• Effects of HTLV‑1 on leukocyte trafficking and migration in ACs compared to healthy individuals
  • arash letafati,1 Kasra Allaei Rouzbahani,2 Sayed‑Hamidreza Mozhgani,3,* Mehdi Norouzi,4 Atefeh Bahavar,5 Maryam Kazemi,6
    1. Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
    2. Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Student Research Committee , Faculty of Medicine , Hormozgan University of Medical Sciences ,Bandar Abbas ,Iran
    3. Non‑Communicable Disease Research Center, Alborz University of Medical Sciences, Karaj, Iran Department of Microbiology and Virology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
    4. Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Research Center for Clinical Virology, Tehran University of Medical Science, Tehran, Iran
    5. Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
    6. cellular and molecular research center,Basic Health sciences Institute, Shahrekord University of Medical sciences,shahrekord, Iran


  • Introduction: Human T-cell lymphotropic virus-1 (HTLV-1) is a retrovirus that can cause various diseases, including HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP), adult T-cell leukemia/lymphoma (ATLL). Tax and HBZ are integral proteins in HTLV-1 infection and the progression of associated cancers. Tax plays a crucial role in viral replication and evading the immune system by manipulating various cellular signaling pathways. It facilitates the proliferation of T-cells, induces genomic instability, and hampers immune responses, ultimately leading to the transformation of infected cells. Conversely, HBZ, known as the basic leucine zipper protein, contributes to viral persistence and immune evasion. It supports cell survival, modulates the expression of host genes, and regulating various cellular processes [1, 2]. Cell migration can play a role in the spread of infectious agents, including HTLV-1. Infected T cells can also migrate to other tissues, such as the central nervous system, where they can cause inflammation and damage [3, 4]. In HTLV-1 infected individuals, the virus can induce changes in the expression of genes involved in cell migration and adhesion [5–7]. Cell motility plays a crucial role in tumor invasion and metastasis, and involves the reorganization of the cellular skeleton. Cellular actin is a primary mechanism involved in cell motility [8]. Infected T cells can cross the BBB by a process known as transmigration [9, 10]. Once inside the Central Nervous System (CNS), infected T cells can cause inflammation and damage to the neurons and other cells of the CNS. This can lead to the development of neurological complications, such as HAM/TSP. Understanding the role of cell migration in HTLV-1 infection is important for investigation about new therapies and strategies to prevent the spread of the virus. Following this research, the migration pathway’s different genes and their interactions were identified. As a consequence of the analysis, the RT-qPCR method was employed to evaluate seven crucial genes, LFA1, MLCK, RAC1, RAPL, ROCK1, VAV1 and CXCR4 following HTLV-1 infection and their interaction in ACs carriers compared to healthy individuals.
  • Methods: Study population A total of 40 participants were enrolled, with an equal ratio of 20 asymptomatic carrier (ACs) patients and 20 healthy subjects. Among the ACs patients, there were 16 males and 4 females, while the healthy subjects included 16 males and 4 females as well. In ACs subjects, the mean age for males was 48.56 ± 5.81, with a minimum of 38 and a maximum of 58. In healthy subjects, the mean age for females was 48 ± 5.22, with a minimum of 43 and a maximum of 53. Sample collection 6 mL of blood samples were isolated in EDTA anticoagulant sterile tubes. The selection criteria for the participants in the research encompassed individuals with asymptomatic patients with PCR confirmation and healthy individuals who had not taken any medications, did not have a history of autoimmune diseases, or any ongoing infectious illnesses such as HIV, HCV, and HBV, and each participant provided signed consent. ELISA assessment This ELISA test utilized the DIA. PRO kit (HTLV I, II Ab version ULTRA, DIA.PRO, Italy) to detect antibodies against the HTLV-1 virus in the samples. Positive samples went through confirmation test by conventional PCR. Proviral load We used Ficoll density gradient medium (Cedarlane, Hornsby, ON, Canada) to separate peripheral blood mononuclear cells (PBMCs) from blood samples treated with EDTA, and then