A novel medium-throughput biological assay system for HTLV-1 infectivity and drug discovery


1 Department of Microbiology, Faculty of Medicine, Sabzevar University of Medical Sciences, Sabzevar, Iran

2 Department of Virology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Virology Department, Pasteur Institute of Iran, 69 Pasteur Ave 1316943551. Tehran, Iran

4 Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran


Objective(s): Here, a reporter cell line containing two reporter vectors were developed, in order to monitor the Human T-Lymphotropic Virus type1(HTLV-1) infectivity and the cell viability simultaneously.
Materials and Methods: The reporter cell line was constructed by stably transfected baby hamster's kidney cell line (BHK-21), with the genomes expressing two different reporters in separate plasmids.The first reporter gene is transactivated by the HTLV-1 tax protein, while the second reporter is continuously expressed when introduced into a mammalian cell. In order to show its functionality, the effect of the drug mix on HTLV-1 was assayed by this system and was compared to the results obtained by other methods.
Results: HTLV-1 reporter cell line was found to produce high level of luciferase when co-cultured with MT-2 and Hut-102 cells but not with Jurkat cell. Moreover, the combination therapy against HTLV-1 can reduce luciferase expression of the cell when co-cultured with MT-2 and Hut-102 comparable to the ELISA (R=0.932, P-value =0.002). In addition, the results revealed the superiority of the present system over the molecular methods.
Conclusion: The results demonstrated that the biological assay system is a beneficial tool for the medium-throughput anti-HTLV-1 drug screening and inhibitory effect.


