Incidence of TT virus infection in patients with non-A to G liver disease]

The sera of patients with liver disease associated with non-A to G hepatitis were examined for the presence of TTV DNA. These patients included 18 cases with AH, 8 cases with CH, 6 cases with LC, 4 cases with HCC, and 36 cases with blood donors. The detection of TTV DNA was performed as described by Nishizawa et al. TTV DNA was detected in 60.0%, 62.5%, 66%, 50%, 28% of the patients with AH, CH, LC and HCC, respectively. Among the patients with AH, the aminotransferases and total bilirubin values were lower in the TTV DNA-positive than -negative patients. Among the patients with chronic liver disease, however, there were no differences in the blood chemistry results between the TTV DNA-positive and -negative patients. The histological study of the liver tissues from a TTV positive patient with CH showed no evidence of necro-inflammatory reaction, although there was evidence of irregular regeneration in the TTV DNA-positive a patient. These results suggest that TTV infection may modify the pathological condition of the liver disease.     

TT virus infection in patients with hepatocellular carcinoma associated with non-A to G hepatitis: histopathological study]

To study if TTV infection is involved in the development of hepatocellular carcinoma (HCC), we tested the sera of 19 patients with HCC associated with non-A to G hepatitis for the presence of serum TTV DNA, and compared the blood chemistry values and liver histology of the patients in the TTV DNA-positive and -negative groups. Detection of TTV DNA was performed described as Nishizawa, et al method. TTV DNA was detected in the sera of 47.4%. There were no significant differences in the blood chemistry results and other tests between the TTV-positive and -negative patients. Histological examination of the non-tumor regions of the liver showed that there were no significant differences in the number of areas and characteristics of the necro-inflammatory reactions, the degree of staging and irregular regeneration of hepatocyte between the two groups. These results suggest that the development of HCC in patients with non-A to G hepatitis is not associated with TTV infection.     

A novel unenveloped DNA virus (TT virus) associated with acute and chronic non-A to G hepatitis.

In 1997, a novel DNA virus was isolated from the serum of a patient with posttransfusion hepatitis of unknown etiology in Japan, and it was named TT virus (TTV) after the initials of the index patient. TTV is a nonenveloped, single-stranded and circular DNA virus, and its entire sequence of approximately 3.9 kb has been determined. For being a DNA virus, TTV has a wide range of sequence divergence, allowing the classification into at least 16 genotypes separated by a sequence difference of >30% from one another. The nucleotide sequence of the noncoding region of the TTV genome is conserved, whereas that of the coding region is highly variable. TTV strains with extremely high sequence divergence are common in the same individuals, thereby indicating a mixed infection of TTV strains of different genotypes. An association is found between hepatitis of unknown etiology and the TTV genotypes which are detectable by PCR with primers deduced from the N22 region (genotype 1) in the open reading frame 1 encoding the capsid protein. It would be important to select the primers for specific detection of the TTV genotypes associated with clinical diseases, to further evaluate the capacity of TTV to induce acute and chronic liver disease as well as extrahepatic manifestations.     

Detection of TT virus in hemodialysis patients]

Recently a newly discovered DNA virus, transfusion transmitted virus (TTV), was introduced as a cause of post-transfusion hepatitis. We studied the frequency of TTV viremia in 60 hemodialysis patients in Yamaguchi, Japan. TTV DNA was detected by heminested PCR, using primers described by Okamoto et al. TTV DNA was detected in 18 patients (30%). There was no differences in clinical characteristics, including age, gender, history of blood transfusion, and double infection of other hepatitis viruses, between TTV DNA positive patients and negative patients. Also the frequency of TT viremia was not associated with the duration of hemodialysis. These results suggest that the routes of TTV infection may be different from those of infection by HBV, HCV, or HGV.     

