Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

doi:10.4236/aid.2013.33033

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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.

Email: [email protected]

Received August 2nd, 2013; revised August 25th, 2013; accepted September 2nd, 2013

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.

REFERENCES

[1] T. Nishizawa, H. Okamoto, K. Konishi, H. Yoshizawa, Y.

Miyakawa and M. Mayumi, “A Novel DNA Virus (TTV)

Associated with Elevated Transaminase Levels in Post-

transfusion Hepatitis of Unknown Etiology,” Biochemical

and Biophysical Research Communications, Vol. 241, No.

1, 1997, pp. 92-97. doi:10.1006/bbrc.1997.7765

[2] H. Miyata, H. Tsunoda, A. Kazi, A. Yamada, M. A. Khan,

J. Murakami, et al., “Identification of a Novel GC-Rich

113-Nucleotide Region to Complete the Circular, Single-

Stranded DNA Genome of TT Virus, the First Human

Circovirus,” Journal of Virology, Vol. 73, No. 5, 1999, pp.

3582-3586.

[3] I. K. Mushahwar, J. C. Erker, A. S. Muerhoff, T. P. Leary,

J. N. Simons, L. G. Birkenmeyer, et al., “Molecular and

Biophysical Characterization of TT Virus: Evidence for a

New Virus Family Infecting Humans,” Proceedings of

the National Academy of Sciences of the United States of

America, Vol. 96, No. 6, 1999, pp. 3177-3182.

doi:10.1073/pnas.96.6.3177

[4] T. Kamahora, S. Hino and H. Miyata, “Three Spliced

mRNAs of TT Virus Transcribed from a Plasmid Con-

taining the Entire Genome in COS1 Cells,” Journal of

Virology, Vol. 74, No. 21, 2000, pp. 9980-9986.

doi:10.1128/JVI.74.21.9980-9986.2000

[5] H. Okamoto, T. Nishizawa, N. Kato, M. Ukita, H. Ikeda,

H. Iizuka, et al., “Molecular Cloning and Characterization

of a Novel DNA Virus (TTV) Associated with Posttrans-

fusion Hepatitis of Unknown Etiology,” Hepatology Re-

search, Vol. 10, No. 1, 1998, pp. 1-16.

doi:10.1016/S1386-6346(97)00123-X

[6] H. Okamoto, T. Nishizawa, A. Tawara, M. Takahashi, J.

Kishimoto, T. Sai, et al., “TT Virus mRNAs Detected in

the Bone Marrow Cells from an Infected Individual,” Bio-

chemical and Biophysical Research Communications,

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

228

Vol. 279, No. 2, 1998, pp. 700-707.

doi:10.1006/bbrc.2000.4012

[7] M. Irshad, Y. K. Joshi, Y. Sharma and I. Dhar, “Transfu-

sion Transmitted Virus: A Review on Its Molecular Char-

acteristics and Role in Medicine,” World Journal of Gas-

troenterology, Vol. 12, No. 32, 2006, pp. 5122-5134.

[8] L. Kakkola, K. Hedman, H. Vanrobaeys, L. Hedman and

M. Söderlund-Venermo, “Cloning and Sequencing of TT

Virus Genotype 6 and Expression of Antigenic Open

Reading Frame 2 Proteins,” Journal of General Virology,

Vol. 83, No. 5, 2002, pp. 979-990.

[9] Y. E. Khudyakov, M. E. Cong, B. Nichols, D. Reed, X. G.

Dou, S. O. Viazov, et al., “Sequence Heterogeneity of TT

Virus and Closely Related Viruses,” Journal of Virology,

Vol. 74, No. 7, 2000, pp. 2990-3000.

doi:10.1128/JVI.74.7.2990-3000.2000

[10] H. Okamoto, M. Takahashi, T. Nishizawa, M. Ukita, M.

Fukuda, F. Tsuda, et al., “Marked Genomic Heterogene-

ity and Frequent Mixed Infection of TT Virus Demon-

strated by PCR with Primers from Coding and Noncoding

Regions,” Virology, Vol. 259, No. 2, 1999, pp. 428-436.

doi:10.1006/viro.1999.9770

[11] Y. Tanaka, M. Mizokami, E. Orito, T. Ohno, T. Nakano,

T. Kato, et al., “New genotypes of TT virus (TTV) and a

genotyping assay based on restriction fragment length

polymorphism.,” FEBS Letters, Vol. 437, No. 3, 1998, pp.

201-206. doi:10.1016/S0014-5793(98)01231-9

[12] P. Biagini, D. Todd, S. Hino, A. Mankertz and S. Mishiro,

“Eight Report of the International Committee on Taxon-

omy of Viruses,” Elsevier/Academic Press, London, 2004.

