Unchanged high prevalence of antibodies to hepatitis E virus (HEV) and HEV RNA among blood donors with an elevated alanine aminotransferase level in Japan during 1991-2006.

Hepatitis E is rare in Japan but is occurring more frequently than previously thought. To investigate whether de novo subclinical infection of hepatitis E virus (HEV) has recently increased in Japan, HEV RNA was assayed in serum samples obtained from 4019 Japanese voluntary blood donors with alanine aminotransferase (ALT) of > or =61 IU/l, who are likely to have ongoing HEV infection, during 1991-2006. The overall rates of IgG-class antibody to HEV (anti-HEV IgG), anti-HEV IgM/IgA and HEV RNA among 3185 donors in 2004-2006 were comparable with those among 594 donors in 1998 (5.3 vs. 5.2%, 0.2 vs. 0.5%, and 0.2 vs. 0.3%, respectively). Among blood donors with ALT > or = 201 IU/l in three groups according to the year of blood collection (1991-1995 [n = 156], 1996-1999 [n = 116] and 2004-2006 [n = 61]), there were no appreciable differences in the prevalence of anti-HEV IgG (5.8, 4.3, and 6.6%, respectively), anti-HEV IgM/IgA (1.9, 3.4, and 3.3%, respectively) and HEV RNA (1.3, 3.4, and 3.3%, respectively). The eleven HEV isolates obtained in the present study differed from each other by 1.7-22.8% in the ORF2 sequence and segregated into genotype 3 or 4. The occurrence rate of subclinical infection with divergent HEV strains has essentially remained unchanged during 1991-2006 in Japan.

High prevalence of hepatitis B virus infection amongst Tibetans in Nepal.

Tibetans have been living in Nepal since 1959. Study of the prevalence of viral hepatitis among them showed that they have a high prevalence of hepatitis B virus (HBV) infection. Prevalence of total HBV infection and Hepatitis B surface antigen (HBsAg) among them was 61% and 16% compared to 10.0% and 0.7% respectively among the Nepalese. The predominant HBsAg subtype among the Tibetans was 'ayw'. Perinatal and childhood transmission was found important in the spread of HBV infection among the Tibetans.

Heterogeneous distribution of TT virus of distinct genotypes in multiple tissues from infected humans.

TT virus (TTV) DNA was quantitated in the serum and nine autopsy tissues (bone marrow, lymph node, muscle, thyroid gland, lung, liver, spleen, pancreas, and kidney) obtained from each of three TTV-infected subjects by real-time polymerase chain reaction (PCR), which can detect all TTV genotypes. TTV DNA was detected in all examined tissues, with the viral load being equal to or up to 300 times higher than that in the corresponding serum (2.1 x 10(5) to 5.3 x 10(7) copies/g vs 1.2-3.9 x 10(5) copies/ml). Generally, the TTV viral load was higher in the bone marrow, lung, spleen, and liver than in the other tissues, although it varied by individual. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified TTV DNA of 3.3 kilobases (kb) revealed considerable differences among the TTVs in the serum and tissue specimens from each subject. Further, the 3.3-kb amplicons from the serum and tissue specimens from one subject were molecularly cloned, and 30 clones each from the serum and each tissue specimen were subjected to RFLP and sequence analysis (total, 300 clones): the TTV clones were classified into six genotypes including four novel genotypes. The genotypic variability was remarkable: each specimen had one to five TTV genotypes at different frequencies. TTV DNA in replicative intermediate forms and TTV mRNA were detectable in all tissues tested. These results indicate the broad, uneven distribution of TTV genotypes in tissues and suggest that viral replication takes place in multiple tissues at distinct levels in infected individuals.

Molecular epidemiology of TT virus (TTV) and characterization of two novel TTV genotypes in Indonesia.

