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Teimoori R, Sharifi Z, Pourkarim M R, Ajorloo M. Development of a TaqMan Real Time PCR Assay for Quantifying the HTLV-1 Proviral Load Using the HBZ Gene. bloodj 2025; 22 (2) :91-99
URL: http://bloodjournal.ir/article-1-1573-en.html
Development of a TaqMan Real Time PCR Assay for Quantifying the HTLV-1 Proviral Load Using the HBZ Gene
Reihane Teimoori , Zohreh Sharifi , Mahmoud Reza Pourkarim , Mehdi Ajorloo *
Keywords: Viral Load, Real-Time PCR, HTLV-1
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      References:
  1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7415-9. [DOI:10.1073/pnas.77.12.7415] [PMID] []
  2. Naderi M, Talebi S, Buyzan A, Yousefi Nojookambari N, Yazdansetad S. The Interaction of Cell-Human T-cell Leukemia Virus Type 1 (HTLV-1) in the Development of Adult T-cell Leukemia/Lymphoma: The Molecular Aspects of Leukemogenesis. Razi Journal of Medical Sciences. 2022;29(6):25-37.https://rjms.iums.ac.ir/article-1-7259-en.html
  3. Keikha M, Karbalaei Zadeh Babaki M, Marcondes Fonseca LA, Casseb J. The Relevance of HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis in Iran: A Review Study. Reviews in Clinical Medicine. 2019;6(2):60-5. [DOI:10.22038/rcm.2019.38759.1266]
  4. Mohanty S, Harhaj EW. Mechanisms of Innate Immune Sensing of HTLV-1 and Viral Immune Evasion. Pathogens. 2023 May 19;12(5):735. [DOI:10.3390/pathogens12050735] [PMID] []
  5. Letafati A, Bahari M, Salahi Ardekani O, Nayerain Jazi N, Nikzad A, Norouzi F, Mahdavi B, Aboofazeli A, Mozhgani SH. HTLV-1 vaccination Landscape: Current developments and challenges. Vaccine X. 2024 Jul16;19:100525. [DOI:10.1016/j.jvacx.2024.100525] [PMID] []
  6. Herrmann D, Meng S, Yang H, Mansky LM, Saad JS. The Assembly of HTLV-1-How Does It Differ from HIV-1? Viruses. 2024 Sep 27;16(10):1528. [DOI:10.3390/v16101528] [PMID] []
  7. Enose-Akahata Y, Vellucci A, Jacobson S. Role of HTLV-1 Tax and HBZ in the Pathogenesis of HAM/TSP. Front Microbiol. 2017 Dec 21;8:2563. [DOI:10.3389/fmicb.2017.02563] [PMID] []
  8. Giam CZ, Semmes OJ. HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma-A Tale of Two Proteins: Tax and HBZ. Viruses. 2016 Jun 16;8(6):161. [DOI:10.3390/v8060161] [PMID] []
  9. El Hajj H, Bazarbachi A. Interplay between innate immunity and the viral oncoproteins Tax and HBZ in the pathogenesis and therapeutic response of HTLV-1 associated adult T cell leukemia. Front Immunol. 2022 Jul 22;13:957535. [DOI:10.3389/fimmu.2022.957535] [PMID] []
  10. Cassar O, Gessain A. Serological and Molecular Methods to Study Epidemiological Aspects of Human T-Cell Lymphotropic Virus Type 1 Infection. Methods Mol Biol. 2017;1582:3-24. [DOI:10.1007/978-1-4939-6872-5_1] [PMID]
  11. Caterino-de-Araujo A, Campos KR. Defective particles of human T-lymphotropic virus and negative results in molecular assays. Infect Genet Evol. 2021 Dec;96:105141. [DOI:10.1016/j.meegid.2021.105141] [PMID]
  12. Ghasemzadegan H, Shahabi M, Rezaei N, Sharifi Z. Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors. The Scientific Journal of Iranian Blood Transfusion Organization. 2019;16(3):194-200. https://bloodjournal.ir/browse.php?a_id=1248&sid=1&slc_lang=en
  13. Ji H, Chang L, Yan Y, Wang L. Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1. Virol J. 2023 Jan 17;20(1):9. [DOI:10.1186/s12985-023-01970-y] [PMID] []
  14. Eusebio-Ponce E, Anguita E, Paulino-Ramirez R, Candel FJ. HTLV-1 infection: An emerging risk. Pathogenesis, epidemiology, diagnosis and associated diseases. Rev Esp Quimioter. 2019 Dec;32(6):485-496. https://seq.es/abstract/rev-esp-quimioter-2019-october-25-2/
  15. Zhao J, Zhao F, Han W, Xu X, Wang L, Li R, et al. HTLV screening of blood donors using chemiluminescence immunoassay in three major provincial blood centers of China. BMC Infect Dis. 2020 Aug 6;20(1):581. [DOI:10.1186/s12879-020-05282-2] [PMID] []
  16. A Strategy for Screening and Confirmation of HTLV-1/2 Infections in Low-Endemic Areas. Front Microbiol. 2020 Jun 3;11:1151. [DOI:10.3389/fmicb.2020.01151] [PMID] []
  17. Saito M, Matsuzaki T, Satou Y, Yasunaga J, Saito K, Arimura K, et al. In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Retrovirology. 2009 Feb 19;6:19. [DOI:10.1186/1742-4690-6-19] [PMID] []
  18. Waters A, Oliveira AL, Coughlan S, de Venecia C, Schor D, Leite AC, et al. Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: addressing the issue of indeterminate HTLV results. J Clin Virol. 2011 Sep;52(1):38-44. [DOI:10.1016/j.jcv.2011.05.022] [PMID]
  19. Casoli C, Pilotti E, Bertazzoni U. Proviral load determination of HTLV-1 and HTLV-2 in patients' peripheral blood mononuclear cells by real-time PCR. Methods Mol Biol. 2014;1087:315-23. [DOI:10.1007/978-1-62703-670-2_25] [PMID]
  20. U.S. Department of Health and Human Services Food and Drug Administration. Bioanalytical Method Validation Guidance for Industry. Silver Spring, MD: U.S. Department of Health and Human Services, Center for Drug Evaluation and Research (CDER); May 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf








Development of a TaqMan Real Time PCR Assay for
Quantifying the HTLV-1 Proviral Load Using
the HBZ Gene
 ‎
Reihane Teimoori1, Zohreh Sharifi1     , Mahmoud Reza Pourkarim2      , Mehdi Ajorloo1


1Biological Products and Blood Safety Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
2Laboratory of clinical and Epidemiological Virology, Rega Institute, Leuven, Belgium

Received: 2025/02/16
Accepted: 2025/04/21
 





    https://doi.org/10.18502/avr.v34i2.18052

    



Citation:
Teimoori R, Sharifi Z, Pourkarim M.R, Ajorloo M. Development of a TaqMan Real Time PCR Assay for Quantifying the HTLV-1 Proviral Load Using the HBZ Gene. J Iran Blood Transfus. 2025: 22 (2) : 91-99
    



Correspondence: Ajorloo M., Assistant professor of Biological Products and Blood Safety Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821) 88601501             
E-mail: m.ajorloo@tmi.ac.ir		 A B S T R A C T
Background and Objectives
HTLV-1 is the first identified human oncogenic retrovirus, associated with ATL and HAM/TSP diseases. The HBZ gene, one of the key genes of this virus, plays an important role in disease progression due to its low immunogenicity and genetic stability. Despite the progress of diagnostic methods such as ELISA and Western blot, indeterminate results are occasionally reported. Moreover, measuring viral load is critical for prognosis of the disease. In this study, a sensitive system for determining HTLV-1 proviral load based on the HBZ gene was designed and implemented using the TaqMan Real Time PCR method.
Materials and Methods
In this experimental study, primers and probes were designed to amplify a 106 bp region of the HBZ gene. Then, genomic DNA was extracted from PBMCs. After amplification of the HBZ gene, the PCR product was inserted into the TOPO TA vector and used as a standard to develop the TaqMan Real Time PCR method.
Results
The logarithmic dilutions of 1.2×101-108 were prepared from the recombinant plasmid to draw the standard curve. The slope of the standard curve line was -3.2 and R2 was 0.97, indicating the linearity and efficiency of the test. The results of the reproducibility of the reaction at the intra-assay and inter-assay levels (three replicates per step) and the average coefficient of variation CV of the standard samples were below 5.3% and 5.9%, respectively. Also, the Limit of Detection of this test was determined to be 1.2×101 Copies/µL, reflecting the high sensitivity of this assay.
Conclusions 
Given its high sensitivity, rapid analysis and ease of use, the Real Time PCR method represents a suitable approach for determining the proviral load of HTLV-1 and assessing the prognosis of associated diseases.
Key words: Viral Load, Real-Time PCR, HTLV-1
 
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.