TT病毒核酸的检测

目的初步测定献血员、慢性乙型肝炎、慢性丙型肝炎及慢性非甲～戊型，非庚型肝炎患者中ＴＴ病毒的感染情况，分析不同ＴＴ病毒感染者血清中ＴＴ病毒开放读码框架２区部分基因序列。方法以套式聚合酶链反应测定ＴＴ病毒核酸，取献血员（ＴＸ２）、慢性乙型肝炎（ＴＸ３）、慢性丙型肝炎（ＴＸ４）及慢性非甲～戊型，非庚型肝炎患者（ＴＸ１）各１例ＴＴ病毒ＰＣＲ阳性产物，以双脱氧核苷酸链终止法测定核苷酸序列。结果检测２０例献血员、２９例慢性乙型肝炎、３１例慢性丙型肝炎及３２份慢性非甲～戊型，非庚型肝炎标本，ＴＴ病毒核酸阳性者分别占５．０％（１／２０）、６．９％（２／２９）、２９．０％（９／３１）和３４．４％（１１／３２）。与Ｎ２２克隆相比，ＴＸ１、ＴＸ２、ＴＸ３与ＴＸ４在核苷酸水平的同源性分别为９５．４１％、９８．４７％、９７．４５％和９７．９６％。结论本组献血员、慢性乙型肝炎、慢性丙型肝炎及慢性非甲～戊型，非庚型肝炎患者中均存在ＴＴ病毒感染者。从本组不同ＴＴ病毒感染者血清中分离出４株ＴＴ病毒序列，其开放读码框架２区部分基因序列高度保守     

Detection of TT virus in healthy volunteer]

A novel DNA virus, TT virus(TTV), has been reported in Japanese patient with non A to G posttransfusion hepatitis. We sought to determine whether TTV infection occurs in healthy volunteer, and to compared with DNA extraction methods and polymerase chain reaction(PCR) primer for TTV in diagnostic system. Using a nested PCR assay, serum sample of healthy volunteer serve our laboratory in Japan were examined for the presence of TTV DNA. Twenty of 90(22%) healthy volunteer were detected to have TTV sequences in their serum. Also, we found that DNA extraction methods with a modified phenol-chloroform method. Our result suggested that detection of TTV DNA are high ratio of adults in Japan and were necessary to take care of selected using diagnostic systems.     

Detection of TT virus (TTV) in Japanese hemodialysis (HD) patients]

The clinical and epidemiological studies on TT virus (TTV) were achieved in 44 Japanese HD patients. TTV-DNA was detected in 29.5% of HD patients, and the percentage was higher than that reported in normal populations. HBsAg, anti-HBc, anti-HCV and HGV-RNA were positive in 6.8%, 36.4%, 22.7% and 2.3%, respectively. One of three HD patients with TTV had liver disorders. In comparison between TTV positive and negative groups, there were significant differences of anti-HBc and total cholesterol value.     

非甲非戊型肝炎患者TT病毒感染的检测

目的 了解非甲非戊型肝炎患TT病毒感染状况。方法 采用套式PCR方法对44份临床诊断为急慢性非甲非戊型肝炎患的血清标本,进行TT病毒检测。结果 14例TTV-DNA阳性,检出率31．8％(14／44)。对TTV-DNA阳性标本的PCR产物进行克隆后测序,结果表明该序列与日本株相应位置核苷酸序列同源性为98％。结论 推测TT病毒可能为临床急慢性非甲非戊型肝炎病因之一。     

Virological response to interferon therapy in patients dually infected with TT virus and hepatitis C virus]

In 30 chronic hepatitis C patients co-infected with TTV, TTV DNA in the sera immediately after cessation of IFN and 6 months after the end of IFN was examined. Sustained loss of TTV DNA was observed in 12 of 30 (40.0%) patients. In 7 of 30 (23.3%) patients, TTV DNA re-appeared 6 months after becoming undetectable at the end of IFN therapy. In the remaining 11 patients (36.7%), TTV DNA remained detectable during the entire follow-up period. The ALT values correlated only with the presence of HCV RNA regardless of the effect of IFN on TTV replication. This study indicates that IFN therapy is effective against TTV.     