The prevalence of TT virus (TTV) DNA among 244 healthy individuals in 23 cities on 12 islands in Indonesia was determined by polymerase chain reaction (PCR) with primers derived from the coding region (N22), which can detect TTV DNA of genotypes 1-6. By N22 PCR, TTV DNA was detected in 102 (42%) individuals. The amplified PCR products were molecularly cloned and three clones each were subjected to sequence analysis. Three hundred one (98%) of the 306 TTV clones were classified into genotype 1, 2 or 3, and none into genotypes 4-6. The remaining five clones from two individuals (Kt-08 and Kt-10) on Kutai, Kalimantan Island, differed by >30% from known TTV isolates of all 21 genotypes and were tentatively classified into genotypes 22 and 23, respectively. Using primers specific for the new TTV genotype 22 or 23, TTV genotype 22 was detected significantly more frequently in Kutai than in the other 22 cities (41% vs. 5%, P < 0.001). TTV genotype 23 was restricted to Kutai (17% vs. 0%, P < 0.001), suggesting the indigenous nature of this genotype. Analysis of two TTV isolates (Kt-08F and Kt-10F) demonstrated the extreme diversity of TTV and the preservation of the genomic organization and transcription profile.

Hepatic histopathologic range compared with virological studies of hepatitis viruses among autopsy cases in Tokyo.

Few reports exist comparing virological studies on hepatitis viruses with histopathological studies of autopsy cases other than those of liver clinics. Relations between hepatitis virus-related markers and hepatic histopathology were studied in 1044 autopsy cases (779 men and 265 women) at the Medical Examiner's Office, Tokyo. Heart blood was obtained at the autopsy, and the sera were submitted for virus-marker detection of HBV, HCV, and HGV/GBV-C. Hematoxylin and eosin-stained paraffin sections were used for histological assessment. Histopathologically, 463 cases were determined as so-called normal liver; among them 440 cases (95.0%) were negative for all hepatitis virus-related markers, but HBV-DNA was positive in 13 cases, three cases were positive for HCV-RNA (indicating a healthy carrier rate of HCV-RNA of 4.1%), and seven cases were positive for HGV/GBV-C RNA. The incidence of these three virus-related markers was low in cases with fatty liver and micronodular cirrhosis, but in cases with chronic hepatitis, macronodular cirrhosis and hepatocellular carcinoma, the incidence of HBV-DNA and HCV-RNA increased with advancing disease. A positive rate of anti-HBs or anti-HBc (HBV-Ab) or both was found between 30 and 50% in all histopathological groups, and no noticeable relations between the positive rate and microscopical changes were detected. The presence of HGV/GBV-C RNA seemed to be unrelated to hepatic inflammation or generalized inflammatory changes or both occurring together. The decadal age incidence of the virus-related markers and their incidence in various hepatic diseases are also reported.

Inverse relationship between the titre of TT virus DNA and the CD4 cell count in patients infected with HIV.

OBJECTIVES: To investigate the prevalence and relative titre of TT virus (TTV) DNA, and to examine the relationship between the extent of TTV viraemia and the immune status among 144 patients with HIV infection; 178 age- and sex-matched healthy individuals were also studied. METHODS: TTV DNA was detected quantitatively by two distinct polymerase chain reaction (PCR) methods [untranslated region (UTR) and N22]. UTR PCR detects all TTV genotypes, and N22 PCR can primarily detect four major TTV genotypes (1-4). RESULTS: Using UTR PCR and N22 PCR, respectively, TTV DNA was detected significantly more frequently in HIV-infected patients than in controls (99 versus 91%, P < 0.001; 56 versus 27%, P < 0.0001), and the relative titre (10N/ml) was significantly higher in HIV-infected patients [4.5 +/- 1.2 (mean +/- SD) versus 3.1 +/- 0.9, P < 0.0001; 2.6 +/- 1.5 versus 1.5 +/- 0.9, P < 0.0001]. Age, sex, co-infection with hepatitis B or C virus, and risk factors for HIV transmission did not appear to be significant factors associated with the titre of TTV viraemia. However, the titre of TTV DNA was significantly higher in HIV-infected patients with AIDS (P < 0.0001), those with low CD4 T cell count (P < 0.0001), or those with high HIV viral loads (P = 0.0047). CONCLUSION: TTV is highly prevalent and high-titred in HIV-infected patients. The TTV viral load may reflect the degree of immune status of these immunocompromised hosts.

IgM-class antibodies to TT virus (TTV) in patients with acute TTV infection.

