Advances in Infectious Diseases, 2013, 3, 223-229
http://dx.doi.org/10.4236/aid.2013.33033 Published Online September 2013 (http://www.scirp.org/journal/aid)
223
Expression of TTV-ORF2 Protein for Detection of
Anti-TTV IgG Antibodies in Human Sera
Shiwani Singh1, Akanksha Singh1, Dhananjay Singh Mankotia1, Kalpana Luthra2,
Mohammad Irshad1
1Clinical Biochemistry Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India;
2Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
Received August 2nd, 2013; revised August 25th, 2013; accepted September 2nd, 2013
Copyright © 2013 Shiwani Singh et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The present study describes the cloning and expression of ORF-2 region of TTV genome and the use of expressed pep-
tide in developing immunoassay for detection of anti-TTV antibodies in serum. Presence of TTV-DNA in serum was
detected by PCR amplifying N-22 region of ORF-1 of TTV genome. This was followed by genotyping of TTV by
RFLP using N-22 amplicon. Using genotype-1 positive serum as the source of TTV, the ORF-2 region was amplified
by PCR and subsequently cloned and expressed in pET-19b vector. The expressed protein, identified as 20 kDa protein
on SDS-PAGE gel, was purified by affinity chromatography and then used as antigen to develop western blot assay for
detection of anti-TTV antibodies in serum. Analysis of sera for anti-TTV antibodies and their comparison with presence
of TTV-DNA, produced encouraging results. There was a good relation between presence of anti-TTV and TTV-DNA
in these sera samples. Anti-TTV antibodies could be detected in all TTV-DNA positive sera irrespective of the presence
of TTV-genotype. This investigation demonstrates that ORF-2 peptide may be used in developing immunoassay for
identification of TTV infection.
Keywords: TTV; ORF-2; Anti TTV-IgG; Expression; N-22
1. Introduction
Torque Teno Virus (TTV) is an icosahederal, non-enve-
loped, circular, single-stranded, negative sense DNA
virus with 3852 bases in its genome [1-3]. The genome
of TTV contains a highly conserved non-coding region
and the coding region. The coding region consists of
ORF1 that encodes viral capsid protein and ORF2 en-
codes non-structural proteins. Beside ORF1 and ORF2,
the coding region of TTV also has ORF3 and ORF4
whose products are not known [4-7]. The TTV genome
exhibits high diversity and thus, has been classified into
several genotypes [8-11]. More than 40 genotypes of
TTV have been identified till date [12].
TTV is mainly transmitted via the parenteral route and
found in blood and blood products [13,14]. However,
TTV can be transmitted by non-parenteral routes also.
This is evident by its excretion in feces [7,15] and in ex-
haled breath [16]. The association of TTV with crypto-
genic chronic liver diseases [17,18], post-transfusion
hepatitis [5,11,19] and acute hepatitis of unknown etiol-
ogy [7], suggested a possible etiological role of this agent
in the development of both acute and chronic hepatitis.
TTV-DNA has been reported more frequently in patients
with liver cirrhosis and hepatocellular carcinoma [20,21],
though its role in causing the disease is not known. In
several studies, the viral genome has been reported at
comparable prevalence rates in the blood of patients and
healthy persons. This led to the hypothesis that TTV
might be essentially non-pathogenic in nature [22].
Although several reports have been published on
various aspects of TTV including physico-chemical
characteristic of the virus, however, its prevalence, ge-
nomic organization, infective potency and diagnostic
procedures for its detection still need investigations.
Moreover, there are only few reports available that dem-
onstrate cloning and expression of N-22 and ORF1 but
without reaching any final conclusions [8,23-28]. The
present investigation was undertaken to express ORF2
region of TTV genome and used the translation product
to develop a simple immunoassay for detection of
anti-TTV antibodies in serum. This was aimed to have a
simple diagnostic assay for TTV infection in all small

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera 224
diagnostic laboratories.
2. Methodology
2.1. Ethics
The Ethical approval for this study was given by Ethical
Committee of All India Institute of Medical Sciences,
New Delhi, India.
2.2. Patients and Blood Samples
Sera were obtained, with informed consent, from 110
patients including 50 patients with liver diseases, 50 pa-
tients with renal failure and 10 healthy controls.
2.3. Detection of TTV-DNA
Sera were analyzed for the presence and genotyping of
TTV-DNA by detecting N-22 region of TTV genome.