extracted DNA from PBMCs with a blood mini kit (Qiagen, Germany). All DNA standards and samples were amplified in duplex. After that, we used a commercial Real-time-based absolute quantification kit (HTLV-1 RG; Novin Gene, Karaj, Iran) to perform a real-time PCR (Q 6000 machine, Qiagen, Germany) and measure the PVL of HTLV-I in PBMCs. We computed the normalized HTLV-I PVL values as the ratio of HTLV-1 DNA copies/albumin DNA copies/2 × 104, and expressed as the number of HTLV-1 proviruses/104 PBMCs. RNA extraction and cDNA synthesis RNA extraction was conducted by RNJia Kit (ROJE, Iran) as per the manufacturer’s instructions to purify total RNA. Subsequently, RNA elution was treated with RNase-free DNase (Qiagen, Germany), and cDNA was synthesized with 5 μl of the extracted RNA using the RTROSET Kit (ROJE, Iran) as outlined by the manufacturer. Quantitative real‑time PCR Infection was authenticated by PCR on Tax, HBZ of the virus, as well as the mRNA expression of LFA1, MLCK, RAC1, RAPL, ROCK1, VAV1 and CXCR4 via a Real-time qPCR. The sequences of the PCR primers from 5′ to 3′ are shown in Table 1. Primer sequences for Tax, HBZ, and RPLP0 were used according to previous studies [11]. The quantitative Real-time PCR assay was executed as per the manufacturer’s instructions. To determine the expression index, the relative copy number of the mRNA of interest was divided by the relative mRNA copy number of RPLP0, yielding the normalized value of the expression for each gene. Statistical analysis. The statistical software GraphPad-Prism was employed to analyze the data obtained and assess the significance of the relationship between the results. The collected data were analyzed using nonparametric Mann–Whitney and Spearman’s correlation tests. In this study, a P-value lower than 0.05 was deemed to be significant.
  • Results: Proviral load. The mean ± SD of the PVL in AC individuals was 71.15 ± 35.46 copies in PBMC. The median PVL was 63 copies/PBMC with a minimum of 38 and a maximum of 183 copies/ml. Gene expression assessment This preliminary study aims to highlight the significance of mRNA expression associated with T-cell migration and HTLV-1 to shed light on its probable relevance in advancing our understanding of fundamental biological processes for this virus. In this investigation, the average expression of LFA1 mRNA was found to be 0.82 ± 0.13 in ACs patients and 0.35 ± 0.05 in the healthy group. The expression of the LFA1 gene was higher in the ACs patients in comparison to healthy group and this was statistically significant (95% CI, P < 0.0001) (Fig. 1a). The average expression of MLCK mRNA was found to be 1.1 ± 0.14 in ACs patients and 0.30 ± 0.13 in healthy group which was significant (95% CI, P < 0.0001).(Fig. 1b). The mean expression of RAC1 mRNA in the group of ACs and the healthy group was 1.16 ± 0.20 and 0.26 ± 0.04 respectively (P < 0.0001, CI 95%) (Fig. 1c). A significant difference has been seen in RAPL mRNA expression across two group (P < 0.0001, CI 95%). The mean expression of RAB3GAP2 (ACs 1.09 ± 0.21; healthy group 0.5082 ± 0.2417) was pairwise compared by the spearman test (Fig. 1d). The mean ROCK1 mRNA expression in the ACs patients and healthy subjects was reported as 1.64 ± 0.14 and 0.47 ± 0.06 respectively (P < 0.0001, CI 95%) (Fig. 1e). The average expression of VAV1 mRNA was found to be 1.13 ± 0.23 in ACs patients and 0.48 ± 0.15 in healthy group which was significant (P < 0.0001, CI 95%) and the mean expression of CXCR4 mRNA in the group of ACs and healthy group were 0.22 ± 0.03 and 0.14 ± 0.02 respectively (P < 0.0001, CI 95%) (Fig. 1f, g). Also, comparison and correlation of difference expression among targeted genes and correlation is show in Fig. 2 and Table 2 respectively. The mean expression for Tax was 0.72 ± 0.22 with a maximum of 0.40 and minimum of 0.42 For HBZ, mean expression was 0.65 ± 0.21, with maximum expression of 1.09 and minimum of 0.42.
  • Conclusion: Our study has demonstrated the critical role of infected T cell migration in the progression of HTLV-1-associated diseases. Also, this study concluded that studied genes may influence the migration of infected T cells, including the expression levels of adhesion molecules and chemokine receptors. This knowledge presents potential therapeutic targets that may be harnessed for the development of new treatment strategies aimed at modulating and controlling the migration process following HTLV-1 infection.
  • Keywords: HTLV-1, ACs, Cell migration, ATLL, HAM/TSP, MRNA expression