Enhanced detection of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) antibodies in various animal species using oligomeric nucleocapsid protein-based diagnostics

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is a zoonotic pathogen that affects various animal species, often asymptomatically, while causing fatal disease in humans. Current serological tests are limited, which led to the development of novel diagnostic assays targeting the viral nucleoprotein (NP), a conserved and immunodominant protein across SFTSV strains. This study validated the new diagnostics using field monkey sera, employing Enzyme-Linked Immunosorbent Assay (ELISA) with infected cell lysate (ICL-ELISA) as the antigen. Immunoblotting with recombinant NP (rNP) demonstrated 94.6% specificity and 96.3% sensitivity for anti-NP antibody detection. As NP exists in both monomeric and RNA-binding oligomeric forms, rNP was expressed in both configurations and used for ELISA and rapid lateral flow assays (LFA). The oligomeric rNP exhibited superior antigenicity, achieving 97.3% specificity and 92.6% sensitivity, outperforming ICL-ELISA. Additionally, oligomer-enriched rNP was applied to develop an LFA test strip, where rNP conjugated with magnetic nanoparticles enhanced immunocomplex enrichment and clarity. The NP Ab-LFA test achieved 84.2% specificity and 100% sensitivity, successfully detecting anti-NP antibodies in rabbit, mouse, and monkey sera. These innovative diagnostic tools offer reliable methods for detecting SFTSV antibodies, facilitating disease control and mitigating the risk of human outbreaks.