TT virus (TTV) is a human circovirus with a single-stranded, circular DNA genome of 3.8 kb. A method was developed to detect IgM antibodies to TTV as a serological marker for the diagnosis of acute TTV infection. IgM antibodies in test sera were captured by a monoclonal antibody against IgM/µ on a solid support followed by binding of IgM with TTV derived from fecal extract of a TTV carrier. The presence of IgM-specific TTV particles was determined by polymerase chain reaction (PCR) using nucleic acids extracted from the solid support. Anti-TTV IgM was detected in sera from two patients with non-A to G post-transfusion hepatitis, who were positive for TTV DNA during weeks 10-21 and 12-17, respectively, following transfusion. The anti-TTV IgM was detectable after alanine transaminase levels were elevated and TTV DNA was detectable in the patients. The duration of the anti-TTV IgM was short-lived compared with anti-TTV IgG. Anti-TTV IgM was not detected in sera from any of 36 healthy individuals, with no detectable anti-TTV IgG or TTV DNA in their serum. These results indicate that anti-TTV IgM antibodies would be a useful marker to detect acute TTV infection.

Visualization of TT virus particles recovered from the sera and feces of infected humans.

TT virus (TTV) has not yet been cultured or visualized. We attempted to recover and visualize TTV-associated particles from the serum samples and feces of infected humans. Serum samples were obtained from 7 human immunodeficiency virus (HIV)-infected patients. Three patients had a high TTV DNA titer (10(8) copies/ml), three had a low TTV DNA titer (10(2) copies/ml), and one was negative for TTV DNA. Fecal supernatant was obtained from a different TTV-infected subject. The serum samples were fractionated by high-performance liquid chromatography, and TTV DNA-rich fractions were subjected to floatation ultracentrifugation in cesium chloride. Virus-like particles, 30-32 nm in diameter, were found in the 1.31-1.33 g/cm(3) fractions from each of the three serum samples with high TTV DNA titer, but not in any fraction from the four serum samples that either were negative for TTV DNA or had low TTV DNA titer. The TTV particles formed aggregates of various sizes, and immunogold electron microscopy showed that they were bound to human immunoglobulin G. Similar virus-like particles with a diameter of 30-32 nm banding at 1.34-1.35 g/cm(3) were visualized in fecal supernatant with TTV genotype 1a by immune electron microscopy using human plasma containing TTV genotype 1a-specific antibody.

Species-specific TT viruses and cross-species infection in nonhuman primates.

Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively; P = 0.038). These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.

Replicative forms of TT virus DNA in bone marrow cells.

TT virus (TTV) is a human virus consisting of a single-stranded, circular DNA genome of 3.8 kilobases (kb). To examine whether TTV replicates in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs), DNA was extracted from the PBMCs and/or BMCs of six TTV-infected individuals and separated by agarose gel electrophoresis. The TTV DNAs from the PBMCs migrated to the 2.0- to 2.5-kb region. The TTV DNAs from the BMCs migrated to the 2.0- to 2. 5-kb and 3.3- to 6.1-kb regions. The faster-migrating TTV DNAs were sensitive to S1 nuclease, while the slower-migrating TTV DNAs were resistant and their position on the agarose gel shifted to the position of the full genomic size upon digestion with restriction enzyme PstI. Full-length inverted polymerase chain reaction on the slower-migrating, double-stranded TTV DNAs from the BMCs amplified a 3.8-kb product. Replicative intermediate forms of TTV DNA are present in BMCs but not in PBMCs.

Quasispecies of TT virus (TTV) with sequence divergence in hypervariable regions of the capsid protein in chronic TTV infection.

Three hypervariable regions were identified in a central portion of open reading frame 1 of TT virus DNA, which codes for a putative capsid protein of 770 amino acids. TT virus circulates as quasispecies, with many amino acid substitutions in hypervariable regions, to evade immune surveillance of the hosts and to establish a persistent infection.

Little contribution of GB virus C to the development of primary hepatocellular carcinoma in Japan.