N-22 region of ~270 bp was amplified by nested PCR
using NG059, NG063 and NG061 primers [10]. PCR
mixture contained 200 µM of each dNTPs (Qiagen, Ger-
many), 25 pmoles/µl of each primer, 1.5 mM MgCl2 and
1.5 U of Taq Polymerase (Qiagen, Germany) in 25 µl
reaction mixture. First round PCR was performed for 35
cycles and second round PCR for 25 cycles of amplifica-
tion. Initial denaturation was at 95˚C for 5 min, amplifi-
cation conditions were: 95˚C for 30 sec, 55˚C for 1 min
and 74˚C for 1min. Amplicons were detected by agarose
gel electrophoresis followed by EtBr staining. Amplified
products were confirmed by sequencing and phyloge-
netic analysis.
2.4. Amplification of Full Length ORF2 Gene
The amplification of full length ORF2 region of TTV
was carried out using serum positive for TTV genotype-1
(G-1). Following set of primers were designed using
softwares like DNA star and Primer 3plus : Forward—
5’-ATG TTT ATT GGC AGG CAC TA-3’ and Re-
verse—5’-TTA CGT TTC TGC GGC GG-3’ (Acces-
sion no. AF122914). Amplification conditions were as
follows : Initial denaturation was done at 94˚C for 7 min,
followed by 35 cycles of amplification conducting an-
nealing at 58˚C for 45 sec, extension at 74˚C for 45 sec
and final extension at 74˚C for 3 min. The amplicon was
resolved on agarose gel and identified as ~459 bp long
fragment.
2.5. Cloning of Full Length ORF2 Using
TA-Sequencing Vector
The PCR product of ORF2 region (~459 bp) was purified
and cloned using TA sequencing vector. The recombi-
nant vector was then transformed into E. coli (DH5α)
cells that were grown overnight at 37˚C on LB agar plate
containing ampicilin. The colonies were screened by
sequencing and Restriction Digestion using NcoI restric-
tion enzyme. The products were resolved on agarose gel.
Presence of ~3000 bp and ~500 bp fragment confirmed
correct insertion of ORF2 gene in TA vector.
2.6. Cloning of ORF2 in pET19b Expression
Vector
Recombinant plasmid was isolated from E. coli cells and
ORF2 was amplified with primers designed with Nde I
and BamHI restriction enzyme sites in between. Primers
were as follows: Forward 5’-CTG ATT CAT ATG ATG
TTT ATT GGC AGG CAC-3’, Reverse: 5’-ATA TAT
GGA TCC TTA CGT TTC TGC GGC GGC-3’. In each
primer, bold sequences are the site for restriction en-
zymes to facilitate cloning in expression vector. After
digestion with NdeI and BamHI restriction enzymes, the
vector (pET19b) and the amplicon were ligated using T4
DNA ligase (Fermentas, Canada). The recombinant vec-
tor was transformed in E. coli BL21 cells and incubated
at 37˚C for overnight on LB agar (containing ampicilin).
The colonies grown were confirmed by sequencing and
restriction digestion of vector with NdeI and BamHI en-
zymes.
2.7. Expression of ORF2 Gene in E. coli
ORF2 region was expressed as a fusion protein contai-
ning 6 histidine residues at N-terminal of expressed protein.
Expression was carried using IPTG in 1mM concentra-
tion for 4, 8, 12, 16 and 20 hrs at 37˚C. Finally, the culture
was harvested at room temperature. A parallel culture
was set up for control without adding IPTG. Expression
of desired protein was confirmed by running the cell lys-
ate of induced and un-induced culture on 12% SDS-
PAGE. Purification of recombinant ORF2 protein was
done by affinity chromatography using QIAexpress Ni-
NTA fast start protein purification kit, Qiagen (Germany).
2.8. Western Blot Analysis for Identification of
Expression Product
After SDS-PAGE, protein was transferred on PVDF
membrane. The membrane was blocked with 3% BSA in
PBS (pH 7.4) and incubated with primary antibodies
(Mouse anti-His antibodies, Qiagen, Germany) in 1:1000
dilutions for 3 hours at room temperature. Following this,
secondary antibody (Goat-anti-mouse antibodies—HRP
conjugate, Santa Cruz, USA) was added in 1:3000 dilu-
tions and incubated for 2 hours at room temperature. Fi-
nally, blot was developed by adding Diaminobenzidine
(DAB, SRL, India).
