was classified into four grades (AO-A3), and the mean score was 1.33320.650. Of 72 CH patients, two were classified as AO, 49 as Al, 16 as A2, and 5 as A3. Comparison of CH patients with and without TTV DNA revealed no significant differences in histological mean scores, ratios of histological distribution and biochemical findings (Table 5).
Staging criteria
Grading criteria
FO: no fibrosis
AO: no necro-inflammatory reaction Al: Mild necro-intlammatory reaction A2: Moderate necro-inflammatory reaction
Fl: fibrous portal expansion F2: bridging fibrosis (portal-portal of portal-central linkage) F3: bridging fibrosis with lobular distortion (disorganization) F4: cirrhosis
A3: Severe necro-inflammatory reaction
Results Prevalence of TTV infection
Of the 261 samples, TTV DNA was detected in 116 (44.4%) and 228 (87.4%) with primer sets A and B, respectively. In total, it was found in 231 (88.5%) by combining results (primer sets A+B). Among patients with liver diseases, 38 (52.8%), 68 (94.4%) and 68 (94.4%) of 72 with HCV-associated chronic hepatitis (HCV-CH), 31 (54.4%), 52 (91.2%) and 53 (93.3%) of 57 with HCV-associated HCC (HCV-HCC), and 5 (41.7%), 12 (100%) and 12 (100%) of 12 with NBNCHCC were positive for TTV DNA using primer sets A, B, and A+B, respectively. The respective values for BDs were 42 (35.00/o), 96 (80.0%) and 98 (81.7%). TTV DNA was detected in 1331141 (94.3%) patients with liver diseases and 981120 (81.7%) BDs with primer sets A+B, the difference being significant ($0.05). However, the prevalence did not vary significantly between patients with HCV-CH and HCV-HCC, or between HCV-HCC and NBNC-HCC (Table 3). Comparison of patients with and without TTV revealed no significant differences for sex ratio, mean age, family history of hepatitis, history of blood transfusion, alcohol abuse, and serum ALT, ALP, yGTP, and platelet levels (Table 4).
for CH patients
Characteristics of HCC
All 69 patients with HCC were diagnosed histologically, and classified into well, moderately or poorly differentiated. The numbers and maximum diameters of tumors were measured by ultrasonography or computerized tomography. Of 69 patients with HCC, 39 had single and 30 had multiple lesions. The mean maximum diameter was 34.0225.2. Of 69 patients, 29 were classified as well, 36 as moderately, and 4 as poorly differentiated. Comparison of HCC patients with and without TTV DNA did not reveal any significant differences for the numbers of tumors, maximum diameters, tumor differentiations, or biochemical findings (Table 6). Phylogenetic analysis
A phylogenetic tree was constructed using alignment of sequences with primer set A. The TTV strains isolated in this study and previously reported are divided into at least five major genetic groups. Groups 1 and 2 were reported by Okamoto et al. (2), and group 3 by Simmonds et al. (12). The other two groups, 4 and 5, were observed among the Japanese population in this
TABLE 3 Prevalence of TTV DNA among patients and blood donors CH HCV No.
Primer set A Primer set B Total (primer sets A+B)”
12 38 (52.8%) 68 (94.4%) 68 (94.4%)
HCC HCV
NBNC
:: (54.4%) 52 (91.2%) 53 (93.0%)
12 5 (41.7%) 12 (100%) 12 (100%)
BDs
Total
120 42 (35.0%) 96 (80.0%) 98 (81.7%)
261 116 (44.4%) 228 (87.4%) 231 (88.5%)
CH, patients with chronic hepatitis; HCC, patients with hepatocellular carcinoma; HCV, patients with HCV, NBNB, patients without either HCV or HBV, BDs, blood donors. BCombined results using primer sets A and B.
223
T. Kato
et al.
TABLE
4
Comparison
of all patients
with and without
TTV DNA Total
No. Sex (M:F) Mean age_‘SD (year) Family history of hepatitis History of BTF Alcohol abuseb ALT (meantSD) (U/l) ALP (mean?SD) (U/l) yGTP (meantSD) (U/l) Platelet (mean?SD) (/pl)
141 96149 56.52 12.8 11 (7.8%) 47 (33.3%) 33 (23.4%) 84.6275.8 230.42 104.9 75.0254.3 14.2726.96
TTV DNA
p-value
Positivea
Negative
133 (94.3%) 85:48 56.52 12.9 10 (7.5%) 45 (33.8%) 29 (21.8%) 85.8277.7 230.9t 105.4 75.5’54.7 14.19k6.98
8 (5.7%) 7:l 56.9r 12.9 1 (lZ.S”/l,) 2 (25.0%) 4 (50.0%) 65.0121.0 220.95 102.7 67.1~~50.1 15.7026.82
NS NS NS NS NS
NS NS NS NS
BTF, blood transfusion a Combined results using primer sets A and B. b Alcohol abuse; >80 mg/dayx 10 years.