RNA of GB virus C was searched for in sera from 109 patients with primary hepatocellular carcinoma. It was detected in 11 patients (10%), and was more frequent than in 3 of 342 blood donors (0.9%) (P < 0.001). The 11 patients included 4 of 29 patients (14%) with hepatitis B surface antigen and 7 of 74 patients (9%) with antibody to hepatitis C virus. RNA of GB virus C was not detected in any of 4 patients without hepatitis B surface antigen or antibody to hepatitis C virus. These results suggest that GB virus C may contribute little to the development of primary hepatocellular carcinoma in Japan.

Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions.

A nonenveloped, single-stranded, and circular DNA virus designated TT virus (TTV) has been reported in association with hepatitis of unknown etiology. TTV has a wide sequence divergence (approximately 52%), by which it is classified into at least 16 genotypes separated by an evolutionary distance of >0.30. Therefore, the detection of TTV DNA by polymerase chain reaction would be influenced by primers deduced from conserved or divergent regions of the genome. Of the 30 sera from healthy individuals, up to 17% tested positive with primers deduced from coding region, much less frequently than up to 93% testing positive with primers from noncoding region. These differences were not attributable to the sensitivity of detection, because a cloned TTV DNA of genotype 1a was detected sensitively (up to 1 copy per test) with primers deduced from either the coding or the noncoding region of the same genotype. Sera testing positive only with noncoding region primers, or those showing higher titers with noncoding than coding region primers, contained TTV DNA strains with sequence divergence of 47-53% from the TA278 isolate of genotype 1a within the N22 region spanning 222-231 nucleotides. Some of the sera contained two or three TTV DNA strains of distinct genotypes. These results indicate TTV strains with extremely high sequence divergence prevailing in healthy individuals and frequent mixed infection with TTV strains of distinct genotypes.

[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.

Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.

An ELISA was developed for serological determination of the six genotypes of hepatitis B virus (HBV) designated A, B, C, D, E, and F. Monoclonal antibodies were raised against genotype-specific epitopes in the preS2-region product, and labeled with horseradish peroxidase. Hepatitis B surface antigen (HBsAg) in sera was captured by immobilized antibodies against the common determinant, and evaluated for reactivity with genotype-specific monoclonal antibodies labeled with the enzyme. Serological genotyping was in complete accord with genotypes determined by S-gene sequences in a panel of 68 sera containing HBV/HBsAg of different genotypes. Of 514 sera with HBsAg from Japan, 507 (98.6%) were genotyped serologically: genotype A was identified in 24 (4.7%); B in 196 (38.1%); C in 282 (54.9%); D in 2 (0.4%); and F in 3 (0.6%). There were no sera containing HBV of genotype E. Likewise, 425 of 446 (95.3%) sera with HBsAg from Brazil, China, India, Indonesia, Kenya, Korea, Nepal, Papua New Guinea, the Philippines, and Thailand were classified into A (25.6%), B (24.2%), C (33.9%), and D (11.7%) genotypes; there were no sera with HBsAg of genotype E or F among them. Some sera unclassifiable by ELISA revealed mixed infection with HBV of distinct genotypes, or contained HBsAg deprived of genotype-specific epitopes by point mutations. The ELISA would be useful for large-scale surveys, because it allows serological detection of HBV genotypes without sequencing nucleotides.

Infection by an unenveloped DNA virus associated with non-A to -G hepatitis in Japanese blood donors with or without elevated ALT levels.

BACKGROUND: An unenveloped, single-stranded DNA virus named TT virus has been found in association with elevated alanine aminotransferase (ALT) levels in recipients of transfusions and has been detected frequently in patients with acute or chronic hepatitis of non-A to -G etiology in Japan. DNA of the TT virus was searched for in blood donors with or without elevated ALT levels. STUDY DESIGN AND METHODS: A total of 861 blood donors without previous transfusions and who were negative for markers of hepatitis B or C virus infection were tested. DNA of the TT virus was detected by polymerize chain reaction with hemi-nested primers. RESULTS: TT virus DNA was detected in 62 of 280 (22.1% [95% CI: 18.1-26.6]) donors with elevated ALT levels (mean +/- SD, 89.3 +/- 36.4 U/L; range, 61-301 U/L), which is significantly more frequently (p<0.02) than its detection in 91 of 581 (15.7% [95% CI: 13.2-18.4]) donors with normal ALT (< or = 45 U/L). The frequency of TT virus DNA increased with age, in donors with and without elevated ALT. CONCLUSION: The detection of TT virus DNA, at a frequency higher in donors with elevated ALT than in those without, strengthens the association of TT virus with non-A to -G hepatitis.