[13] P. Simmonds, F. Davidson, C. Lycett, L.E. Prescott, D.M.

macdonald, J. Ellender, et al., “Detection of a Novel

DNA Virus (TTV) in Blood Donors and Blood Products,”

The Lancet, Vol. 352, No. 9123, 1998, pp. 191-195.

doi:10.1016/S0140-6736(98)03056-6

[14] D. Prati, Y. H. Lin, C. De Mattei, J. K. Liu, E. Farma, L.

Ramaswamy, et al., “A Prospective Study on TT Virus

Infection in Transfusion-Dependent Patients with Beta-

Thalassemia,” Blood, Vol. 93, No. 5, 1999, pp. 1502-

1505.

[15] C. A. Pinho-Nascimento, J. P. Leite, C. Niel, et al.,

“Torque Teno Virus in Fecal Samples of Patients with

Gastroenteritis: Prevalence, Genogroups Distribution, and

Viral Load,” Journal of Medical Virology, Vol. 83, No. 6,

2011, pp. 1107-1111. doi:10.1002/jmv.22024

[16] K. Chikasue, M. Kimura, K. Ikeda, T. Ohnishi, S. Ka-

wanishi, T. Iio, et al., “Detection of Torque Teno Virus

DNA in Exhaled Breath by Polymerase Chain Reaction,”

Acta Medica Okayama, Vol. 66, No. 5, 2012, pp. 387-

397.

[17] R. Tuveri, F. Jaffredo, F. Lunel, B. Nalpa, S. Pol, C.

Feray, et al., “Impact of TT Virus Infection in Acute and

Chronic, Viral- and Non Viral-Related Liver Diseases,”

Journal of Hepatology, Vol. 33, No. 1, 2000, pp. 121-127.

doi:10.1016/S0168-8278(00)80168-3

[18] E. Odemis, F. Gurakan, K. Ergunay, A. Yuce, H. Ozen

and N Kocak, “TTV Infection in Children with and with-

out Liver Disease,” Indian Journal of Gastroenterology,

Vol. 23, No. 4, 2004, pp. 135-137.

[19] H. Tanaka, H. Okamoto, P. Luengrojanakul, T. Chainu-

vati, F. Tsuda, T. Tanaka, et al., “Infection with an Un-

enveloped DNA Virus (TTV) Associated with Posttrans-

fusion Non-A to G Hepatitis in Hepatitis Patients and

Healthy Blood Donors in Thailand,” Journal of Medical

Virology, Vol. 56, No. 3, 1998, pp. 234-238.

doi:10.1002/(SICI)1096-9071(199811)56:3<234::AID-J

MV10>3.0.CO;2-E

[20] P. Pineau, M. Meddeb, R. Raselli, L. X. Qin, B. Terris, Z.

Y. Tang, et al., “Effect of TT Virus Infection on Hepato-

cellular Carcinoma Development: Results of a Euro-

Asian survey,” The Journal of Infectious Diseases, Vol.

181, No. 3, 2000, pp. 1138-1142. doi:10.1086/315321

[21] K. Kooistra, Y. H. Zhang, N. V. Henriquez, B. Weiss, D.

Mumberg and M. H. M. Noteborn, “TT Virus-Derived

Apoptosis-Inducing Protein Induces Apoptosis Preferen-

tially in Hepatocellular Carcinoma-Derived Cells,” Jour-

nal of General Virology, Vol. 85, No. 6, 2004, pp. 1445-

1450. doi:10.1099/vir.0.79790-0

[22] M. Pistello, A. Morrica, F. Maggi, M.L. Vatteroni, G.

Freer, C. Fornai, et al., “TT Virus Levels in the Plasma of

Infected Individuals with Different Hepatic and Extra-

hepatic Pathology,” Journal of Medical Virology, Vol. 63,

No. 2, 2001, pp. 189-195.

doi:10.1002/1096-9071(20000201)63:2<189::AID-JMV1

014>3.0.CO;2-H

[23] Y. Tanaka, E. Orito, T. Ohno, T. Nakano, K. Hayashi, T.

Kato, et al., “Identification of a Novel 23kda Protein En-

coded by Putative Open Reading Frame 2 of TT Virus

(TTV) Genotype 1 Different from the Other Genotypes,”

Archives of Virology, Vol. 145, No. 7, 2000, pp. 1385-

1398. doi:10.1007/s007050070097

[24] A. Handa, B. Dickstein, N. S. Young and K. E. Brown,

“Prevalence of the Newly Described Human Circovirus,

TTV, in United States Blood Donors,” Transfusion, Vol.

40, No. 2, 2000, pp. 245-251.

doi:10.1046/j.1537-2995.2000.40020245.x

[25] F. Tsuda, H. Okamoto, M. Ukita, T. Tanaka, Y. Akahane,

K. Konishi, et al., “Determination of Antibodies to TT

Virus (TTV) and Application to Blood Donors and Pa-

tients with Post-Transfusion Non-A to G Hepatitis in Ja-

pan,” Journal of Virological Methods, Vol. 77, No. 2,

1999, pp. 199-206. doi:10.1016/S0166-0934(98)00154-2

[26] S. Y. Lo, K. F. Peng, H. C. Ma, J. H. Yu, Y. H. Li, H. H.

