Introduction: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne member of the bunyaviricetes class, with infections resulting in up to 40% mortality. Knowledge of anti-SFTSV adaptive immunity is limited to the detection of SFTSV-specific antibodies while the presence and role of virus-specific T cells during SFTSV infection have never been characterized. The aim of this work is to develop and validate a rapid and scalable, whole blood cytokine release assay (CRA) to quantify SFTSV-specific T cell responses directly from <3 mL of fresh blood without complex in vitro T cell manipulation.
Methods: We designed overlapping 15-mer peptides (10 AA overlap) covering the whole proteome of SFTSV and 2 selected Hemaphysalis Longicornis salivary antigens. The synthesized 819 peptides were organized into 7 different peptide pools (RdRp1=208, RdRp2=207, Glycoprotein=213, Nucleoprotein=47 and Non-structural=57; Longistatin=30, HL34=57). The peptide pools were first utilized in IFN-γ ELISPOT assays using peripheral blood mononuclear cells (PBMC) purified from individuals previously hospitalized for SFTSV infection (N=4) and healthy controls (N=9), both ex vivo and after in vitro expansion. Following which whole blood of acute SFTSV patients (N=8) recruited from Jeju in 2025 were longitudinally collected spanning acute infection and convalescence, and analyzed using the whole blood CRA. Geographically matched healthy individuals were also recruited as controls (N=9).
Results: ELISPOT performed on PBMCs directly ex vivo and after in vitro expansion demonstrated that multiple SFTSV peptide pools stimulated IFN-γ secretion in 4 out of 4 confirmed SFTSV patients. In contrast, only 2 out of 9 healthy controls had detectable SFTSV-specific cells. Intracellular cytokine staining (ICS) confirmed that SFTSV peptide-stimulated IFN-γ producing cells were CD8+ T cells. In addition, using the whole blood CRA, we detected SFTSV-specific cells in all 8 recruited SFTSV patients during convalescence and none in the geographically matched healthy controls.
Conclusions: We have demonstrated that overlapping peptide pools spanning the full SFTSV proteome can reliably detect multispecific SFTSV-reactive T cells both directly ex vivo and following in vitro expansion. Notably, SFTSV-specific T cells can be quantified using a streamlined assay that avoids complex in vitro manipulation, including PBMC isolation, cryopreservation, and thawing. Whole-blood CRA–based quantification of SFTSV-specific T cells during acute infection is currently underway. This platform will subsequently be applied to define correlates of protection, support prevalence studies, and inform future vaccine trials, ultimately guiding the development of next-generation SFTSV-specific therapeutics and preventive strategies to mitigate this biothreat to U.S. and partner-nation forces.