Determination of antibodies to TT virus (TTV) and application to blood donors and patients with post-transfusion non-A to G hepatitis in Japan.

Recently, a nonenveloped single-stranded DNA virus named TT virus (TTV) has been reported in association with non-A to G post-transfusion as well as sporadic acute and chronic liver disease. A method was developed for the detection of antibody to TTV (anti-TTV) by means of immune precipitation and detection of TTV DNA by the polymerase chain reaction. The test serum was incubated with TTV, recovered from feces of a carrier, and after incubation, the formed immune complexes were precipitated with goat antiserum to human IgG. TTV DNA was sought for by the polymerase chain reaction in both precipitate and supernatant. The detection of TTV DNA in the precipitate, but not in the supernatant, was considered to represent anti-TTV in the test serum. Of the 44 healthy blood donors in Japan, anti-TTV was detected in one of the six (17%) with TTV DNA and 11 of the 38 (29%) without TTV DNA. In the two patients with post-transfusion non-A to G hepatitis, free anti-TTV developed as they cleared TTV in serum. Anti-TTV complexed with TTV in serum, detectable by precipitating sera with goat anti-human IgG and testing for TTV DNA, elicited while the patients had elevated alanine transaminase levels. The determination of anti-TTV would be useful for detecting resolved infection in surveys for exposure to TTV in the general population, and for establishing the mechanism of liver injury associated with TTV infection.

Distinct genotypes of a nonenveloped DNA virus associated with posttransfusion non-A to G hepatitis (TT virus) in plasma and peripheral blood mononuclear cells.

TT virus (TTV) is a nonenveloped, single-stranded DNA virus with little sequence homology to known viruses, and associated with elevated transaminase levels in the patients with posttransfusion hepatitis of unknown etiology. The DNA of TTV was detected, by semi-nested polymerase chain reaction, in peripheral blood mononuclear cells (PBMC) from the 30 healthy individuals with circulating virus in plasma. A sequence of 222 bases was determined on 6-10 TTV DNA clones each from plasma and 6 clones -each from PBMC from eight individuals selected at random from this group. TTV can be classified into genotypes separated by an evolutionary distance > 0.30, which can be divided further into subtypes separated by that of 0.15. Three individuals possessed two different TTV variants of distinct genotypes, with predominant genotypes different between plasma and PBMC. Another possessed TTV of the same genotype in both the plasma and PBMC, but clones with a subtype not seen in plasma were observed in PBMC. A third individual had TTV variants with or without a deletion mutation, and those with the deletion mutation abounded only in PBMC. The remaining three individuals were infected with TTV with the same sequence both in plasma and PBMC. These results indicate that TTV variants with phylogenetic differences could infect the same individual, and that some variants would have a predilection for PBMC. It remains to be seen, however, if TTV replicates in PBMC or whether it has been sequestered before its evolution in the host.

Epidemiology of hepatitis C virus infection in Nepal.

Prevalence of hepatitis C virus (HCV) infection in Nepal was studied by assaying sera from different population groups for anti-HCV by the second generation enzyme immunoassay method and for HCV RNA by polymerase chain reaction. The anti-HCV was positive in 0.6% of 2,860 healthy adults. HCV infection was responsible for 1.3% of acute viral hepatitis. Only drug addicts (DA) are known to have a very high incidence of the infection. The number of intravenous drug abusers (IDA) in Nepal have increased considerably since 1991 when buprenorphine (tidigesic) was introduced in the local market. About 72% of the drug addicts were found to be IDA and 94% of the IDA were anti-HCV positive. It is concluded that though the prevalence of HCV infection in the community is low, and at present it accounts for only a small number of acute and chronic liver diseases, the presence of a large number of DA in the country with high incidence of HCV infection may result in the emergence of HCV as an important cause of chronic liver disease in Nepal in future.