TABLE
5
Comparison
of CH patients
with and without
TTV DNA Total
Positive”
a Combined
IF-value
TTV DNA Negative
No. Histology Staging FO:Fl:F2:F3:F4 MeaniSD Grading AO:Al :A2:A3 Mean&SD
17143171312 1.028kO.872
17:39:7:3:2 1.029-tO.897
0:4:0:0:0 1.000r0
NS NS
2:49:16:5 1.333-tO.650
2~45~16~5 1.35320.664
0:4:0:0 1.000t0
NS NS
ALT (mean?SD) (U/l) ALP (mean?SD) (U/l) y GTP (mean&SD) (U/l) Platelet (meant SD) (/,ul)
98.3294.9 173.8~50.0 63.4k50.3 18.4625.20
100.3k97.1 174.6250.6 64.6251.4 18.33k5.20
62.3t28.7 160.0+42.2 42.52 12.5 20.7Oz5.51
NS NS NS NS
results using primer
72
68 (94.4%)
4 (5.6%)
sets A and B
study. On bootstrap analysis for evaluation of the statistical reliability of the tree, the clusters for groups l4 exhibited values of 97.9%, 96.1%, 97.9% and 96.1%, respectively (Fig. 2). The reliability of the cluster named group 5 could not be calculated, because only one strain included in this group was isolated in this study. Distribution of groups revealed that group 1 is
the predominant group in Japan, and that no diseasespecific group exists. The strains in group 3, reported by Simmonds et al. (12), were not observed in this study (Table 7). A phylogenetic tree using alignment sequences with primer set B did not allow division into these groups, because mean pairwise genetic distances between sequences were smaller than with primer set
Fig. 2. Phylogenetic tree drawn with an alignment of the sequences using primer set A with TTV strains divided into 5 major groups. This tree includes the 116 strains isolatedfrom Japanese in this study, the 5 strains reported by Okamoto et al., and the 23 strains reported by Simmonds et al. The names of the strains are indicuted on the right. N22 was reported by Nishizuwa et al. (I). TA278, TXOII, TSO03, and NAO04 were reported by Okamoto et al. (2). AF060545 - AF060549, AF072738 AF072749, AF079.537 - AF079543 were reported Simmonds et al. (12). Strains numbered with “JaCHC-“, “JaHCC-“, “JaNBNC-“, and “JaBD-” were isolated from patients with HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The percentages indicated in parentheses ure the results of bootstrap analysis. The root of the neighbor-joining tree was tentatively taken us the midpoint of the longest path. The length of the horizontal bars indicate the numbers qf nucleotide substitutions per site. 224
TTV infection in Japan
SW
Group 1 (97.9%)
W W
Group 4
?__JaBDSO
(Group 3
I
I
I_
-
JaHCC35 AR)79541 AFO7Q543
b 0.100
Group 5 JaNBNClO
225
T Kato et al. TABLE
6
Comparison
of HCC patients
with and without
TTV DNA Total
HCC
Positive”
Negative
HCV
NBNC
No.
57
12
69
65 (94.2%)
Tumor number (single:multiple) Tumor diameter (mean?SD) (mm) Tumor histology (well:moderately:poorly)
29:28 33.2224.4 2712713
10:2 37.52 18.9 2:9: 1
39:30 34.0t25.2 29~3614
36~29 33.6225.9 27~3414
ALT (mean?SD) (U/l) ALP (mean-t-SD) (U/l) y GTP (mean?SD) (U/l) Platelet (mean_CSD) (/PI)
75.32455 284.0288.7 88.0t57.8 9.21T5.23
HCC, patients with hepatocellular carcinoma; HCV patients a Combined results using primer sets A and B.
TABLE
47.OL35.1 315.3k200.2 82.8248.7 13.23k7.30
patients
without
70.6e46.2 289.9-c 115.4 86.8-56.1 9.8625.92
4 (5.8%) 3:1 4O.O-c8.2 2:2:0
NS NS NS
66.82 14.0 281.8k113.9 91.8264.0 10.70t3.38
NS NS NS NS
either HCV or HBV
7
TTV genotypes
of patients
Group Group Group Group Group Total
and blood donors
1 2 3” 4 5
BDs
Total
CH HCV
HCC HCV
NBNC
27 (71.1%) 9 (23.7%) 0 2 (5.3%) 0
24 (77.4%) 7 (22.6%) 0 0 0
3 (60.0%) 1 (20.0%) 0 0 1 (20.0%)
28 (64.3%) .9 (21.4%) 0 5 (11.9%) 0
38
31
5
42
CH, patients with chronic hepatitis; HCC, patients or HBV; BDs, blood donors. a Group 3 was reported by Simmonds et al.
with hepatocellular
A, and the heterogeneity of the amplified sequences was too small for genotyping (data not shown).
