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

Oz virus, an emerging tick-borne thogotovirus, has been reported to cause fatal human infection in Japan. However, its ecology and geographic distribution remain largely unknown. In this study, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS ELISA) for detecting Oz virus antibodies in animals and used it to conduct a seroepidemiological survey of wild boars (<i>Sus scrofa</i>) in Miyazaki Prefecture, Japan. Recombinant Oz virus nucleoprotein was expressed in <i>E. coli</i> and used as both the capture and detection antigen. Relative to the neutralization test, the DAgS ELISA showed a sensitivity of 72.2%, a specificity of 88.2%, and an overall concordance rate of 79.0%. We used this assay to examine 1045 wild boar serum samples collected between November 2022 and May 2025, finding a seroprevalence of 33.5%. The seroprevalence did not significantly differ by sex, age, or region, but showed significant seasonal variation, peaking in summer (<i>p</i> < 0.0001). Oz virus RNA was detected by quantitative RT-PCR in one serum sample (0.09%). Phylogenetic analysis of the partial Oz virus glycoprotein gene showed that this strain shared 98.8% nucleotide identity with the EH8 strain, which was the first Oz virus isolate obtained from ticks in Ehime Prefecture. These findings suggest that wild boars in Miyazaki are frequently exposed to Oz virus and that ticks in the region harbor the virus. However, no human cases have been reported to date. The DAgS ELISA developed in this study provides a practical tool for serological surveillance in animals. Continuous monitoring of animal populations is warranted to clarify the epidemiology of Oz virus in the region and to identify potential reservoir species.