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13 Yap PL. The viral safety of intravenous immune globulin. Clin Exp
Immunol 1996; 104: 35–42.
14 Garson JA, Preston FE, Makris M, et al. Detection by PCR of hepatitis
C virus in factor VIII concentrates. Lancet 1990; 335: 1473.
15 Simmonds P, Zhang LQ, Watson HG, et al. Hepatitis C quantification
and sequencing in blood products, haemophiliacs, and drug users.
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among South African patients swith hemophilia: evidence implicating
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outbreak of hepatitis A in Irish haemophilia A patients. Vox Sang 1994;
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to patients with hemophilia by factor VIII concentrates treated with
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Presence of a newly described human DNA virus (TTV) in patients
with liver disease
Nikolai V Naoumov, Elena P Petrova, Mark G Thomas, Roger Williams
Summary
Background A newly described DNA virus, named
transfusion-transmitted virus (TTV), was recently detected
with high prevalence in Japanese patients with fulminant
hepatitis and chronic liver disease of unknown aetiology. We
investigated the presence of this virus in patients with liver
disease in the UK to find out whether TTV infection is
associated with liver damage.
Methods We used semi-nested PCR to amplify TTV DNA from
serum samples from 126 adults, of whom 72 were patients
with a range of chronic liver diseases, 24 had spontaneous
resolution of infection with hepatitis C virus (HCV), and 30
were normal controls. Direct DNA sequencing and
phylogenetic analysis were used to characterise the TTV
isolates.
Findings We detected TTV DNA in 18 (25%) of the 72
patients with chronic liver disease, which was not different
from the 10% prevalence in normal controls (p=0·15). The
rate of TTV DNA was similar among patients with various
liver diseases. The majority of TTV-positive cases had no
biochemical or histological evidence of significant liver
damage. TTV DNA sequencing of nine isolates showed the
same genotypic groups as in Japan: three patients were
infected with genotype 1, which showed 4% nucleotide
divergence, and six patients were infected with genotype 2
with 15–27% divergence.
Interpretation The high prevalence of active TTV infection in
the general population, both in the UK and in Japan, and the
lack of significant liver damage, suggest that TTV, similar to
hepatitis G virus (HGV), may be an example of a human virus
with no clear disease association.
Lancet 1998; 352: 195–97
Institute of Hepatology, Department of Medicine
(N V Naoumov MRCPath, E P Petrova PhD, Prof R Williams FRCP); and
Departments of Biology and Anthropology (M G Thomas PhD),
University College London Medical School, London, UK
Correspondence to: Dr Nikolai V Naoumov, Institute of Hepatology,
University College London Medical School, London WC1E 6HX, UK
(e-mail: n.naoumov@ucl.ac.uk)
THE LANCET • Vol 352 • July 18, 1998
Introduction
Following the discovery of hepatitis C virus (HCV), it
became apparent that a proportion of cases with acute and
chronic hepatitis are still negative for all known viral
markers,1 which prompted investigations to search for new
hepatitis agents. In 1997, investigators in Japan isolated a
DNA clone of a novel human virus using a representationdifference analysis from serum samples from a patient
(TT) with post-transfusion hepatitis of unknown aetiology
and designated TTV for transfusion-transmitted virus.2
Preliminary data show that TTV is an unenveloped, singlestranded DNA virus with 3739 nucleotides.3 Two genetic
groups of this virus have been identified, differing by 30%
in nucleotide sequences. TTV DNA was detected in 47%
of patients with fulminant non-A-G hepatitis and 46% of
patients with chronic liver diseases of unknown aetiology,
suggesting that TTV may be the cause of some cryptogenic
liver diseases.3 So far, TTV has been described only in
Japanese patients.
The aim of this study was to gain information about the
prevalence of this newly described virus in the UK and its
association with liver damage. We used semi-nested PCR
with primers directed to conserved regions of the TTV
genome and tested serum samples from healthy individuals
and patients with a range of chronic liver diseases.
