Evaluation of Proviral Load by Monitoring the HBZ Gene in Individuals Infected with HTLV-۱

Background: Human T-cell lymphotropic virus type ۱ (HTLV-۱) is the sole human oncogenic retrovirus, responsible for diseases such as adult T-cell leukemia/lymphoma (ATL) and HTLV-۱-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Approximately ۵% of those infected with HTLV-۱ develop ATL, typically after decades of viral latency. Higher viral loads are correlated with more severe diseases due to increased expression of genes like HBZ, crucial for immune regulation and cellular proliferation. Accurate measurement of proviral load is therefore essential for understanding disease progression and evaluating treatment effectiveness. This study aims to measure the proviral load in HTLV-۱ infected individuals. Methods: This experimental study involved genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of ۵۰ blood donors who tested positive for HTLV-۱ via Western blot. Primers for the HBZ gene were designed using bioinformatics software. The HBZ gene, obtained through cloning, was utilized to construct a standard curve for the Real-Time PCR assay. The copy number of the HBZ gene vector was calculated, and serial dilutions (۱۰۱ Copy/µL – ۱۰۷ Copy/µL) were prepared to create a linear standard curve. Proviral load in samples was measured using SYBR Green Real-Time PCR, with the ERV۳ gene used for normalization. Statistical analysis of the correlation between proviral load and variables such as age and gender was performed using SPSS software version ۲۸. Results: Proviral load was evaluated using the Kolmogorov-Smirnov test, revealing a non-normal distribution; hence, the median and interquartile range (IQR) were used. The median proviral load in HTLV-۱ positive samples was ۱۸.۰۱ Copies/۱۰۶ PBMC, with an IQR of ۸.۲۷ – ۴۹.۸۹ Copies/۱۰۶ PBMC. The slope of the standard curve for the Real-Time PCR test was -۳.۲ (slope = -۳.۲) and R² = ۰.۹۸, indicating the linearity and efficiency of the reaction. Additionally, the limit of detection (LOD) for the test was ۱۲ × ۱۰۱ Copy/µL, indicating the minimum concentration that can be detected with accuracy and reliability.

کلیدواژه ها:

Proviral load ، HBZ gene ، HTLV-۱ ، Real-Time PCR ، ERV۳ gene ، bioinformatics ، Kolmogorov-Smirnov test ، SPSS software