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Journal of Clinical Microbiology, May 2001, p. 2022-2024, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.2022-2024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High Prevalence of TT Virus (TTV) and TTV-Like
Minivirus in Cervical Swabs
Claudia
Fornai,
Fabrizio
Maggi,
Maria Linda
Vatteroni,
Mauro
Pistello, and
Mauro
Bendinelli*
Virology Section and Retrovirus Center,
Department of Biomedicine, University of Pisa, Pisa, Italy
Received 16 October 2000/Returned for modification 30 January
2001/Accepted 6 March 2001
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ABSTRACT |
Genomes of TT virus (TTV) and TTV-like minivirus DNA were detected
in 80% and 61% of cervical swabs from healthy women, respectively, regardless of concurrent human papillomavirus infection. These results
show that the potential exists for sexual transmission of TTV and
related viruses.
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TEXT |
TT virus (TTV) is a recently
described DNA virus of humans that shows a number of unusual properties
(9, 11). From the clinical microbiology standpoint, a most
remarkable feature of TTV is its widespread presence in the blood of
infected individuals
often at high titers and for prolonged or
indefinite periods of timein the apparent total absence of associated
pathological effects (1). For example, in recent studies
of the general population of Tuscany, Italy, the prevalence of subjects
who carry TTV in the blood was on the order of 90%, and viremia loads
ranged between 7.7 × 102 and 6.2 × 108 DNA copies per ml of plasma, as determined with an open
reading frame 1-targeted real-time PCR assay (8, 12).
Similarly high prevalence rates have been reported from other
countries, whenever detection methods of comparable sensitivity were
used (5, 10). Thus, two aspects of TTV biology appear
especially intriguing: the routes of interindividual transmission that
permit such a dissemination and the type(s) of relationship with the
infected organism that permits TTV to replicate extensively with no
apparent damage to the host. A clear understanding of the body sites
where TTV dwells and persists can help shed light on both of these aspects.
Here we examined for the presence of TTV DNA 50 consecutive cervical
swabs sent to our laboratory for human papillomavirus (HPV) testing
between October 1999 and May 2000. The specimens were collected from
apparently healthy women using routine procedures for prophylactic
screening of cervical cancer. Tests were performed without knowledge of
cytology and clinical data. Each swab was soaked and vortexed gently in
sterile phosphate-buffered saline, which was then stored in aliquots at
70°C until use. HPV detection and genotyping were carried out by
amplifying a segment of the L1 gene with consensus primers MY09 and
MY11 and subsequent restriction fragment length polymorphism analysis,
as reported previously (2). Thirty-eight swabs were found
to be HPV DNA positive; of these, 20 carried low-risk HPV and 12 carried high-risk HPV, while 6 HPV isolates were not typed (Table
1).
TTV detection was carried out using a TaqMan real-time PCR assay
(8, 12) that, being targeted to a highly conserved segment of the nontranslated region (UTR) of the viral DNA, detects a wider
range of genotypes than most TTV detection methods described to date,
including the ones used by us in previous studies (6). Forward and reverse primers were 5'-GTGCCGIAGGTGAGTTTA-3'
(positions 177 to 194) and 5'-AGCCCGGCCAGTCC-3'
(positions 226 to 239), respectively. The probe was
5'-TCAAGGGGCAATTCGGGCT-3' (positions 205 to 223), which was
labeled with 6-carboxy-fluorescein and 6-carboxy-tetramethyl-rhodamine at the 5' and 3' ends, respectively, and had a propynilic group bound
to each thymidine to increase the annealing temperature. The procedures
used for quantification of copy numbers and evaluation of intra- and
interassay precision and reproducibility have been previously described
(12). Maximum intra- and interassay variation in the
threshold cycle was about 3%, and specificity was confirmed by
repeatedly sequencing the products of amplification. The lower limit of
sensitivity of the assay was 1.0 × 103 copies/µg of
DNA. All cervical swabs were tested in triplicate twice from
independent DNA extractions. Samples positive in only one replicate or
with a coefficient of variation of 50% or greater constituted less
than 2% of samples tested. These samples were reextracted and tested
again in triplicate.
Nucleotide sequence accession numbers.
Sequences of a 265-bp
UTR segment from the TTV-like minivirus (TLMV) isolates listed in Fig.
1 were submitted to GenBank (accession no. AF312400 to AF312420).
