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Clinical and Diagnostic Laboratory Immunology, September 2000, p. 845-849, Vol. 7, No. 5
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Prevalence and Phylogenetic Analysis of TT Virus
DNA in Western India
V. A.
Arankalle,*
S. S.
Gandhe,
T. M.
Deshmukh,
M. S.
Chadha, and
A. M.
Walimbe
Hepatitis Division, National Institute of
Virology, Pune, India
Received 6 December 1999/Returned for modification 8 May
2000/Accepted 31 May 2000
 |
ABSTRACT |
In western India, TT virus (TTV) DNA positivity varied from 6.7%
(5 of 75) in chronic hepatitis patients to 24.4% (10 of 41) in
hemophiliacs; 7.4% (4 of 54) of voluntary blood donors had circulating
TTV DNA. Phylogenetic analysis revealed a predominance of genotype 1a.
In India, TTV is transmitted mainly by nonparenteral routes and is not
an important cause of chronic liver diseases.
| |
TEXT |
Recently, a novel DNA virus termed
TT virus (TTV) on the basis of the initials (T.T.) of one of the
infected patients investigated was found to be associated with
posttransfusion non-A to -G hepatitis cases in Japan (15).
Although the virus shares some of the characteristics of parvoviruses,
significant sequence similarity to the known parvoviruses has not been
observed (17). TTV was found to be highly prevalent in
Japan, with significantly higher proportions among patients with
parenteral risk, patients with fulminant hepatic failure of non-A to -G
etiology, and patients with chronic liver diseases (CLDs) of unknown
etiology (17). A recent study from Japan (10)
showed that rates of detection of TTV DNA did not differ statistically
between patients with non-A to -E hepatitis and patients with hepatitis
A, B, or C or controls. The clinical characteristics were comparable
for patients with or without TTV DNA.
In order to investigate (i) whether TTV represents the causative agent
for the residual non-A to -G hepatitis and (ii) the extent of
transfusion-associated transmission of this virus, studies are being
conducted in different parts of the world (4, 7, 8, 14, 18, 19,
21). The sequence data generated document the existence of
several genotypes of TTV (7, 8, 14, 17, 21, 22). So far, no
information is available from India. To ascertain the extent of TTV
infection among Indian patients, we studied certain categories of
individuals from western India. These included voluntary blood donors
(n = 54), paid plasma donors from a commercial
plasmapheresis unit (n = 31), hemophiliacs
(n = 41), patients suffering from CLDs (n = 75) (including 54 who were hepatitis B virus [HBV] DNA positive
and 21 who were HBV DNA as well as HCV RNA negative), patients
undergoing hemodialysis (n = 24), and symptomless
hepatitis B surface antigen (HBsAg) carriers with consistently normal
serum alanine aminotransferase levels for a period of 3 years
(n = 83). Aliquots of stored (
20°C) serum samples
were utilized for TTV DNA screening.
All serum samples were screened for the presence of TTV DNA by nested
PCR. DNA isolation was carried out using DNAZOL reagent (GIBCO-BRL Life
Technologies) according to the manufacturer's instructions, followed
by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min for first- and second-round PCRs. Primers representing part of open
reading frame 1 (ORF1), as described by Simmonds et al.
(21), were used. Primers for the first round of PCR were
A5430 (5'-CAG ACA GAG GAG AAG GCA ACA TG-3') and A5427 (5'-TAC CAY TTA GCT CTC TAT TCT WA-3'). Primers for the
second round of PCR were A8761 (5'-GGM AAY ATG YTR TGG ATA GAC
TGG-3') and A5432 (5'-CTA CCT CCT GGC ATT TTA CCA-3').
Amplified DNA fragments (278 bases) were minicolumn purified (Wizard;
Promega). Both strands of column-purified PCR products were sequenced
using a Taq dye terminator cycle sequencing kit (Perkin-Elmer) and an automatic sequencer. Twenty TTV DNA-positive samples were sequenced. These included three samples each from voluntary blood donors, hemophiliacs, and HBsAg carriers; five from
patients suffering from CLDs; four from paid plasma donors; and two
from patients undergoing hemodialysis.
Phylogenetic analysis was based on the comparison of a 171-nucleotide
fragment of ORF1. MEGA (11) and PHYLIP version 3.5c (6) software was employed to determine the phylogenetic
status of different TTV isolates. For analysis with MEGA, the
Jukes-Cantor algorithm was utilized, employing the neighbor-joining
method. The reliability of different phylogenetic groupings was
evaluated by using the bootstrap test (1,000 bootstrap replications)
available in MEGA. For PHYLIP program-based analysis, the Jukes-Cantor
algorithm was used, employing the neighbor-joining method with and
without midpoint rooting. For evaluation of the results obtained,
bootstrap analysis was performed (SEQBOOT; 1,000 bootstrap replications).