TT virus(TTV) infection in patients with acute hepatitis]

A novel DNA virus, TT virus(TTV), has been reported in patients with posttransfusion hepatitis of unknown etiology. However association between TTV and acute hepatitis has not been shown. We investigated the prevalence of TTV in acute hepatitis. TTV-positive rates in acute hepatitis A, B, C, cytomegalovirus infection, Epstein-Barr virus infection, and acute hepatitis of unknown etiology were 15.3%, 21.8%, 60.0%, 0%, 10.0%, 22.6%, respectively. There were no significant differences in TTV prevalence between each etiology and healthy blood donors(20.8%). Clinical data were similar between patients with or without TTV. In this study we could not find any difference in the prevalence of TTV between acute hepatitis with known etiologies and that with unknown etiology. TTV did not affect the clinical features of acute hepatitis with known etiologies.     

Prevalence of TT virus in patients with fulminant hepatic failure in Japan]

A novel virus(TTV) was isolated from patients with post-transfusion hepatitis of unknown etiology. We studied the prevalence of TTV in 26 patients with fulminant hepatic failure. Serum samples at admission from seven(27%) of the 26 patients were positive for TTV. TTV was also detected in 29(27%) of the 106 healthy blood donors. There were no differences in the positive rate between patients with fulminant hepatic failure and healthy blood donors. There were no differences in clinical findings, or duration from onset to coma in patients with and without TTV. However, the outcome of the patients with TTV was significant worse than those without. TTV may not cause severe hepatitis such as fulminant hepatic failure.     

The prevalence of TTV infection in non-A to G chronic liver disease, especially non-A to G hepatocellular carcinoma, and the clinical significance of TTV infection]

We examined the prevalence of TT virus (TTV) infection in non-B, non-C, non-G chronic liver disease, in particular, in hepatocellular carcinoma (HCC), and the clinical significance of TTV infection. Among 829 cases with chronic liver disease, 30 cases (4%) had non-B, non-C, non-G chronic liver disease. The percentage of TTV-DNA positive cases among these 30 cases with non-B, non-C, non-G chronic liver disease was 57% (17/30), significantly higher than the percentage TTV-DNA positivity (14%; 5/107) among blood donors (P < 0.01). All the TTV-DNA positive cases were still positive for TTV-DNA throughout the follow-up period (4 +/- 2 years; 1-7 years), thus demonstrating that TTV infection is persistent. These findings suggest that TTV may be one of the causes of non-B, non-C, non-G chronic liver disease. When the 30 cases of non-B, non-C, non-G chronic liver disease were divided into a TTV-DNA positive group and TTV-DNA negative group and the clinical data between the two groups compared, it was found that the serum ALP and serum gamma-GTP levels in some cases in the TTV-DNA positive group were higher than those in the TTV-DNA negative group. Twelve cases of non-B, non-C, non-G HCC were divided into two groups (TTV-DNA positive and TTV-DNA negative), and the clinical data between the two groups compared. All the four cases of HCC which were not associated with liver cirrhosis were TTV-DNA positive. However, with respect to the age at the time of onset of the HCC, no significant differences were noted between the cases of HCC associated with liver cirrhosis and those not associated with liver cirrhosis. In spite of older patients, many cases of HCC associated with TTV infection are not associated with liver cirrhosis.     

Susceptibility of TT virus to interferon therapy]

A novel single stranded DNA virus, TT virus, associated with posttransfusion hepatitis was identified. The impact of the virus on development of chronic liver diseases and the susceptibility of the virus to interferon therapy has not been investigated. We retrospectively analyzed blood samples from 16 patients infected with both hepatitis C virus and TT virus who were treated by interferon. TT virus DNA was detected by nested polymerase chain reaction before, during and after interferon. Eight of these 16 patients became negative for TT virus DNA after such therapy but the virus re-appeared in one patient after cessation of therapy. All nine patients in whom interferon eliminated HCV exhibited normalization of alanine aminotransferase activity irrespective of persistence of TTV. In contrast, five of the seven patients who showed persistence of HCV after interferon had high levels of alanine aminotransferase (P = 0.034). These results suggest that TTV is sensitive to interferon and eradicated in a half of treated patients, but persists without liver injury for a long period in some patients.     

