岡本 宏明

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 (10(N)ml) was significantly higher in HIV-infected patients [4.5 +/- 1.2 (mean +/- SD) versus 3.1 +/- 0.9, Pc 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. <(c)> 2001 Lippincott Williams & Wilkins.

The sera of 38 patients with nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH), were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-l PCR), and various histological features of the liver biopsy specimens were compared among those who were positive or negative for TTV infection. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 37 (97%) of the 38 patients. In contrast, N22 PCR which detects primarily TTV genotypes 1-4, detected TTV DNA in 18 patients (47%). In the liver biopsy specimens, moderate to many acidophilic bodies, moderate to marked focal/ spotty necrosis of hepatocytes and marked stellate, pericellular or perivenular fibrosis were observed significantly more frequently among those who were positive for TTV DNA by N22 PCR, than among those who were negative by N22 PCR. Twelve patients (32%) were positive for TTV genotype 1. Moderate to marked vacuolation of nuclei, moderate to many acidophilic bodies, and moderate to marked focal/spotty necrosis as well as marked stellate, pericellular or perivenular fibrosis were found significantly more frequently in the TTV genotype 1-positive group than in the TTV genotype 1-negative group. These results suggest that certain TTV genotypes including genotype 1 influence the necrosis and inflammation of hepatocytes and liver fibrosis in NAFLD patients. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

The sera of 43 patients with alcoholic liver disease (ALD) who abstained from. alcohol for 4 weeks, were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-1 PCR). The clinical course of the TTV DNA-positive and -negative groups was compared. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 40 patients (93%). N22 PCR which detects primarily TTV genotypes 1-6, detected TTV DNA in 17 patients (40%). The alanine aminotransferase (ALT) level 4 weeks after the start of abstinence was significantly higher and the rate of change in ALT {[(ALT on admission - ALT 4 weeks after abstinence)/(ALT on admission)] x 100} was lower in the patients who were positive by N22 PCR, than in those who were negative by N22 PCR. Twelve patients (28%) were positive for TTV genotype 1. In the TTV genotype 1-positive group, the ALT 4 weeks after the start of abstinence was significantly higher, and the improvement rates of ALT, gamma -glutamyl transpeptidase and alkaline phosphatase levels were lower than those in the TTV genotype 1-negative group. These results suggest that certain genotypes of TTV may interfere with the improvement of liver function following the start of abstinence in ALD patients. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

TT virus (TTV) is a human circovirus with a single-stranded, circular DNA genome of 3.8 kb. A method was developed to detect IBM antibodies to TTV as a serological marker for the diagnosis of acute TTV infection. ISM antibodies in test sera were captured by a monoclonal antibody against IgM/mu 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 ISM 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 market to detect acute TTV infection. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

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 highperformance liquid chromatography, and TTV DNA-rich fractions were subjected to floatation ultracentrifugation in cesium chloride. Virus-like particles, 30-32 nm in diameter, mere 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 mere 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) mere visualized in fecal supernatant with TTV genotype 1a by immune electron microscopy using human plasma containing TTV genotype 1a-specific antibody. (C) 2000 Academic Press.

Although replicative forms of TT virus (TTV) DNA have been found in the liver and bone marrow cells, mRNAs of TTV have not yet been detected in these tissues. The entire nucleotide sequence of a TTV clone [TYM9 (3759 bases)] isolated front a patient with high TTV viremia (10(6) copies/ml) was determined, and the poly(A)(+) RNAs from bone marrow cells were subjected to reverse transcription-polymerase chain reaction with primers specific for the TYM9 sequence. Sequence analysis of the amplified products revealed the presence of three distinct species of spliced TTV mRNAs [2.9, 1.2, and 1.0 kilobases (kb)] with common 5' and 3' termini as well as splicing to bind nucleotide (nt) 181 to nt 283. The shorter mRNAs of 1.2 kb and 1.0 kb possessed another splicing to join nt 681 with nt 2341 or nt 2579. The transcription profile of TTV found in an infected human corroborates that observed in vitro. (C) 2000 Academic Press.