Fisher's exact test and chi-square tests were used for comparison of
two proportions. Odds ratios (ORs) were calculated for the assessment
of risk of TTV infection in different categories in comparison with
voluntary blood donors. The software EPI INFO (version 6.02) was used
to carry out the computations.
Prevalence of TTV DNA.
Table 1
documents the TTV DNA positivity among different groups screened in
nested PCR. Voluntary blood donors exhibited 7.4% (4 of 54)
positivity. None of these 54 voluntary blood donors were positive for
HBsAg or antibodies to HCV (data not shown). The prevalence of TTV DNA
among voluntary blood donors has been shown to vary from 1% in the
United States (3) and 1.9% in the United Kingdom
(21) to 10.7% in the United States (5) and 12%
in Japan (17). Considering recent reports (5, 12) of underreporting of TTV on the basis of PCR assays utilizing the
primers used by Simmonds et al. (21), higher exposure rates may be found among Indian populations following the use of more efficient primers. All the same, this first report from India documents
circulation of TTV for the last 10 years at least.
Of the 83 symptomless HBsAg carriers (a subset of the voluntary blood
donors screened earlier), 7 were found to be TTV DNA
positive (8.4%).
Thus, HBsAg-positive or -negative voluntary blood
donors were not
significantly different (
P > 0.9) with respect
to TTV
DNA positivity. A significantly higher proportion (
P <
0.05) of hemophiliacs (10 of 41; 24.4%) were found to have
circulating
TTV DNA when compared to voluntary blood donors, patients
suffering
from CLDs, and HBsAg carriers. Based on the estimation of
ORs,
hemophiliacs appeared to be at a fourfold-higher risk of TTV
infection
(Table
1).
The TTV DNA positivity among CLD patients (5 of 75; 6.7%) and
voluntary blood donors was not significantly different (
P >
0.5). Fifty-four of 75 patients suffering from CLDs were HBV DNA
positive. All five TTV DNA-positive CLD patients belonged to the
HBV
DNA-positive subset of CLD patients examined. The comparable
prevalence
rates among CLD patients and voluntary blood donors
suggest that TTV
may not be pathogenic in most infected individuals,
confirming
observations from the United Kingdom (
14), Taiwan
(
8), and recently, Japan (
10). However, these
observations
are in contrast to other reports from Japan
(
17), the United
States (
3), and Thailand
(
19). Interestingly, none of the
21 HBV DNA- and HCV
RNA-negative CLD patients were TTV DNA positive.
However, TTV DNA
positivity among HBV DNA-positive and HBV DNA-
and HCV RNA-negative CLD
patients was not significantly different
(
P > 0.5).
Thus, TTV may not represent the major etiologic agent
for non-B, non-C
chronic hepatitis in western India. In fact,
all five TTV DNA-positive
patients suffering from CLDs were also
HBV DNA positive. No clinical
follow-up of these patients is available
to address disease severity
and outcome among patients positive
for both HBV and TTV or for HBV DNA
alone.
To assess the extent of transfusion-associated transmission of the
virus, TTV DNA positivity was evaluated for three risk
groups, i.e.,
patients undergoing hemodialysis, hemophiliacs,
and paid plasma donors
from a blood products manufacturing organization
(who have a very high
prevalence of anti-HIV [
2], anti-HCV
[
9], and anti-HEV [
1] antibodies).
Compared to that among
voluntary blood donors, TTV DNA positivity among
paid plasma donors
(4 of 31; 12.9%) and patients undergoing
hemodialysis (2 of 24;
8.3%) was not significantly different
(
P > 0.5), indicating that
the risk of TTV infection
in these categories was not high. The
fourfold-higher risk of TTV
infection in hemophiliacs could be
associated with intermittent use of
imported blood
products.
The data also emphasize that the parenteral route may not be an
important mode of transmission of TTV in the Indian population
and that
other modes may be operative to an appreciable extent.
In this
connection it is important to note that TTV DNA has been
detected in
feces and bile samples and a possibility of fecal-oral
transmission was
suspected (
16,
23). A recent study attributing
an outbreak
of enterically transmitted non-A, non-E viral hepatitis
to TTV is
noteworthy (
13). Among the rural populations of Nigeria,
Gambia, Brazil, and Ecuador (
20), TTV DNA positivity was
found
to be 7 to 74%. An environmental source of TTV infection,
comparable
to those for other enteric infections, has also been
suggested
(
4). The predominant mode of TTV transmission
among Indian
populations remains to be
elucidated.
Based on the limited data, infection of the population under study with
dual or multiple variants of TTV does not appear to
be
frequent.
Phylogenetic analysis.
A wide variation among different TTV
sequences from Japan led to classification of different isolates of the
virus (17), with those separated by an evolutionary distance
of 0.3 constituting types (1 and 2) and those separated by a distance
of >0.15 representing subtypes (1a, 1b, 2a, 2b, and 2c). Subsequently,
additional genotypes (types 3 and 4) were discovered (22,
23).