2.9. Blot Assay for Anti-TTV Antibodies Using
ORF2 Protein as Antigen
The protein was transfered from gel to PVDF membrane.

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera 225
The membrane was blocked with 3% BSA. Human sera
in different dilutions (1:100, 1:1000, 1:3000 and 1:5000)
were used for detection of IgG antibodies against TTV
infection. Anti-TTV antibodies were detected by secon-
dary antibody (anti-human IgG HRP conjugated antibody,
Qiagen, Germany) added in 1:6000 dilutions in PBST.
Secondary antibodies were detected with substrate (DAB,
SRL, India).
ing vector and was confirmed by sequencing. Retrieved
sequences were BLAST analyzed and showed 100%
similarity with ORF2 sequence of TTV genotype-1
(JA20 isolate) (Figure 1).
ORF-2 region of TTV genotype-1 was amplified and
cloned in expression plasmid containing histidine tag. It
expressed the protein at 8, 12, 16 and 20 hour post incu-
bation at 37˚C (Figure 2). Optimal expression of protein
was obtained after 16 hr incubation at 37˚C. ORF-2 of
JA-20 isolate often produces a truncated protein of mo-
lecular mass ~20 KDa. The protein was purified by affin-
ity chromatography and was 1 mg/ml in concentration.
Expression was confirmed by immuno-blotting with anti-
His antibodies (Figure 3). The ORF2 protein was evalu-
ated for its immunogenic nature by detection of anti-TTV
antibodies in human sera (Figure 4) with different dilu-
tions. In order to detect presence of antibodies in different
patient group, 1:1000 dilution of human sera were used.
3. Results
Using N-22 region of TTV genome for detection and
genotyping of TTV, a total number of 110 patient’s sera
were analyzed. TTV viraemia (TTV-DNA) was detected
in 25 of 50 (50%) patients with liver disease, 28 of 50
(56%) patients with renal disease and 2 of 10 (20%) sera
from healthy control. All positive samples were sub-
jected to sequencing. Retrieved sequences were BLAST
analyzed with known TTV sequences available in the
database. Genotype 1 was found to be the predominant
genotype among studied group followed by detection of
genotype 2 in few cases. Since genotype 1 was the pre-
dominant genotype, JA-20 isolate of TTV genotype-1
was used as a reference isolate for amplification of ORF2
region. ORF2 was amplified and cloned in TA sequenc-
The result of blot assay demonstrated that TTV-DNA
negative samples, from both disease groups as well as
healthy controls, could not develop the Western blot,
showing the absence of anti-TTV antibodies in these sera.
Since, genotype 1 and 2 are prevalent in Indian popula-
tion [29], only sera positive for TTV-DNA of genotype 1
Figure 1. Sequence alignment of cloned ORF2 region with known TTV sequences using BLAST analysis. All the sequences
retrieved after sequencing of cloned ORF2 were aligned using BLAST and shows 100% similarity with known TTV se-
uences of genoty pe 1. q

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera 226
Figure 2. SDA-PAGE analysis of expressed ORF2 protein.
ORF2 protein was expressed at 37˚C for 16 hrs with 1mM
IPTG. Gel picture shows expression of ORF2 protein in
induced bacterial culture in comparison to un-induced cul-
ture.
Figure 3. Western blot analysis using anti-His antibodies.
Expressed protein was confirmed by Western blot using
anti-His antibodies and was compared with uninduced bac-
terial culture.
and 2 were used for antibody detection. Although, OFR2
used for Western Blot assay was derived from genotype1
(G1), however, all these samples developed the blot in-
dicating presence of anti-TTV antibodies in them.
Therefore, it is suggested that ORF2 protein is not geno-
type specific and can detect antibodies in all TTV-DNA
positive samples.
A total number of 110 sera samples including 50 sera
Figure 4. Western blot analysis using Human Sera. ORF2
protein was used as antigen to dete c t anti-TTV antibodies in
human sera. Expressed protein was transferred on PVDF
membrane and was reacted with human sera in different
dilutions. 1:1000 dilution was used for further analysis of
different sera samples.
from patient with liver disease, 50 sera from renal dis-
eases and 10 sera from healthy controls were analyzed
for anti-TTV antibodies. These samples were first ana-
lyzed for the presence of TTV-DNA by PCR. Analysis of
these TTV-DNA positive samples shows the presence of
anti-TTV antibodies in 25 of 25 (100%) cases with liver
disease, 28 of 28 (100%) cases with renal disease and 2
of 2 (100%) sera from healthy control (Table 1). Thus,
all the samples positive for TTV-DNA also showed the
presence of anti-TTV antibodies. These results indicate a
successful development of Western blot assay using
ORF2 expressed protein for detecting anti-TTV antibod-
ies in human sera. There is a good scope of this protein
for use in developing a simpler immunoassay for the di-
agnosis of TTV infection.