Discussion Although the global distribution of TTV has been documented in previous reports, the incidence of TTV detection is not so frequently documented (2,12-14). TTV viremia was detected with a prevalence of 38.8 47.5% in Japan, 20.638.5% in the UK, and 4.&27.3% in North America among patients with chronic liver disease (2,13,14). Among blood donors, it was also detected at rates of 11.7% in Japan, 1.9% in the UK, and 1.O% in North America (2,12,14). According to our data, however, the prevalence of TTV seems to be significantly higher than previously reported. Our present findings suggest that: 1) TI’V is very prevalent among patients with liver diseases and even among blood donors in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development and progression of HCC. One possible explanation for the differences in
226
70.4245.0 289.5t 114.5 87.1256.1 9.9125.80
with HCV, NBNB,
o-value
T-IV DNA
carcinoma;
HCV; patients
81 (70.4%) 26 (22.6%) 0 7 (6.1%)
1 (0.9%) 115
with HCV, NBNB. patients
without
either HCV
prevalence of TTV in previous reports is the geographic variation in expansion of TTV The other possibility is the difference in the sensitivity of primers. Thus far, several primers for detection of TTV have been reported. The initial primer set, designed by Nishizawa et al. (I), detects TTV strains in genetic group 1, but not the other genetic groups. Although the primer set A used in this study, reported by Okamoto et al. (2), can detect TTV strains in different genetic groups, the sensitivity is relatively low. Simmonds et al. utilized another set of primers with a similar sensitivity to set A (11). In contrast, the primer set B used in our present study has high sensitivity, demonstrating most patients with liver diseases and BDs to be infected with TTV (4). Unfortunately, the region amplified with this primer set is not appropriate for genotyping because of the lower heterogeneity. The sequence in the targeted region may be essential for virus function, and therefore largely conserved. Using the region amplified by the primer set A, TTV strains could be divided into at least five major genetic groups. Two groups were reported by Okamoto et al., one group by Simmonds et
TTV infection in Japan
al., and the remaining two groups were newly discovered (2,12). An assessment of the association with disease indicated the absence of any genetic specificity. However, there are some TTV strains detected with primer set B but not A and not classified into groups, and they may be related to liver diseases. To evaluate the relationship between genetic groups and diseases conclusively, development of a detection system for TTV with higher sensitivity and availability for genotyping is needed. Among patients with HCV-CH, no association between TTV infection and histological score or serum ALT level could be found. This suggests that TTV infection cannot impact on liver damage in HCV infection. Among patients with HCC, the numbers and maximum diameters of tumors, and their histological differentiation also did not significantly differ with reference to TTV infection, suggesting no relation of TTV with progression of HCC. Moreover, the prevalence of TTV was not significantly different between HCV-CH and HCV-HCC, or between HCV-HCC and NBNCHCC. These findings indicate that the existence of TTV seems to affect the development of HCC in patients with HCV and that TTV is not the main causative agent of HCC among patients with NBNC-HCC. Thus, TTV seems not to be associated with HCC. Certainly, the prevalence of TTV in patients with liver diseases was significantly higher than that of BDs Q~0.05) as in a previous report (2), but the significance of this remains unclear. It may reflect only the risk of being exposed to TTV On the other hand, hepatotropism, with concentrations of TTV lO-loo-fold higher in liver tissue than in serum, and correlation between TTV titer and serum ALT level have been suggested (2). From our data, the association between TTV and hepatitis cannot be disproved completely. The relation between TTV titer and liver damage could not be estimated. A part of TTV infection with a high titer of TTV in liver tissue or in serum may relate to liver injury. To elucidate the association between TTV and liver damage, an accurate quantitative assay for TTV and further studies on a larger number of patients are required. In summary, the present study revealed a high prevalence of TTV infection in patients with liver diseases as well as in BDs. TTV does not seem to be associated with liver damage due to HCV and hepatocarcinogenesis.
Acknowledgements The authors are grateful to Dr. M. Mayumi, Jichi Medical School, for providing the primers NG059, NG061 and NG063, and to Dr. S. Mishiro, Toshiba General Hospital, for the primers T801 and T935. M. Mizokami was supported in part by a grant from the Japanese Ministry of Health and Welfare, Health Science Research Grants (Non-A, Non-B Hepatitis Research Grants), and by the Viral Hepatitis Research Foundation of Japan.
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