Methods
Study population
The study population comprised 126 adults (83 male, 43 female)
in six groups (table 1). The first group of 33 patients had chronic
hepatitis C, all seropositive for anti-HCV by enzyme immunoassay
(Abbott Diagnostics, Maidenhead, UK) and HCV RNA by
Amplicor assay (Roche Diagnostics, Basle, Switzerland). 17 of
these patients had acquired HCV through well-documented blood
transfusion; 13 had a long period of intravenous drug use, and in
three patients the source of infection was not evident.
Since the preliminary data suggested that TTV is transmitted
mainly via a parenteral route,3 we selected as a second
group 24 patients with past exposure to HCV via blood
transfusion or intravenous drug use, but with spontaneous
resolution of HCV infection and repeatedly normal liver-function
tests. All were seropositive for anti-HCV, confirmed by an
immunoblot assay (LIA [Innogenetics, Ghent, Belgium]),
but HCV RNA was undetectable in the serum on several
occasions.
195
EARLY REPORTS
Group
TTV DNA positive
Chronic hepatitis C (n=33)
Blood transfusion (n=17)
IV drug use (n=13)
Sporadic (n=3)
Asymptomatic anti-HCV positive/HCV RNA negative cases (n=24)
Chronic hepatitis B (n=10)
Non-B, non-C liver diseases (n=13)
Liver-transplant recipients non-A-G (n=16)
Healthy controls (n=30)
7 (21%)
4
2
1
3 (12%)
2 (20%)
5 (38%)
4 (25%)
3 (10%)
Table 1: Prevalence of TTV infection in patients with chronic
liver diseases and healthy controls
The third group comprised ten patients with chronic hepatitis B
who were seropositive for hepatitis B e antigen and HBV DNA
exceeding 2000 pg/mL (Digene assay, Murex, Dartford, UK).
In the fourth group were 13 patients with liver diseases
seronegative for all HBV and HCV markers. For 11 of these
patients, the aetiology was established as alcoholic cirrhosis,
autoimmune hepatitis, haemochromatosis, hydatid cyst, or liver
metastases. The other two patients had raised serum
aminotransferases of unknown aetiology.
The fifth group comprised 16 patients who had undergone
orthotopic liver transplantation for acute liver failure or cirrhosis of
unknown aetiology (n=9) or autoimmune liver disease (n=7). The
serum samples tested for TTV were taken 5–9 years after
transplantation, and the liver-graft histology was assessed at the
same time. Serum markers for HBV, HCV, as well as testing for
hepatitis G virus (HGV) by reverse-transcription PCR (Boehringer
Mannheim, Mannheim, Germany) were negative in all patients in
this group. The control group included 30 healthy individuals who
were medical and laboratory personnel with normal liver-function
tests.
Detection of TTV
We extracted the total DNA from 100 µL serum using
QIAamp Blood kit (QIAGEN Ltd, Crawley, UK) and
resuspended it in 50 µL elution buffer. TTV DNA was
amplified by semi-nested PCR with TTV-specific primers
derived from two conserved regions of the published
sequences.3. For the first round, 10 pmol primer A (sense
primer 5'-ACA GAC AGA GGA GAA GGC AAC ATG3') and 10 pmol primer B (antisense primer 5'-CTG GCA
TTT TAC CAT TTC CAA AGT T-3') were used in
50 µL PCR mixture containing 1⫻PCR buffer, 0·2 mM
deoxynucleoside triphosphates, 2 mM magnesium
chloride, and 0·5 U Taq polymerase (Gibco BRL, Paisley,
UK). The amplification was for 35 cycles at 94°C for 30 s,
58°C for 30 s, and 72°C for 45 s, followed by 7 min at
72°C after the last cycle in a Perkin Elmer 2400
thermocycler. The second round was with another sense
primer C (5'-GGC AAC ATG TTA TGG ATA GAC
TGG-3') and the same antisense primer B. 1 ␮L of the first
round PCR was transferred into another tube and
amplified for 25 cycles at 94°C for 30 s, 58°C 30 s, 72°C
for 30 s, with a final extension step at 72°C for 7 min
giving a 272 bp amplification product. The amplicons were
electrophoresed in 2% agarose gel, stained with ethidium
bromide, and photographed under ultraviolet light.
