INTRODUCTION

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TT virus (TTV) is a newly discovered human virus that was first detected in the serum of a Japanese patient (initials, T.T.) with posttransfusion hepatitis (12, 13). The TTV genome is constituted by a single-stranded, circular DNA of negative polarity (9, 13). The TTV nucleotide sequence (3,818 to 3,853 nucleotides) does not show a significantly high homology to the sequence of any other virus. Several TTV isolates have been entirely sequenced (4, 6, 8, 9, 14), revealing a high degree of divergence among strains and the existence of at least 16 genotypes, which are separated by an evolutionary distance greater than 0.30 (15).

Although TTV DNA titers closely correlated with aminotransferase levels in the sera of some patients during posttransfusion hepatitis (12), no clear association between TTV infection and human liver disease has been established at this time. Very high prevalences (62 to 96%) of TTV infection have been found in healthy populations of Japan (15, 20) as well as in developing Asian, African, and South American countries (1, 11, 17).

As was initially demonstrated, TTV transmission occurs through the parenteral route (12). However, very high prevalences in healthy populations indicate the existence of other routes of transmission.

Coinfection with multiple TTV strains has been described for people exposed to blood and blood products (3, 5, 21), as well as for patients with liver disease (2, 7, 22). Here we show that such a coinfection is a common event in Brazilian health care workers and that a healthy person can be coinfected by at least seven strains.

	MATERIALS AND METHODS

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Population studied. In 1999, all persons working in a public hospital of the city of Rio de Janeiro, Brazil, were invited to receive immunization against hepatitis B. At the occasion of the first-dose injection, blood samples from 1,104 people were collected. Testing of the DNA of TTV was done with serum samples obtained from 191 subjects, selected among persons aged 19 to 50 years who declared not having received blood transfusion.

Extraction and amplification of DNA. Two hundred and fifty microliters of serum was treated with 0.5 mg of proteinase K per ml in the presence of 0.2 M NaCl-0.25% sodium dodecyl sulfate for 4 h at 37°C. DNA was then extracted by phenol-chloroform and precipitated by ethanol. The pellet was dried and resuspended in 50 µl of distilled water. DNA was amplified by a single round of PCR performed with 5 µl of DNA in a 50-µl reaction mixture. AmpliTaq Gold polymerase (Perkin-Elmer Applied Systems, Foster City, Calif.) and primers T801 and T935 were used according to the protocol described by Takahashi et al. (20). Ten microliters of PCR products was loaded onto a 2% agarose gel, electrophoresed, and stained with ethidium bromide to visualize bands with an expected length of 199 bp.

Molecular cloning and nucleotide sequencing. PCR products were cloned into the pCR2.1-TOPO vector (TOPO TA cloning kit; Invitrogen, San Diego, Calif.). Recombinant plasmids were purified and insertion DNAs were sequenced by using Cy5-labelled forward and reverse M13 primers and the reagents provided in the Autoread Sequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Sequencing products were analyzed on an ALFexpress automated sequencer (Amersham Pharmacia).

Computer analysis of TTV sequences. Alignment of multiple nucleic acid sequences was performed with the University of Wisconsin Genetic Computer Group program PILEUP. Phylogenetic analysis was performed with the program GROWTREE from the same package. Genetic distances between TTV strains were calculated by using the program Dnadist from the package PHYLIP, version 3.5c.

	RESULTS

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Seroprevalence of TTV DNA. One hundred ninety-one health care workers (47 men, 144 women) of a public hospital of the city of Rio de Janeiro, Brazil, were enrolled in this study. Serum samples were analyzed for the presence of TTV DNA. One hundred twenty-five samples were positive, corresponding to a prevalence of 65.4%.

Phylogenetic analysis. PCR products were cloned, and sequences of 159 bases (nucleotides 26 to 184) surrounding the TATA signal region localized upstream of open reading frame 2 (ORF2) were determined for 100 clones derived from 31 individuals. One clone from each of 23 subjects was sequenced, while 7 to 19 clones were sequenced from eight individuals. A total of 59 different sequences was thus obtained.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree of 69 TTV isolates. The Jukes-Cantor algorithm was used for distance determination, and the tree was constructed with the neighbor-joining method. Ten sequences available in the databases are identified by their GenBank accession numbers, followed by the names of the isolates. The genotypes of seven of them (AB008394, AF122915, AF122913, AF122914, AF122916, AF122921, AB017613) are known and indicated at the right. Fifty-nine sequences, whose names begin by the letters SC, are from this work. Different clones from a single patient are distinguished by a number after the hyphen. Fourteen sequences contain a single-base deletion at position 140.

