Evaluation of Oz virus susceptibility in several vertebrate cell lines and its application in efficient isolation from field-collected ticks.

Oz virus (OZV) is a tick-borne virus first isolated from Amblyomma testudinarium ticks collected in Ehime, Japan, in 2013. The first fatal case of OZV infection in human was reported in Ibaraki, Japan, in 2022. To better understand how tick-borne viruses spread and persist in nature, it is important to isolate epidemic strains and further assess their genetic markers and replication kinetics. Therefore, the development of efficient virus isolation and culture methods is urgently required. In this study, we screened cell lines, highly susceptible to OZV. The original OZV strain EH8 was inoculated into eight vertebrate cell lines, and the viral titers were measured on day 7 post-inoculation. The result showed that BHK-21, Vero, HuH-7, and Huh7.5.1-8 cells exhibited viral titers exceeding 107 PFU/mL. Among these, BHK-21 cells were selected for virus isolation from ticks collected from OZV-endemic regions. A new OZV strain, 23AT5, was successfully isolated from A. testudinarium ticks collected in July 2023 at the site where OZV was first discovered. Genetic analysis revealed that strain 23AT5 shares a high genomic sequence identity with strain EH8, indicating that these strains are phylogenetically closely related. This current study marks the isolation of a closely related strain from the same location a decade later, suggesting that the virus transmission cycle may have been stably maintained in the area over time. This study provides insights into the natural history of OZV and highlights the importance of continuous surveillance, that further address knowledge gaps regarding OZV geographical distribution and natural hosts.