Lin, et al., “Prevalence of TT Virus DNA in Eastern Tai-

wan Aborigines,” Journal of Medical Virology, Vol. 59,

No. 2, 1999, pp. 198-203.

doi:10.1002/(SICI)1096-9071(199910)59:2<198::AID-J

MV12>3.0.CO;2-2

[27] J. Qiu, L. Kakkola, F. Cheng, C. Ye, M. Söderlund-Ven-

ermo, K. Hedman, et al., “Human Circovirus TT Virus

Genotype 6 Expresses Six Proteins Following Transfec-

tion of a Full-Length Clone,” Journal of Virology, Vol.

79, No. 10, 2005, pp. 6505-6510.

doi:10.1128/JVI.79.10.6505-6510.2005

[28] C. Ott, L. Duret, I. Chemin, C. Trépo, B. Mandrand and F.

Komurian-Pradel, “Use of a TT Virus ORF1 Recombinant

Protein to Detect Anti-TT Virus Antibodies in Human

Sera,” Journal of General Virology, Vol. 81, No. 12,

2000, pp. 2949-2958.

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

229

[29] M. Irshad, S. Singh, K. Irshad, S. K. Agarwal and Y. K.

Joshi, “Torque Teno Virus: Its Prevalence and Isotypes in

North India,” World Journal of Gastroenterology, Vol. 14,

No. 39, 2008, pp. 6044-6051. doi:10.3748/wjg.14.6044

[30] E. M. de Villiers and H. zur Hausen, “TT Viruses—The

Still Elusive Human Pathogens,” Springer, Berlin, 2009,

pp. v-vi.

[31] E. M. de Villiers, M. Bulajic, C. Nitsch, D. Kecmanovic,

M. Pavlov, A. Kopp-Schneider, et al., “TTV Infection in

Colorectal Cancer Tissues and Normal Mucosa,” Interna-

tional Journal of Cancer, Vol. 121, No. 9, 2007, pp.

2109-2112. doi:10.1002/ijc.22931

[32] I. Jelcic, A. Hotz-Wagenblatt, A. Hunziker, H. Zur

Hausen and E. M. de Villiers, “Isolation of Multiple TT

Virus Genotypes from Spleen Biopsy Tissue from a

Hodgkin’s Disease Patient: Genome Reorganization and

Diversity in the Hypervariable Region,” Journal of Vi-

rology, Vol. 78, No. 14, 2004, pp. 7498-7507.

doi:10.1128/JVI.78.14.7498-7507.2004

[33] H. zur Hausen and E. M. de Villiers, “TT Viruses: Onco-

genic or Tumor-Suppressive Properties?” Current Topics

in Microbiology and Immunology, Vol. 331, 2009, pp.

109-116. doi:10.1007/978-3-540-70972-5_7

[34] H. Zheng, L. Ye, X. Fang, B. Li, Y. Wang, X. Xiang, et

al., “Torque Teno Virus (SANBAN Isolate) ORF2 Pro-

tein Suppresses NF-kappaB Pathways via Interaction with

IkappaB Kinases,” Journal of Virology, Vol. 81, No. 21,

2007, pp. 11917-11924. doi:10.1128/JVI.01101-07

[35] M. A. Peters, D. C. Jackson, B. S. Crabb and G. F.

Browning, “Chicken Anemia Virus VP2 Is a Novel Dual

Specificity Protein Phosphatase,” Journal of Biological

Chemistry, Vol. 277, 2002, pp. 39566-39573.

doi:10.1074/jbc.M201752200

[36] T. Chen, E. Väisänen, P. S. Mattila, K. Hedman and M.

Söderlund-Venermo, “Antigenic Diversity and Seropreva-

lences of Torque Teno Viruses in Children and Adults by

ORF2-Based Immunoassays,” Journal of General Virol-

ogy, Vol. 94, No. 2, 2013, pp. 409-417.

doi:10.1099/vir.0.046862-0

[37] L. Kakkola, H. Bondén, L. Hedman, N. Kivi, S. Moisala,

J. Julin, et al., “Expression of All Six Human Torque

Teno Virus (TTV) Proteins in Bacteria and in Insect Cells,

and Analysis of Their IgG Responses,” Virology, Vol.

382, No. 2, 2008, pp. 182-189.

doi:10.1016/j.virol.2008.09.012

[38] F. Tsuda, M. Takahashi, T. Nishizawa, Y. Akahane, K

Konishi, H. Yoshizawa, et al., “IgM-Class Antibodies to

TT Virus (TTV) in Patients with Acute TTV Infection,”

Hepatology Research, Vol. 19, No. 1, 2001, pp. 1-11.

doi:10.1016/S1386-6346(00)00086-3