As evidenced by the dendrogram shown in Fig.
1, the
59 isolates analyzed in the present study fall into six major
genotypes.
The majority of the isolates (36 of 59; 61%), including 19 of
20 Indian isolates, belonged to genotype 1. This genotype was
further divided into two branches. Branch 1a (28 isolates) included
all
19 Indian isolates, 2 German isolates, 1 isolate each from
China and
Thailand, and 5 isolates from Japan. Both of the isolates
from Germany,
one isolate each from Thailand and the United Kingdom,
and four
isolates from Japan constituted genotype 1b.
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FIG. 1.
Phylogenetic analysis based on a 171-nucleotide
fragment of the ORF1 genes of 59 TTV isolates. These included one
isolate from China (TTVCHN1 [accession no. AB079173]), 13 isolates
from Germany (C2, H3, A3, D3, F5, F4, D5, A4, H1, H4, A1, C1, and Be7
[AF108946 to AF108958]), 20 isolates from India (present study,
indicated in boldface [AF177443 to AF177462]), 20 isolates from Japan
(TA278, G104901, TY96117, N22, TX011, TS003, NA004, CMR12, MONG969,
JAM18, JAM21, JAM28, JAM29, JAM53, JAM59, MONG268, MONG301, TC1, CK7,
and EN3 [AB008394, AB011489, AB011494, AB017767, AB017769 to AB017771,
AB017877, AB017881, AB017886 to AB017893, AB018850, AB018961, and
AB018963]), 2 isolates from Thailand (TTV33 and TTV36 [AF078114 and
AF078115]), and 3 isolates from the United Kingdom (BLDN9, BLDN20, and
BLDN17 [AF072749, AF079541, AF079543]). Percent bootstrap supports
(1,000 replicates) are shown by numbers at respective nodes. Genotypes
(in boxes) and subgenotypes are marked in boldface. Each dash
represents 0.004833 substitution per nucleotide.
|
|
Genotype 2 included eight isolates and was subdivided into types 2a,
2b, and 2c. One Indian isolate grouped along with genotype
2c sequences
represented mainly by the German isolates, whereas
genotype 2a
consisted of one isolate from Japan. Isolates from
Japan and Germany
belonged to genotype
2b.
Genotype 3 was characterized by isolates from Germany and Japan. This
genotype was divided into 3a (one German isolate), 3b
(two German
isolates), and 3c (all four isolates from
Japan).
Genotype 4 included two isolates from the United Kingdom and one
isolate from Germany, whereas three isolates from Japan constituted
genotype 5. Two isolates from Japan belonged to genotype
6.
Table
2 shows a comparison of percent
nucleotide identities (PNI) and evolutionary distances (ED) among the
six major genotypes.
Minimum and maximum percent nucleotide divergences
(PND) were
noted between genotypes 2 and 4 (PND, 23.6 ± 1.8; ED,
0.29 ± 0.03)
and 1 and 6 (PND, 40.6 ± 1.6; ED, 0.66 ± 0.04), respectively.
Within individual genotypes, the PNI varied from
98 in genotype
6 to 86.8 ± 7.5 in genotype 3, with the ED varying
from 0.01 to
0.15 ± 0.09, respectively.
Among subgenotypes, minimum divergence was noted between 5a and 5b
(PND, 12 ± 1; ED, 0.12 ± 0.001). Subgenotypes 3a and 3b
were maximally divergent from each other (PND, 21.5 ± 0.5; ED,
0.25 ± 0.003).
Overall, TTV seems to be prevalent in India and to be transmitted
mainly by nonparenteral routes. However, the causal relationship
of the
virus and liver disease remains to be established. The
use of highly
efficient primers for PCR may increase the prevalence
rates of TTV DNA
in different categories as well as the identification
of additional
genotypes. Most TTV isolates investigated in the
present series
belonged to genotype 1a. Symptomless, apparently
healthy voluntary
blood donors and patients suffering from CLDs
were infected with the
same genotype. Genotypes 3, 4, 5, and 6
were not identified in the
patients investigated. The significance
of the prevalent genotype with
respect to the severity of disease,
singly or in association with other
hepatitis viruses, needs to
be
investigated.
| |
ACKNOWLEDGMENTS |
We thank D. A. Gadkari for critical evaluation of the
manuscript, L. P. Chobe for timely help, and R. Gangakhedkar for
helpful suggestions. We thank the Director, Bio-informatics Centre,
Department of Biotechnology, University of Pune, for assistance with
computer programs and access to the GenBank database.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Hepatitis
Division, National Institute of Virology, Indian Council of Medical
Research, 20-A, Dr Ambedkar Rd., Pune, India 411001. Phone: 91 212 627301. Fax: 91 212 622669. E-mail: aarankalle{at}hotmail.com.
| |
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Clinical and Diagnostic Laboratory Immunology, September 2000, p. 845-849, Vol. 7, No. 5
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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