Coinfection with multiple TTV isolates. Several clones derived from the same serum were sequenced to determine if coinfection (or superinfection) with multiple TTV strains occurred. This was performed for eight individuals whose demographic, professional, serological, and clinical data are shown in Table 1.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   Alignment of partial nucleotide sequences of 36 TTV variants found in eight health care workers. The number of clones showing identical sequences is given. The sequence of prototype isolate TA278 of genotype 1a (13) is indicated at the top. Dashes represent the same nucleotides as in the TA278 isolate. In two clones from subject SC319, an insertion of an A nucleotide occurs at position 45; slashes indicate the absence of this nucleotide in the other sequences.

	DISCUSSION

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TTV is a virus with a wide nucleotide sequence divergence (6, 15, 18, 23). The PCR assays developed soon after the discovery of the virus were not able to amplify DNA of all genotypes, and the TTV prevalences in the populations of different countries have therefore been dramatically underestimated. Recently, improved PCR protocols and new sets of primers have led to increased rates of TTV DNA detection. The PCR method employed in this study has thus allowed the detection of TTV in 92% of the serum samples of a group of Japanese blood donors (20), a prevalence much higher than the 23% obtained with the same samples by using the previously designed primers NG059, NG061, and NG063 (13). Another independent study has shown that the method allows the detection of five- to sixfold more TTV-positive samples than two other protocols (7). Using PCR primers whose design was based on NG059, NG061, and NG063, but which were degenerated to allow the detection of a higher number of TTV variants, we recently reported a TTV seroprevalence of 62% in blood donors living in the city of Rio de Janeiro, Brazil (11). The prevalence (65.4%) obtained now in a group of 191 health care workers living in the same city was not significantly different.

Although the DNA segment analyzed here is relatively short and is more conserved than other regions of the TTV genome (4, 14), no two sequences were identical when derived from different persons. Eighty-nine percent of the genetic distances between two of our strains were higher than 0.10 (17% higher than 0.30), with a maximum value at 0.49. These findings demonstrate the large genetic diversity of TTV strains circulating in Brazil. At the moment, few TTV genotypes have been sequenced in the genome region under study. This made it difficult to ascribe a genotype, from the 16 previously described (15), to each of our samples. However, it is interesting that 14 sequences, with a single-nucleotide deletion at position 140, constituted a separate genotype (Fig. 1). Examination of the nucleotide sequences deposited in Genbank by using the BLAST program revealed that two sequences (AF109811 and AF109812), from Chinese TTV isolates, presented the 1-nucleotide deletion at position 140. These two sequences presented an overlapping of only 72 bp with ours. On this small genome segment, a very high homology (93 to 98%) was observed between the Chinese sequences and the 14 Brazilian sequences belonging to the separate genotype.

The ORF2 translation initiation codon was initially localized to position 107 (9, 13). However, further studies suggested that another ATG codon, at position 263, is preferentially recognized as the start codon (4, 6, 14). The existence of a single-nucleotide deletion at position 140 in 14 sequences, which would introduce a frameshift mutation, reinforced this hypothesis. However, it is noteworthy that there was no case in which all TTV clones derived from the same individual contained that deletion. Therefore, a complementation between wild-type and deletion mutant strains cannot be excluded.

Mixed infections of TTV have been reported in individuals at high risk for infection with parenterally transmitted viruses, such as intravenous drug users (9), hemophiliacs (9, 21), and hemodialysis patients (3, 5), as well as in patients with liver disease (2, 7, 9, 22). Recently, coinfections with two or three TTV strains have been reported to be in some healthy Japanese individuals (15, 16). Here we show that such a mixed infection is a common event in healthy Brazilian people, at least in health care workers. Our results confirm and extend previous observations showing that infection by a given genotype is not protective against the superinfection by another type (5, 15). Furthermore, we show that the number of TTV isolates infecting an individual can be high. For example, for subject SC894, the nucleotide sequences of 19 clones were determined and 11 distinct sequences were obtained (Table 2). Genetic distances between two TTV sequences present in the same serum could be very close or very divergent (up to 0.45 for subject SC314). Although the mutation rate of the TTV genome as well as the duration of infection in the persons under study are unknown, it is likely that very close sequences (genetic distance < 0.02) derived from a single source of infection (quasispecies). On the contrary, there is no doubt that subject SC314 was infected from multiple sources. Using a cutoff value of 0.10, it was demonstrable that TTV strains belonging to at least seven genotypes or subtypes may infect the same healthy person (Table 2).

A recent study has demonstrated similar prevalences of TTV infection in medical workers and in age-matched controls (10). It remains to be determined if the phenomenon of multiple infection occurs at a comparable frequency in both groups. In a previous paper, we showed that the TTV seroprevalence increased continuously with age (19). Here, the only individual (SC189) for whom all TTV clones were identical was the youngest of a group of eight persons. She was 22 years old, whereas all others were 40 to 50 years old. Further studies are necessary to determine if an accumulation of TTV strains occurs during the life of healthy individuals or if such a tendency is restricted to health care workers.

Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper have been submitted to the GenBank database under accession no. AF216433 through AF216491.