Detection and typing of TT virus DNA genotype by the PCR-RFLP method

TT virus (TTV) is a novel single-stranded DNA virus that was identified in patients with post-transfusion-hepatitis of non-A-G type in Japan in 1997. We developed a new detection and typing method for TTV DNA strains, by combining polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) techniques. TTV DNA was amplified by nested-PCR with TTV-specific mixed primers derived from the conserved open reading frame 1 (ORF1) region of the published TTV DNA sequences. Using the enzymes Hae III, Dra I, Eco RI and Pst I, we were able to distinguish between the four TTV genotypes. In 200 serum samples obtained from healthy pregnant women at an outpatient clinic, the positive rate of TTV detection was 27.0%, almost identical to the positive rate of the detection of ORF1-PCR in Japanese healthy individuals. The strains detected in the positive serum samples were genotype 1a (9.3%), genotype 1b (11.1%), genotype 2 (51.9%), genotype 3 (7.4%) and other genotypes (1.9%). This results indicate that genotype 1 and genotype 2 were the two major strains in the Japanese population. Moreover, 10 serum samples (18.5%) presented RFLP patterns in which some genotypes were mixed.     

Molecular virology of TT virus]

In 1997, a novel DNA virus was isolated from the serum of a patient with posttransfusion hepatitis of unknown etiology in Japan, and it was named TT virus (TTV) after the initials of the index patient. TTV is a nonenveloped, single-stranded and circular DNA virus, and its entire nucleotide sequence of 3.9 kb has been determined. For being a DNA virus, TTV has a wide range of sequence divergence, allowing the classification into at least 16 genotypes separated by an evolutionary distance of > 0.30. Nucleotide sequence of the noncoding region is conserved, whereas the coding region sequence is highly variable. TTV strains with extremely high sequence divergence prevail in infected individuals. An association of TTV genotypes, detectable by PCR with N22 primers or genotype 1-specific primers, with hepatitis of unknown etiology suggest that TTV of restricted genotypes would be clinically important.     

Similar frequency of TT virus infection in patients with liver enzyme elevations and healthy subjects

TT virus (TTV) is a novel DNA virus that has been identified in patients with post-transfusional hepatitis of unknown aetiology. However, its pathogenic role in liver injury remains unclear. To determine its frequency and clinical impact in cryptogenic liver diseases, we investigated the TTV prevalence in patients with liver enzyme elevations of unknown aetiology and in healthy subjects. Fifty-four patients (33 male, 21 female) who have been followed up for elevated ALT/AST levels of unknown aetiology and 118 healthy subjects (99 male and 19 female) were included in the study. TTV DNA was investigated by the polymerase chain reaction. Other possible causes of transaminase elevation were excluded in detailed biochemical and serological tests. A liver biopsy was performed in 45 patients. TTV DNA was detected in 46 patients with liver enzyme elevations (85.1%) and in 94 healthy subjects (79.6%). There was no statistical difference between the groups (p = 0.51). Histological examination of the liver revealed no specific change in TTV DNA positive patients that could be attributed to this virus infection. These results showed that TTV is a common virus in patients with liver enzyme elevation of unknown aetiology and even among healthy subjects in our geographical area. TTV infection is therefore widespread in the general population and does not seem to be associated with liver damage.     

72例肝炎患者TT病毒DNA检测及部分基因序列分析

目的 了解肝炎患者中的TTV的感染情况并对部分分离株进行基因分型。方法 设计通用引物，采用半套式聚酶链反应检测肝炎患者血清标本中TTVDNA，并对部分PCR产物进行直接测序和序列分析。结果 在72例不同肝炎患者血清中，共检出TTVDNA阳性血清39份，总检出率为54.16%.其中,在非甲-非庚型肝炎患者中,TTVDNA阳性率为87.5%;在甲-庚型肝炎患者TTVDNA的阳性率(50.0%).对4株TTV序列分析结果显示,它们与日本分离株TA278、NA004、TKB555、PT3最高的同源性分别为97.7%、99.1%、96.8%、91%。经系统发育分析。其中有1株属G1型，有3株属G2型。结论 本次研究的肝炎人群中，TTV感染率较高，。感染的TTV颁发于G1及G2两个不同的基因型，TTV可能是非甲-非庚肝炎致病因素之一。     