Grading and staging
of chronic hepatitis
HBV and
TTV
HCV and
TTV
Non-A-G
post-OLT
and TTV
TTV alone
Alcohol
misuse
and TTV
None
Mild
Moderate
Severe
Cirrhosis
1
1
-
4
1
-
2
2
-
1
1
-
1
Total
2
5
4
2
1
Table 2: Liver histology in 14 patients with TTV infection
196
We carried out direct sequencing of the amplicons with
ABI 310 automated sequencer (PE Applied Biosystems)
using primer C as a sequencing primer. Phylogenetic
analysis was done with sequences between positions 1964
and 2178 of the published TTV genome (accession
number AB008394). We used maximum-likelihood and
distance-based methods with the test version 4.0d63 of
PAUP*, written by David L Swofford, and PHYLIP4
software packages, respectively. The dataset was bootstrap
resampled 1000 times to ascertain support for major
branches of the tree.
For statistical analyses, we used ␹2 test with Yates
correction or Fisher’s exact test, as appropriate.
Results
TTV DNA was detected in 18 (25%) of 72 patients with
chronic liver diseases—not significantly different from the
prevalence in normal controls: three (10%) of 30 (table 1,
␹2=2·069, p=0·15). When we did this analysis for the
individual groups studied, we found TTV infection more
frequently in patients with different liver diseases who were
seronegative for HBV and HCV markers: five (38%) of 13
patients (group 4, table 1) compared with normal controls
(p=0·042, Fisher’s exact test). We got similar results when
taking into account all patients in this series who had liver
diseases with non-B, non-C aetiology (group 4 plus livertransplant recipients in group 5; table 1), since nine (31%)
of 29 such patients were positive for TTV DNA (p=0·045,
Fisher’s exact test). The prevalence of TTV infection did
not differ between patients with chronic hepatitis C,
patients with spontaneous resolution of HCV infection
(serum HCV RNA negative), patients with chronic
hepatitis B, the group with non-B, non-C liver diseases,
and the group of liver-transplant recipients (table 1).
Of the 24 individuals with TTV DNA in serum, the
majority (15 [63%] of 24) have had either blood
transfusions or a period of intravenous drug use. Those
without a history of parenteral procedures included six
patients with chronic liver diseases and the three normal
individuals. All but three of TTV-positive patients in our
study were of European descent.
A large proportion of TTV-positive patients (14 [58%]
of 24) had normal liver-function tests; these 14 included
eight patients with chronic liver diseases, three with
spontaneous resolution of HCV infection, and the three
healthy controls. Of the 14 patients for whom liver
histology was available, ten showed no significant liver
damage—either no inflammation in four patients, or mild
portal inflammation without interface hepatitis in six
patients (table 2). The two patients in the non-B, non-C
Patient 3
Patient 5
Patient 9
Japan 1a
Patient 6
Patient 8
Patient 2
Patient 7
Patient 1
Japan 2a
Patient 4
Phylogenetic tree of TTV isolates identified in nine patients in
UK and relation to representatives of two main genotype groups
reported in Japan3
A maximum-likelihood tree was calculated with assumption of a constant
rate of evolution (tips of all branches being equidistant from root) and
nucleotide substitution model of Hasegawa and colleagues.14
THE LANCET • Vol 352 • July 18, 1998
EARLY REPORTS
group who had raised aminotransferases of unknown
aetiology were positive for TTV DNA. Liver histology
showed no inflammation or other liver pathology in one of
them, and chronic hepatitis with moderate activity in the
other (table 2).