Fecal supernatant or serum containing TT virus (TTV) of genotype la (10(5) copies/ml) from patients with acute TTV infection was inoculated intravenously into two naive chimpanzees. Serum samples were obtained weekly and tested for TTV DNA by genotype 1-specific polymerase chain reaction. TTV DNA was detected in chimpanzee 228 at weeks 5-15 after inoculation with 0.5 mi of serum, and in chimpanzee 234 at weeks 7-19 after inoculation with 1 mi of fecal supernatant. The TTV DNA titer peaked at weeks 12 and 13 in chimpanzee 228 and at weeks 14-16 in chimpanzee 234. Mild biochemical and histological changes in biopsied liver samples were observed in both chimpanzees in association with the reduction in TTV titer. TTV DNA was transient in chimpanzee 228, but in chimpanzee 234 it reappeared at week 21 and persisted through week 30. These results indicate that TTV in feces is infectious and suggest that ITV has hepatitis-inducing capacity. (C) 2000 Academic Press.

By means of polymerase chain reaction with a primer pair (NG133-NG147) deduced from the untranslated region (UTR) of TT virus (TTV), TTVs with markedly distinct genomic lengths were recovered from sera of humans and nonhuman primates, and their entire nucleotide sequences were determined. A human TTV [TGP96 of 2908 nucleotides (nt)] was obtained that was about 900 nt shorter than heretofore reported TTVs (3787-3853 nt). Likewise, TTVs of chimpanzee occurred in two distinct genomic sizes [Pt-TTV6 (3690 nt) and Pt-TTV8-II (2785 nt)]. Two TTVs of Japanese macaque [Mf-TTV3 (3798 nt) and Mf-TTV9 (3763 nt)] were comparable in genomic length, but only 55% similar in sequence. These five human and nonhuman primate TTVs, along with TTVs of tamarin [So-TTV2 (3371 nt)] and douroucouli [At-TTV3 (3718 nt)], were compared over the entire nucleotide sequence. Although the seven TTVs were only less than or equal to 55% similar, they share a common genomic organization with two open reading frames (ORFs), designated ORF1 (654-735 amino acids) and ORF2 (91-152 amino acids). The N-terminal sequences of ORF1 proteins were rich in arginine, and sequence motifs necessary for transcription and replication were conserved among them all. Like the human prototype TTV (TA278), all seven TTVs from various animals possessed in common two 15-nt sequences (CGAATGGCTGAGTTT and AGGGGCAATTCGGGC) in the UTR that were covered by NG133 and NG147, respectively. These primers would be instrumental in research on TTVs in previously unexamined species for defining their virological characteristics and evolutionary relationships. (C) 2000 Academic Press.

Peripheral blood mononuclear cells (PBMC) harbored TT virus (TTV) of genotypes (3 and 4) different from those (1 and 2) of free virions in plasma of the same individuals, PBMC may act as a reservoir, and TTV of particular genotypes might have tropism for hematopoietic cells.

BACKGROUND: The potentially hepatotropic flavivirus-like virus, GB virus type C (GBV-C)/HGV, has been detected in a few patients with acute and chronic hepatitis and in a certain proportion of blood donors and recipients of blood or blood components. STUDY DESIGN AND METHODS: Sera from 2979 pregnant Japanese women were examined for the presence of GBV-C/HGV RNA by nested RT-PCR. Mothers who were positive for viral RNA and their 34 infants were followed and tested for infection. RESULTS: Of the 2979 women, 32 (1.1%) were positive for GBV-C/HGV RNA. Twenty-six (76.5%) of 34 babies born to these women were positive for the virus when first tested. A significantly higher titer of viral RNA was observed in mothers whose infants were infected than in those whose infants were uninfected (mean +/- SD, 10(6.3) +/- 0.9 VS. 10(4.6) (+/-) (0.9)/mL; p < 0.001). Twenty-three (96%) of 24 babies born to mothers whose serum viral titers were 10(6) mL or more were infected with the virus. Infants delivered by elective caesarean section had a lower risk (OR, 0.13; 95% CI, 0.02-0.82) than those delivered vaginally or by emergency caesarean section. No other risk factors for mother-to-infant transmission were confirmed. CONCLUSIONS: GBV-C/HGV is frequently transmitted from mothers to infants in the general population. The most critical factor is the titer of viral RNA in the maternal serum. By the use of elective caesarean section in women with high titers of viral RNA, vertical transmission of the virus may be lessened.

