One method for treatment of JCV and BKV-related diseases is by adoptive transfer of virus-specific T-cells. Precursor frequency of JCV-capsid protein VP1-specific T-cells was very low compared to other viruses. Optimized assays revealed JCV-specific T cells with high avidity, peptide-specificity and high functional reactivity in long-term cultures. Additionally, novel, naturally processed JCV-VP1-derived CD8+ T cell epitopes were identified. These new data contribute to the development of therapies that, due to their cross-reactivity, target both polyomaviruses-related diseases simultaneously.