We did DNA sequencing analysis in nine TTV-positive
samples, which verified that the amplicons represent the
TTV genome. Alignment with the published TTV
sequences3 showed that three of these isolates belong to
TTV genotype 1, and that six of the nine belong to
genotype 2. The nucleotide divergence of TTV in the UK
from the sequences in Japan was between 3·7% and 4·1%
for the three patients infected with genotype 1a, whereas
for genotype 2a, there was greater variability—between
15% and 27% (figure). Both maximum-likelihood and
distance-based methods gave the same basic tree topology.
Furthermore, bootstrap support for the two major lineage
cluster was 100% in both cases.
Discussion
This study, the first to our knowledge for the UK, shows
that the DNA virus, recently described in Japan and
named TTV,2,3 is present in the UK with a similar
prevalence and the same viral genotypes as in Japan,
suggesting that TTV has a worldwide distribution. The
prevalence of TTV DNA in the general population appears
to be high—about 10%, as recorded in this study, and in
blood donors in Japan.3 This rate of TTV infection in
normal people is at least three times higher than the
prevalence of HGV agent in the general population—1·7%
in the USA5 and 3·2% in Scotland (UK).6 The majority of
patients with TTV infection had a history of blood
transfusion or intravenous drug use, confirming the
importance of the parenteral route of transmission.
However, we also detected TTV in a large proportion of
individuals with “community-acquired” infection—38% in
our series—so the role of non-parenteral transmission
requires further investigation.
Although in our series the prevalence of TTV infection
was slightly higher in liver-disease patients with non-B,
non-C aetiology than in healthy controls, this result does
not necessarily imply a causative role for TTV. More
relevant is the fact that the majority of TTV-positive cases
had normal liver-function tests and only minor changes in
liver histology. The present study suggests, therefore, that
TTV is not the causative agent of chronic liver diseases of
unknown aetiology, and that neither does it affect the
degree of liver damage when present in coinfection with
HBV or HCV.
As a single-stranded DNA virus, TTV was suggested to
belong to Parvoviridae family, though it has no aminoacid
similarity to the known parvoviruses.3 The parvovirus B19
is the only autonomous human pathogenic parvovirus
currently recognised.7 Experimental infection with B19 in
healthy adult volunteers was shown to cause early
haematological changes with a slight decrease in
haemoglobin, lymphopenia, and thrombocytopenia; and,
in a second-phase illness, erythematous rash consistent
with an adult form of erythema infectiosum.8 In children,
B19 infection may cause acute hepatitis at the time of
erythema infectiosum, which resolves without chronic liver
damage.9 Parvovirus B19 has also been suggested as a
possible causative agent of fulminant liver failure in
THE LANCET • Vol 352 • July 18, 1998
association with aplastic anaemia in paediatric cases.10
During a recent B19 epidemic in Denmark, only two
adults were identified with mild liver dysfunction due to
acute B19 infection in a general practice incorporating
1200 patients.11 Whatever the relation between TTV and
parvovirus B19, our data show that TTV is unlikely to be
the long-sought pathogen that causes non-A, non-B, nonC hepatitis.
The scenario for TTV seems to be similar to that for the
GB virus C/HGV because the clinical impact of infection
with this agent is increasingly doubtful. In 73% of cases,
HGV infections were not accompanied by hepatocellular
injury, and an additional 16% were associated with only a
minor rise in alanine aminotransferase.12,13 If our findings
that TTV has no pathogenic role for liver diseases are
confirmed, it may be another example of a human virus—
initially identified as a genomic sequence with a high
prevalence in the general population—that has no clear
disease association. These findings should emphasise the
importance of a cautious interpretation of the role of new
viral genomes discovered by molecular biological
techniques.
Contributors
Nikolai Naoumov was the principal investigator and was responsible for the
design of the study and writing of the paper. Nikolai Naoumov, Elena
Petrova, and Mark Thomas carried out the laboratory work and did data
interpretation. Mark Thomas carried out the phylogenetic analysis. Roger
Williams was responsible for the series of patients and contributed to the
writing of the paper.
Acknowledgments
Elena Petrova is a recipient of a NATO Postdoctoral Fellowship from the
Royal Society, UK. Mark Thomas is supported by the Melford Charitable
Trust, UK.
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