The results of TTV testing are shown in Table
1. Overall, 80% of the
cervical swabs examined were found positive, thus indicating
that TTV
infection of the feminine genital tract is extremely
common in this
select group of women. TTV loads varied widely
among individual
samples, ranging between 1.3 × 10
3 and 1.7 × 10
8 copies per µg of total DNA. When the swabs were
grouped according
to the results of HPV analysis, TTV prevalence rates
and titers
were similar. Thus, although the numbers of patients
examined
were too low to draw firm conclusions, it would appear that
TTV
infection is not facilitated by concurrent HPV infection (Table
1).
While this study was in progress, Takahashi et al. (
13)
reported on the existence of a new virus, related but clearly distinct
from TTV, in the plasma of Japanese voluntary blood donors. Due
to its
small estimated size (less than 30 versus 30 to 50 nm)
and shorter
genome (approximately 2.9 versus 3.8 kb) relative
to that of TTV, the
virus was designated TLMV. Early data have
suggested that TLMV may be
as widespread as TTV, while modes of
transmission and pathogenicity
have yet to be investigated (
13,
14). We tested 33 of the
above-discussed cervical swabs (27
TTV positive and 6 TTV negative) for
the presence of TLMV using
a nested PCR assay targeted to a segment of
the UTR of the viral
DNA, which, based on the sequences available in
gene banks at
the time the assay was developed, was well conserved and
specific
for TLMV. The following oligonucleotide primers were used:
external
primers, 5'-ATTTGAATTGCCGACCACA-3' (positions 30 to
48) and 5'-CGCCAGACTGATCTAGCCCGAA-3'
(positions 292 to 313);
internal primers, 5'-ATTGCCGACCACAAACTGACA-3'
(positions 36 to 56) and 5'-AGCCCGAATTGCCCCTAGTC-3' (positions
280 to
300). Bands of the expected size (265 nucleotides [nt])
were detected
in 20 samples (61%), regardless of concurrent infection
with HPV
(Table
1) and TTV (not shown). Sequence alignment of
12 such bands with
the CLUSTALW program confirmed that they belonged
to TLMV, while
phylogenetic relationships, estimated by using
DAMBE (version 4.0.9 [
15]) and the neighbor-joining method,
showed a wide
genetic heterogeneity (Fig.
1). TLMV
isolates detected
in plasma samples have also been shown to be
genetically highly
heterogeneous (
13,
14). Interestingly,
in addition to the
265-nt TLMV bands, four cervical swabs yielded bands
of approximately
400 nt, which, when sequenced, were found to belong to
the YONBAN
strain of TTV. This strain is currently grouped together
with
TTV, but its high degree of divergence has led researchers to
propose that it might represent a separate viral species
(
14).
Our TLMV primers had been designed before UTR
sequences of the
YONBAN strain had become available, but comparison
with subsequently
published YONBAN sequences showed that the internal
antisense
primer is highly conserved between TLMV and YONBAN. This
might
explain why the TLMV PCR had permitted amplification of YONBAN
virus from some swabs.
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FIG. 1.
Phylogenetic analysis of the TLMV isolates based on a
265-bp UTR segment (nt 36 to 300). Isolate NLC023 (13) was
used as an outgroup. The bar indicates the number of nucleotide
substitutions per site. The sequences from cervical swabs are marked
with  , while reference sequences are indicated by the isolate name
only. Sequences obtained in our laboratory from seven plasma samples
( ) and two liver biopsy specimens ( ) are also shown.
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|
In conclusion, the present findings show that cervical specimens may
contain a wide array of TTV and related viruses. Whether
this reflects
active replication of such viruses in the female
genital tract, for
example by lymphoid cells (
7), or exudation
from plasma
remains to be established. Information about sexual
transmission of TTV
and related viruses is scarce. Recently, Inami
et al. (
4)
detected TTV DNA in 6 of 10 human semen samples
tested. Together with
the latter results, the present data indicate
that prerequisites exist
for TTV and related viruses being commonly
transmitted via the sexual
route. Viral sequences obtained from
sexual partners should be compared
in order to shed light on the
importance of this relative to other
modes of transmission. Furthermore,
in some studies the high prevalence
of TTV infections in infants
was attributed to close contact with
infected mothers (
3).
Exposure to virus-containing vaginal
fluids during delivery may
contribute to perinatal transmission of
these widespread
viruses.
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ACKNOWLEDGMENTS |
This work was supported in part by grants from the Ministero della
Università e Ricerca Scientifica.
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FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Biomedicina, Università di Pisa, Via San Zeno 37, I-56127 Pisa,
Italy. Phone: 39 (50) 559.440. Fax: 39 (50) 559.455. E-mail:
bendinelli{at}biomed.unipi.it.
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Journal of Clinical Microbiology, May 2001, p. 2022-2024, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.2022-2024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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