Detection of TT virus (TTV) in non-B, non-C hepatitis patients with hepatocellular carcinoma, and clinical features of these patients]

A novel human DNA virus, TTvirus (TTV), was identified from a patient with posttransfusion hepatitis of unknown etiology. It is thought to be a new hepatitis virus, but the clinical significance of this virus is uncertain. We investigated the frequency of TTV viremia by PCR in 39 non-B, non-C hepatitis (NBNC) patients with hepatocellular carcinoma (HCC), and clinical features of these patients. TTV viremia was detected in 20 (51.3%) of 39 NBNC hepatitis patients with HCC. Liver cirrhosis (LC) were found in 11 (55%) of 20 TTV-positive patients and 16 (84%) of 19 TTV-negative patients (p < 0.05). The levels of AST, LDH, LAP, gamma GTP in TTV-positive patients were significantly higher than those in TTV-negative patients (p < 0.05). (AST: 58 +/- 26 vs 42 +/- 23 IU/l, LDH: 468 +/- 127 vs 366 +/- 123 IU/l, LAP: 339 +/- 242 vs 206 +/- 80 IU/l, gamma GTP: 207 +/- 207 vs 105 +/- 107 IU/l) These results suggest clinical differences between TTV-positive and TTV-negative patients in NBNC hepatitis patients with HCC.     

Persistent TT virus infection does not contribute to the development of non-A to -G hepatocellular carcinoma. A case-control study of 19 patients in Japan

We tested the sera of patients with non-A, non-B, non-C, non-G (non-A to -G) hepatocellular carcinoma (HCC) for the presence of TT virus (TTV) DNA and clinicopathologically elucidated the relationship between TTV infection and hepatocarcinogenesis. The study cohort consisted of 19 patients with non-A to -G HCC. Detection of TTV DNA was performed by the nested polymerase chain reaction according to a previously published method. TTV DNA was detected in the sera of 9 (47.4%) of the 19 patients with non-A to -G HCC. The clinical background factors and blood biochemical parameters of the TTV-DNA-positive and -negative HCC patients did not significantly differ. Three TTV-DNA-positive and 2 TTV-DNA-negative patients underwent surgical resection of the HCC. The histological findings in the non-cancerous liver tissue of the TTV-DNA-positive and -negative patients did not significantly differ. In conclusion, TTV infection does not affect the features of non-A to -G HCC.     

Detection of hepatitis B virus DNA sequences in liver in HBsAg seronegative patients with liver disease with and without anti-HBc antibodies

Excluding studies from Brechot and co-workers, little support has been found for a role of the hepatitis B virus in the pathogenesis of HBsAg seronegative patients with predominantly chronic liver diseases, including primary liver cancer. In this study liver DNA from 59 predominantly British patients (four cases with paired biopsies, 6-12 months apart) with different, mostly chronic, liver diseases was analysed by molecular hybridization. All were seronegative for HBsAg and serum hepatitis B virus DNA (dot blot hybridization) and their liver diseases were believed to be unrelated to hepatitis B virus infection. Hepatitis B virus DNA was detected in liver of 11 (18.6 per cent) patients; nine had episomal (3.2 Kb) DNA and eight had higher molecular weight bands suggesting integrated forms. Six patients were also seronegative for anti-HBc. Patients of UK and non-UK origin were equally represented. Hepatitis B virus DNA was detected in serum of six of nine patients tested using the polymerase chain reaction. The detection of hepatitis B virus DNA in liver and in serum by this assay in a significant proportion of patients with chronic liver disease, hitherto unsuspected of being hepatitis B virus-related, suggests a possible role for this virus in low- as well as high-prevalence countries.