An enzyme-linked immunosorbent assay (ELISA) has been described for serological determination of hepatitis B virus genotypes, using monoclonal antibodies (mAb) against seven distinct epitopes (b, m, k, s, u, f and g) on the preS2-region products of hepatitis B surface antigen (HBsAg). The usefulness of this method for serological detection of genotype E, however, was theoretical, because no HBsAg samples of this genotype were included in the original test panel. Moreover, the predicted serotype of genotype E (bksufg) closely resembled that of genotype D (bksu, bksuf or bksug). Four HBsAg samples of genotype E were tested by the original described ELISA. The epitope g, predicted to be present in these samples by amino acid sequences, was not detected when HBsAg of genotype E was captured on a solid phase by mAb to the common determinant 'a' of HBsAg and then reacted with mAb to g (5156) labeled with horseradish peroxidase. However, the four examples of HBsAg of genotype E were captured by mAb 5156 to g on a solid phase; they were then detected by labeled mAb to the common determinant 'a'. Since epitopes f and g co-occurred on HBsAg of genotype E, HBsAg samples of this genotype were also detected, by 'sandwiching' them between immobilized mAb to g and labeled mAb to f. By contrast, HBsAg of genotype D in 90 sera was not reactive when sandwiched between mAb to f and g. Thus, this modified ELISA enables the serological determination of all six genotypes of HBsAg and, by inference, of hepatitis B virus. (C) 2000 Elsevier Science B.V. All rights reserved.

TT virus (TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide, TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically, ITV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb, TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb, Both faster and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease, They migrated at 3.7 to 4.0 kb after digestion with EcoRI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum mere found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus stranded DNAs, These results suggest that TTV replicates in the liver via a circular double-stranded DNA.

TT virus (TTV) is a human virus consisting of a single-stranded, circular DNA genome of 3.8 kilobases (bb). 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. (C) 2000 Academic Press.

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-tap 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 m's. 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 ere 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 la. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype la aas 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 m's are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.

TT virus (TTV) has a wide range of sequence divergence by which it is classified into at least 16 genotypes. A TTV isolate of genotype 12 (TJN01) and another of genotype 13 (TJN02) were sequenced in the entire genome, and compared with the reported TTV isolates. TJN01 and TJN02 had genomic lengths of 3787 and 3794 nucleotides (nt), respectively, which were shorter by 66 and 59 nt than the prototype TTV isolate of genotype 1 (TA278). TJN01 and TJN02 shared the nucleotide sequence with TA278 merely in 53.9% and 55.2%, respectively. They possessed two major open reading frames (ORFs) and the noncoding region with a GC-rich region forming stem-loop structures, which are characteristic of TTV. However, their amino acid sequences in ORF1 were similar to that of TA278 in only 35.4 and 34.0%, respectively; TJN01 was 45.4% similar to TJN02. Comparison with TTV isolates of the same genotype identified hypervariable regions in ORF1 of TJN01 and TJN02, as in the prototype TTV of genotype 1. However, quasispecies were barely observed in them. Furthermore, sequences of hypervariable regions scarcely changed during 2-5.5 years in both TJN01 and TJN02. These results indicate that TTV of genotypes 12 and 13 are much different from the prototype TTV of genotype 1.

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.