 
Type of Study: Research | Subject: Virology
References
1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7415-9. [DOI:10.1073/pnas.77.12.7415] [PMID] []
2. Naderi M, Talebi S, Buyzan A, Yousefi Nojookambari N, Yazdansetad S. The Interaction of Cell-Human T-cell Leukemia Virus Type 1 (HTLV-1) in the Development of Adult T-cell Leukemia/Lymphoma: The Molecular Aspects of Leukemogenesis. Razi Journal of Medical Sciences. 2022;29(6):25-37.https://rjms.iums.ac.ir/article-1-7259-en.html
3. Keikha M, Karbalaei Zadeh Babaki M, Marcondes Fonseca LA, Casseb J. The Relevance of HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis in Iran: A Review Study. Reviews in Clinical Medicine. 2019;6(2):60-5. [DOI:10.22038/rcm.2019.38759.1266]
4. Mohanty S, Harhaj EW. Mechanisms of Innate Immune Sensing of HTLV-1 and Viral Immune Evasion. Pathogens. 2023 May 19;12(5):735. [DOI:10.3390/pathogens12050735] [PMID] []
5. Letafati A, Bahari M, Salahi Ardekani O, Nayerain Jazi N, Nikzad A, Norouzi F, Mahdavi B, Aboofazeli A, Mozhgani SH. HTLV-1 vaccination Landscape: Current developments and challenges. Vaccine X. 2024 Jul16;19:100525. [DOI:10.1016/j.jvacx.2024.100525] [PMID] []
6. Herrmann D, Meng S, Yang H, Mansky LM, Saad JS. The Assembly of HTLV-1-How Does It Differ from HIV-1? Viruses. 2024 Sep 27;16(10):1528. [DOI:10.3390/v16101528] [PMID] []
7. Enose-Akahata Y, Vellucci A, Jacobson S. Role of HTLV-1 Tax and HBZ in the Pathogenesis of HAM/TSP. Front Microbiol. 2017 Dec 21;8:2563. [DOI:10.3389/fmicb.2017.02563] [PMID] []
8. Giam CZ, Semmes OJ. HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma-A Tale of Two Proteins: Tax and HBZ. Viruses. 2016 Jun 16;8(6):161. [DOI:10.3390/v8060161] [PMID] []
9. El Hajj H, Bazarbachi A. Interplay between innate immunity and the viral oncoproteins Tax and HBZ in the pathogenesis and therapeutic response of HTLV-1 associated adult T cell leukemia. Front Immunol. 2022 Jul 22;13:957535. [DOI:10.3389/fimmu.2022.957535] [PMID] []
10. Cassar O, Gessain A. Serological and Molecular Methods to Study Epidemiological Aspects of Human T-Cell Lymphotropic Virus Type 1 Infection. Methods Mol Biol. 2017;1582:3-24. [DOI:10.1007/978-1-4939-6872-5_1] [PMID]
11. Caterino-de-Araujo A, Campos KR. Defective particles of human T-lymphotropic virus and negative results in molecular assays. Infect Genet Evol. 2021 Dec;96:105141. [DOI:10.1016/j.meegid.2021.105141] [PMID]
12. Ghasemzadegan H, Shahabi M, Rezaei N, Sharifi Z. Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors. The Scientific Journal of Iranian Blood Transfusion Organization. 2019;16(3):194-200. https://bloodjournal.ir/browse.php?a_id=1248&sid=1&slc_lang=en
13. Ji H, Chang L, Yan Y, Wang L. Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1. Virol J. 2023 Jan 17;20(1):9. [DOI:10.1186/s12985-023-01970-y] [PMID] []
14. Eusebio-Ponce E, Anguita E, Paulino-Ramirez R, Candel FJ. HTLV-1 infection: An emerging risk. Pathogenesis, epidemiology, diagnosis and associated diseases. Rev Esp Quimioter. 2019 Dec;32(6):485-496. https://seq.es/abstract/rev-esp-quimioter-2019-october-25-2/
15. Zhao J, Zhao F, Han W, Xu X, Wang L, Li R, et al. HTLV screening of blood donors using chemiluminescence immunoassay in three major provincial blood centers of China. BMC Infect Dis. 2020 Aug 6;20(1):581. [DOI:10.1186/s12879-020-05282-2] [PMID] []
16. A Strategy for Screening and Confirmation of HTLV-1/2 Infections in Low-Endemic Areas. Front Microbiol. 2020 Jun 3;11:1151. [DOI:10.3389/fmicb.2020.01151] [PMID] []
17. Saito M, Matsuzaki T, Satou Y, Yasunaga J, Saito K, Arimura K, et al. In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Retrovirology. 2009 Feb 19;6:19. [DOI:10.1186/1742-4690-6-19] [PMID] []
18. Waters A, Oliveira AL, Coughlan S, de Venecia C, Schor D, Leite AC, et al. Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: addressing the issue of indeterminate HTLV results. J Clin Virol. 2011 Sep;52(1):38-44. [DOI:10.1016/j.jcv.2011.05.022] [PMID]
19. Casoli C, Pilotti E, Bertazzoni U. Proviral load determination of HTLV-1 and HTLV-2 in patients' peripheral blood mononuclear cells by real-time PCR. Methods Mol Biol. 2014;1087:315-23. [DOI:10.1007/978-1-62703-670-2_25] [PMID]
20. U.S. Department of Health and Human Services Food and Drug Administration. Bioanalytical Method Validation Guidance for Industry. Silver Spring, MD: U.S. Department of Health and Human Services, Center for Drug Evaluation and Research (CDER); May 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
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