| Abstract |
Bovine major histocompatibility (BoLA) presents antigenic peptides to CD4+ T-cells and is thought to play an essential role in controlling disease progression during bovine leukemia virus (BLV) infection. BoLA-DRB3 alleles are associated with resistance and susceptibility to this disease progression in BLV-infected cattle. For example, BLV-infected cattle carrying BoLA-DRB3*0902 do not develop persistent infection and have low proviral load while BLV- infected cattle carrying BoLA-DRB3*1501 have a tendency to develop persistent infection and have high proviral load. The cytokine microenvironment has been considered as one of the major factors in antitumor and antiviral immunity. Moreover, disturbance in the regulation of the cytokine network plays an essential role in the pathogenesis of persistent viral infections and oncogenesis. Although previous reports have been conducted to clarify the cytokines profile during different stages of BLV infection, the precise mechanisms of the resistance are still not known. To gain knowledge of the immunological mechanisms, we aimed to establish quantitative RT-PCR (RT-qPCR) for measurement of cytokines mRNA expression in peripheral blood mononuclear cells (PBMC) from BLV-infected cattle with resistant and susceptible BoLA-DRB3 alleles. PowerUPTM SYBRTM Green Master Mix (Applied Biosystems) was used for RT-qPCR to measure cytokines mRNA expression and the specificity of primers was confirmed using total RNA extracted from PBMC. For the selection of BLV-infected cattle, we clarified BLV status in one farm. RFLP-PCR was used to identify cattle carrying BoLA-DRB3*0902 (resistant allele) and cattle carrying BoLA-DRB3*1501 (susceptible allele). According to BLV analysis, 61/123 cattle (50%) are BLV-infected. Based on results of RFLP-PCR, 5/100 cattle are carrying BoLA-DRB3*0902 and 41 cattle are carrying BoLA-DRB3*1501. Cattle carrying BoLA-DRB3*0902 had significantly lower proviral load compared to those carrying BoLA-DRB3*1501. Designed primer sets allowed the usage of the same annealing temperature for all primers except for PD-1. Results of conventional and
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quantitative PCR using SYBR Green showed amplification of a single specific PCR product, confirmed by sequencing and melting curve analysis. In conclusion, our results showed that polymorphism in BoLA-DRB3 is an important indicator for the proviral load. The difference in antigen presentation generated by the BoLA-DRB3 polymorphism results in immunological differences which can be evaluated by quantitative analysis of cytokines. RT-qPCR system using SYBR Green dye strategy provided a sensitive, easily transferable and cost-effective method for cytokine analysis. |