4. Discussion
TTV is a DNA virus with global prevalence and very
little evidence of being a potent pathogen associated with
some known diseases. Its relatively recent discovery and
association with certain carcinoma causes an interest to
study this virus in more detail [30-33]. TTV infection is
diagnosed by detecting TTV-DNA in blood. Its genome
is not very complex and there are several regions that
may be cloned and expressed for producing a protein for
developing immunoassay for its serodiagnosis. ORF2
region is used as an option to clone and express it to meet
this aim, particularly because it produces nonstructural
protein and may help in developing immunoassays for
detection of antibodies in sera against this non-structural

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera 227
Table 1. Detection of anti-TTV antibodies in Liver and re-
nal disease cases.
Disease group No. of
Cases (n)*
TTV-DNA
Positive
Anti-TTV
Positive % Positive
Liver Disease 50 25 25 50
Renal Disease 50 28 28 56
Healthy Control 10 2 2 20
*n: number of cases studied. Anti-TTV antibodies were detected in Liver
and Renal diseases cases as well as Healthy controls. All the sera sample
were first analyzed for the presence of TTV-DNA by amplifying N22 region
and then genotyped using different restriction enzyme [10]. All samples that
were found positive of TTV-DNA also shows the presence of anti-TTV
antibodies in them by using expressed protein as antigen.
protein for diagnosis of TTV infection.
Full length ORF2 gene of TTV genotype-1 was ampli-
fied using primers designed from JA20 isolate [3]. The
selection of genotype-1 was done for the reason that this
genotype showed significantly high prevalence in Indian
populations [29]. ORF2 of JA-20 isolate of TTV geno-
type-1 produces a truncated protein as compared to the
one produced by full length genomic expression of
TA278 isolate.
As discussed earlier, ORF2 region encodes a protein of
near about 20 KDa [8] which is a non-structural protein
and has been reported to induce host immune response
against TTV [8,34-36]. Although, earlier attempts in
several studies focused on the expression of N22 region
[26] and ORF1 [28,37], however, their protein product
could not prove successful antigenic agent to induce host
immune response or for their use in developing immu-
noassays for sero-detection of TTV infection. As a result,
there was a focus exclusively on ORF2 this time, assum-
ing that it may play a significant role in host immunity
against TTV infection.
The Western blot technique was developed and used
for analysis of anti-TTV antibodies in sera. This study
included a total number of 110 sera from healthy control
as well as patients belonging to different types of liver
diseases and renal ailments. The results of Western blot
demonstrated a distinct difference in antibody positive
and antibody negative sera. Results show the prevalence
of anti-TTV antibodies in sera from TTV DNA positive
healthy controls and patient groups. The results were
encouraging and comparable to those reported earlier in
several other studies [8,24,28]. In all these sera,
TTV-DNA and anti-TTV were detected simultaneously.
From these results, it is evident that serological assays
can be used for detection of TTV infection in small and
routine diagnostic laboratories. The reports from few
other studies have shown the presence of IgM antibodies
[8]. However, these were detected to be the short lived
antibodies [37,38] and could not be frequently found in
sera by different techniques used. Possibly, the screening
of sera samples in this study had a similar problem where
presence of IgM cannot be ruled out.
TTV infection is an infection posing several chal-
lenges to the understanding of biologist and virologist. A
large amount of work has already been done on the virus
for its structural, molecular and epidemiological aspects.
However, its role in causation of disease, promoting
pre-existing infection or damaging cellular integrity and
its functional fabrics, is still mysterious. Moreover, its
role in causing or promoting malignant cell transforma-
tion faces another question mark to its disease causing
potency. This study has touched some of these aspects in
the direction of developing a model for its easy and early
diagnosis in large population to peep into more intrica-
cies related to pathogenicity of TTV.
5. Acknowledgements
The authors thank and appreciate the financial aid pro-
vided by ICMR, New Delhi, India to conduct this study.
Authors are also thankful to Mrs. Suman Rawat for pre-
paring this manuscript.
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