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; 77:7415-7419.
2. Sarvari J, Norozian H, Fattahi MR, Pirbonyeh N, Moattari A. The role of interferon gamma gene polymorphism (+874A/T, +2109A/G, and -183G/T) in response to treatment among hepatitis C infected patients in Fars Province, Southern Iran. Hepat Month 2014; 14:e14476.
3. Hedayati-Moghaddam MR, Tehranian F, Bayati M. Human T-lymphotropic virus Type I (HTLV-1) infection among Iranian Blood Donors: first case-control study on the risk factors. Viruses 2015; 7:5736-5745.
4. Azarpazhooh MR, Hasanpour K, Ghanbari M, Rezaee SA, Mashkani B, Hedayati-Moghaddam MR, et al. Human T-lymphotropic virus type 1 prevalence in northeastern Iran, Sabzevar: an epidemiologic-based study and phylogenetic analysis. AIDS Res Hum Retroviruses 2012; 28:1095-10101.
5. Rafatpanah H, Hedayati-Moghaddam MR, Fathimoghadam F, Bidkhori HR, Shamsian SK, Ahmadi S, et al. High prevalence of HTLV-I infection in Mashhad, Northeast Iran: A population-based seroepidemiology survey. J Clin Virol 2011; 52:172-176.
6. Nerurkar VR, Achiron A, Song KJ, Melland RR, Pinhas-Hamiel O, Melamed E, et al. Human T-cell lymphotropic virus type I in Iranian-born Mashhadi Jews: genetic and phylogenetic evidence for common source of infection. J Med Virol 1995; 45:361-366.
7. Rafatpanah H, Torkamani M, Valizadeh N, Vakili R, Meshkani B, Khademi H, et al. Prevalence and phylogenetic analysis of HTLV-1 in a segregated population in Iran. J Med Virol 2016; 88:1247-1253.
8. Bazarbachi A. Tax fingerprint in adult T-cell leukemia. Blood 2016; 127:1737-1738.
9. Bazarbachi A, Hermine O. Treatment of adult T-cell leukaemia/lymphoma: current strategy and future perspectives. Virus Res 2001; 78:79-92.
10. Taylor JM, Nicot C. HTLV-1 and apoptosis: role in cellular transformation and recent advances in therapeutic approaches. Apoptosis 2008; 13:733-747.
11. Mahmoudi M, Azadmanesh K, Shokrgozar MA, Journeay WS, Laurent S. Effect of nanoparticles on the cell life cycle. Chem Rev 2011; 111:3407-3432.
12. Gervaix A, West D, Leoni LM, Richman DD, Wong-Staal F, Corbeil J. A new reporter cell line to monitor HIV infection and drug susceptibility in vitro. Proc Natl Acad Sci U S A 1997; 94:4653-4658.
13. Richman DD. Human immunodeficiency virus. Playing chess with reverse transcriptase. Nature 1993; 361:588-589.
14. Hirsch V, Riedel N, Kornfeld H, Kanki PJ, Essex M, Mullins JI. Cross-reactivity to human T-lymphotropic virus type III/lymphadenopathy-associated virus and molecular cloning of simian T-cell lymphotropic virus type III from African green monkeys. Proc Natl Acad Sci U S A 1986; 83:9754-9758.
15. Naderi M, Paryan M, Azadmanesh K, Rafatpanah H, Rezvan H, Mirab Samiee S. Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood. J Clin Virol 2012; 53:302-307.
16. Yoshida A, Piroozmand A, Sakurai A, Fujita M, Uchiyama T, Kimura T, et al. Establishment of a biological assay system for human retroviral protease activity. Microbes Infect 2005; 7:820-824.
17. Chiba-Mizutani T, Miura H, Matsuda M, Matsuda Z, Yokomaku Y, Miyauchi K, et al. Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs. J Clin Microbiol 2007; 45:477-487.
18. Hossain MJ, Perez S, Guo Z, Chen LM, Donis RO. Establishment and characterization of a Madin-Darby canine kidney reporter cell line for influenza A virus assays. J Clin Microbiol 2010; 48:2515-2523.
19. Wunder F, Rebmann A, Geerts A, Kalthof B. Pharmacological and kinetic characterization of adrenomedullin 1 and calcitonin gene-related peptide 1 receptor reporter cell lines. Mol Pharmacol 2008; 73:1235-1243.
20. Spenlehauer C, Gordon CA, Trkola A, Moore JP. A luciferase-reporter gene-expressing T-cell line facilitates neutralization and drug-sensitivity assays that use either R5 or X4 strains of human immunodeficiency virus type 1. Virology 2001; 280:292-300.
21. Astier-Gin T, Portail JP, Lafond F, Guillemain B. Identification of HTLV-I- or HTLV-II-producing cells by cocultivation with BHK-21 cells stably transfected with a LTR-lacZ gene construct. J Virol Methods 1995; 51:19-29.
22. Jewell NA, Mansky LM. Construction and characteri-zation of deltaretrovirus indicator cell lines. J Virol Methods 2005; 123:17-24.
23. Samaneh Saffar KA, Taghi Golmohammadi, Majid Golkar, Mahdi Amminian, Hesam Mirshahabi, Hooshang Rafatpanah. Production of recombinant Human T Lymphotropic Virus type 1 Tax protein in Rosetta (DE3) bacterial host. J Paramed Sci 2010; 1:35-42.
24. Fattahi Abdizadeh M, Makvandi M, Samarbafzadeh A, Azadmanesh K. Examination of a reporter vector for HTLV-1 infectivity using MT2, a HTLV-1 producer cell line. Jundishapur J Microbiol 2013; 6:e8257.
25. Azadmanesh K, Amini S, Arashkia A, Kazanji M. Evaluation of stimulatory effects of HTLV-I Tax protein on CREB and NFkB related signaling pathways using two newly designed beta galactosidase based reporter plasmids. Yakhteh Med J 2005; 6:218-225.
26. Coffin JM. The discovery of HTLV-1, the first pathogenic human retrovirus. Proc Natl Acad Sci U S A 2015; 112:15525-15529.
27. Ma Z, Hao P, Yao X, Liu C, Tan J, Liu L, et al. Establishment of an indicator cell line to quantify bovine foamy virus infection. J Basic Microbiol 2008; 48:278-283.
28. Moyo B, Mukanganyama S. Antiproliferative activity of T. welwitschii extract on jurkat T cells In Vitro. Bio Med Res Int 2015; 2015:817624.
29. Mahieux R, Hermine O. In vivo and in vitro treatment of HTLV-1 and HTLV-2 infected cells with arsenic trioxide and interferon-alpha. Leuk Lymphoma 2005; 46:347-355.
30. Connor LM, Marriott SJ. Sequences flanking the cAMP responsive core of the HTLV-I tax response elements influence CREB protease sensitivity. Virology 2000; 270:328-336.
31. Okada M, Jeang KT. Differential requirements for activation of integrated and transiently transfected human T-cell leukemia virus type 1 long terminal repeat. J Virol 2002; 76:12564-12573.
32. Ishitsuka K, Yamano Y, Utsunomiya A, Uchimaru K. [A survey of HTLV-1 carrier clinics in Japan]. [Rinsho ketsueki]  Japan J Clin Hematol 2015; 56:666-672.
33. Copeland KF, Haaksma AG, Derse D, Goudsmit J, Heeney JL. Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system. J Virol Methods 1993; 45:161-167.
34. Hachiya A, Aizawa-Matsuoka S, Tanaka M, Takahashi Y, Ida S, Gatanaga H, et al. Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 using CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5). Antimicrob Agents Chemother 2001; 45:495-501.
35. Araya N, Sato T, Coler-Reilly A, Yagishita N, Yamano Y. Developing novel treatments for HTLV-1-associated myelopathy (HAM) by investigating molecular pathomechanisms. Nihon Rinsho Men'eki Gakkai kaishi = Japan J Clin Immunol 2016; 39:207-212.
36. Yamano Y, Sato T, Ando H, Araya N, Yagishita N. [The current and future approaches to the treatment of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)]. Nihon Rinsho Japan J Clin Med 2012; 70:705-713.
37. Rafatpanah H, Rezaee A, Etemadi MM, Hosseini RF, Khorram B, Afsahr L, et al. The impact of interferon-alpha treatment on clinical and immunovirological aspects of HTLV-1-associated myelopathy in northeast of Iran. J Neuroimmunol 2012; 250:87-93.
38. Heraud JM, Mortreux F, Merien F, Contamin H, Mahieux R, Pouliquen JF, et al. The efficacy of combined therapy of arsenic trioxide and alpha interferon in human T-cell leukemia virus type-1-infected squirrel monkeys (Saimiri sciureus). Antiviral Res 2006; 70:132-139.
39. Hartoonian C, Rasekhian M, Edalat R, Arashkia A, Azadmanesh K. IFN-α(2b) reduces released particles of Human T-lymphotropic Virus-I from HTLV-I transformed cell line. Retrovirology 2011; 8:P30.
40. Al-Saleem J, Kvaratskhelia M, Green PL. Methods for Identifying and Examining HTLV-1 HBZ Post-translational Modifications. Methods Mol Biol 2017; 1582:111-126.
41. Rajaei T, Farajifard H, Rafatpanah H, Bustani R, Valizadeh N, Rajaei B, et al. Role of IL-21 in HTLV-1 infections with emphasis on HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Med Microbiol Immunol 2017; 206:195-201.
42. Gross C, Thoma-Kress AK. Reporter systems to study HTLV-1 transmission. Methods Mol Biol 2017; 1582:33-46.