Development and application of NanoLuc-based LIPS assay for antibody detection of orthohantavirus infections and vaccine responses.

Orthohantavirus (OHV), recognized by the WHO as prioritized pathogens with a high risk of Public Health Emergency of International Concern, could cause severe diseases such as hemorrhagic fever with renal syndrome (HFRS), and poses a persistent public health threat, particularly in China, where Hantaan virus (HTNV) and Seoul virus (SEOV) are endemic. Moreover, newly discovered species such as Luxi virus and Fugong virus pose a potential threat, further underscoring the need for improved surveillance. To address the limitations of current methods, e.g., narrow detection windows and biosafety constraints, we developed a NanoLuc-based luciferase immunoprecipitation system (LIPS) assay targeting nucleoproteins for ten OHVs. The assay was systematically evaluated with comparison to ELISA and colloidal gold assays through analyses of sensitivity, specificity, concordance, and statistical coefficients, using 142 serum samples from HFRS patients, vaccinated individuals, other patients (SFTS patients and Scrub typhus), and healthy controls. When applied to clinical HFRS patients and vaccinated individuals, our LIPS assay demonstrated excellent sensitivity (97.10%), high specificity (98.63%), a broad dynamic range (4.05 × 107-fold dilution), and perfect concordance (97.89%) with reference tests, while revealing dose- and sex-specific antibody responses. Our findings establish LIPS as a reliable, high-throughput tool for sero-epidemiological investigation and vaccine evaluation. By enabling precise antibody profiling and expanding surveillance capabilities, this assay provides a critical advance in preparedness and control of OHV threats in endemic regions.