Ex Vivo Propagation of Pinctada Birnavirus Using Mantle Tissue Fragment Culture: Application for Measuring Replication at Different Temperatures, TCID50 Assay, and UV Sensitivity

Pinctada birnavirus (PiBV) is the causative agent of summer atrophy in pearl oyster (Pinctada fucata (Gould)). The disease, which induces mass mortality in juveniles less than 1 year old and abnormalities in adults, was first reported in Japan in 2019. Research on the disease has been hindered by the lack of cell lines capable of propagating PiBV. We established an ex vivo method for PiBV propagation using mantle tissue, the primary infection site of the virus. The method was used to investigate the proliferation characteristics of the virus at different culture temperatures and the sensitivity of the virus to UV radiation. The marginal zone of the mantle was found to be the most suitable for PiBV replication in terms of both viral yield and reproducibility. PiBV showed optimal propagation at an incubation temperature of 25 °C, with minimal to no increase at 15 °C or 32.5 °C. Using the tissue culture infectious dose 50 (TCID50) measurement system developed in this study, we found that PiBV propagation was no longer detectable after UV irradiation at 6150 J/m2 or higher. The tissue fragment culture method developed in this study is expected to facilitate both ex vivo experiments and PiBV research.