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


The present study describes the cloning and expression of ORF-2 region of TTV genome and the use of expressed peptide 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.

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S. Singh, A. Singh, D. Mankotia, K. Luthra and M. Irshad, "Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera," Advances in Infectious Diseases, Vol. 3 No. 3, 2013, pp. 223-229. doi: 10.4236/aid.2013.33033.

Conflicts of Interest

The authors declare no conflicts of interest.


[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 Posttransfusion 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 Containing 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 Posttransfusion Hepatitis of Unknown Etiology,” Hepatology Research, 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,” Biochemical and Biophysical Research Communications, 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, “Transfusion Transmitted Virus: A Review on Its Molecular Characteristics and Role in Medicine,” World Journal of Gastroenterology, Vol. 12, No. 32, 2006, pp. 5122-5134.
[8] L. Kakkola, K. Hedman, H. Vanrobaeys, L. Hedman and M. Soderlund-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 Heterogeneity and Frequent Mixed Infection of TT Virus Demonstrated 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 Taxonomy 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. Kawanishi, 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 without Liver Disease,” Indian Journal of Gastroenterology, Vol. 23, No. 4, 2004, pp. 135-137.
[19] H. Tanaka, H. Okamoto, P. Luengrojanakul, T. Chainuvati, F. Tsuda, T. Tanaka, et al., “Infection with an Unenveloped DNA Virus (TTV) Associated with Posttransfusion 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-JMV10>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 Hepatocellular 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 Preferentially in Hepatocellular Carcinoma-Derived Cells,” Journal 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 Extrahepatic Pathology,” Journal of Medical Virology, Vol. 63, No. 2, 2001, pp. 189-195. doi:10.1002/1096-9071(20000201)63:2<189::AID-JMV1014>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 Encoded 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 Patients with Post-Transfusion Non-A to G Hepatitis in Japan,” 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 Taiwan Aborigines,” Journal of Medical Virology, Vol. 59, No. 2, 1999, pp. 198-203. doi:10.1002/(SICI)1096-9071(199910)59:2<198::AID-JMV12>3.0.CO;2-2
[27] J. Qiu, L. Kakkola, F. Cheng, C. Ye, M. Soderlund-Venermo, K. Hedman, et al., “Human Circovirus TT Virus Genotype 6 Expresses Six Proteins Following Transfection 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.
[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,” International 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 Virology, 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: Oncogenic 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 Protein 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. Vaisanen, P. S. Mattila, K. Hedman and M. Soderlund-Venermo, “Antigenic Diversity and Seroprevalences of Torque Teno Viruses in Children and Adults by ORF2-Based Immunoassays,” Journal of General Virology, 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

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