Risk of spread of Megalocytivirus pagrus1 (infectious spleen and kidney necrosis virus) from frozen fillets

The emergence of infectious spleen and kidney necrosis virus (ISKNV) is a significant threat to global aquatic food security by causing large scale mortality in the aquaculture of tilapia ( Oreochromis niloticus ) and mandarin fish ( Siniperca chuatsi ). ISKNV (Genotype II) is a genogroup of Megalocytivirus pagrus1 , along with RSIV (Genotype I) and TRBIV (Genotype III). Their recent listing as WOAH-notifiable diseases highlights the need to assess ISKNV spread pathways to support quantitative risk assessments to prevent exotic pathogen incursions. The objectives were to evaluate the risk of ISKNV introduction from the trade in frozen seafood products by determining viability after freezing and the median infectious dose (ID ₅₀ ). An albino rainbow shark ( Epalzeorhynchos frenatum ) challenge model was used with juvenile fish held at 27°C in an aerated freshwater flow-through aquaculture system. Six donor fish were injected with ISKNV with tissues collected after clinical signs appeared and used immediately or stored at –20 °C for seven days. Tissue pools (challenge inocula) were prepared consisting of snout, eyes, and brain (Pool A), caudal skin and muscle (Pool B), or peritoneal viscera (Pool C). Naïve albino rainbow sharks were challenged by intraperitoneal (IP) injection with a clarified tissue homogenate or by bath immersion for one hour. Each treatment group was completed in triplicate, with the frozen immersion bath treatment repeated six times. Fish were sampled for the detection of ISKNV by qPCR at the time of morbidity/death or on day 14. Negative control fish all survived without detection of ISKNV. All tissue pools caused infection and disease via IP injection or immersion, whether used fresh or frozen, showing ISKNV remains infectious after seven days at −20 °C. From dose titration experiments by IP injection, the ID ₅₀ was 42 ISKNV genome equivalents (95% CI: 19-98) estimated by probit regression. This study is the first to investigate the potential for ISKNV spread via frozen fish fillets, a commodity frequently traded in international markets. The findings provide evidence to inform import risk assessments and highlight the need for further investigation into spread pathways involving uncooked, frozen fish products.