Development of a Double-Antigen Sandwich ELISA for Oz Virus and a Seroepidemiological Survey in Wild Boars in Miyazaki, Japan

This study reports the development and use of a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS ELISA) to study Oz virus infection in wild boars in Miyazaki Prefecture, Japan. Recombinant Oz virus nucleoprotein was produced in Escherichia coli and used as both the capture and detection antigen in the assay. The performance of the DAgS ELISA was assessed by comparison with an 80% plaque reduction neutralization test, and the cutoff value was determined using receiver operating characteristic analysis. In addition, viral RNA was examined by quantitative RT-PCR, and a partial glycoprotein gene sequence from a positive sample was analyzed phylogenetically. The DAgS ELISA showed a sensitivity of 72.2%, a specificity of 88.2%, and an overall agreement of 79.0% with the neutralization test, supporting its use for large-scale serological surveys. When applied to 1045 wild boar serum samples collected between 2022 and 2025, the assay identified an overall seroprevalence of 33.5%. Seropositivity did not differ by sex, age, or region, but clear seasonal differences were observed, with the highest prevalence in summer. Oz virus RNA was detected in only one sample (0.09%), and sequence analysis showed high similarity to a previously reported tick-derived strain. These results suggest that wild boars are frequently exposed to Oz virus, but active infection appears to be uncommon.
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