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Simple Summary: Red sea bream iridovirus (RSIV) has been reported to be susceptible to various marine fish species in Asian countries such as South Korea, and it causes significant economic losses due to massive mortalities in rock bream (Oplegnathus fasciatus) in particular. However, studies on the impact of the virus shed from RSIV-infected rock bream on other fish species are limited. Here, we investigated the dynamics of the virus by simulating the natural conditions of RSIV infection in rock bream and examining its release into seawater and the risk of horizontal transmission through cohabitation with other fish species. Our data demonstrated that the virus shed from RSIV-infected rock bream caused high mortality rates in surrounding fish species through horizontal transmission. Our findings suggest the potential for RSIV-infected rock bream in farms to spread the disease to neighboring farms. Additionally, our results demonstrated a high correlation between RSIV load and viral shedding ratio in rock breams, as well as between RSIV load and histopathological infection grades. Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream (Oplegnathus fasciatus) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream (Pagrus major), and flathead grey mullet (Mugil cephalus) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture. [ABSTRACT FROM AUTHOR]

• To produce a highly concentrated megalocytivirus culture, primary cells, established cell line and persistently infected cell line were developed and investigated their abilities in this study. • Red sea bream fin culture was amenable to establish a cell line (PMF). • A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days in a batch culture system (PI-PMF-B) after infecting with megalocytivirus and produced highly concentrated virus (108.33-8.94 viral particles/ml). • A semi-batch culture system (PI-PMF-S) was more efficient and convenient to produce the virus by media exchanges without subculturing. • This is the first study for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells. Megalocytivirus infection is a major threat in rock bream aquaculture in Korea. To produce a highly concentrated megalocytivirus, primary cells, established cell line and persistently infected cell line were used in this study. Megalocytivirus was inoculated in primary fin cell cultures of red sea bream (Pagrus major), rock bream (Oplegnathus fasciatus), olive flounder (Paralichthys olivaceus) and black sea bream (Acanthopagrus schlegelii) and produced at similar concentrations of 108.99 − 9.88 viral particles/mL in all cultures while produced 107.31 viral particles/mL in grunt fin (GF) cell line. Since only red sea bream fin culture was amenable to subculturing for more than 100 times, it was established into Pagrus major fin (PMF) cell line. A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days as a batch culture system (PI-PMF-B) after infecting with megalocytivirus. Virus in supernatant of PI-PMF-B was maintained at high concentrations throughout over 50 consecutive subcultures in a relatively narrow range from 108.33 to 108.94 viral particles/mL with high level of CPE. For a more efficient and convenient production, a semi-batch culture system (PI-PMF-S) was developed in which culture media were exchanged at intervals of 3 days without subculturing for more than 50 media exchanges. Despite low virus productivity in a single cell (specific virus productivity, SVP), total cell number was increased in PI-PMF-S, allowing us to efficiently obtain a much higher concentration of virus (108.56 to 109.75 viral particles/mL) than in PMF-B. This is the first study to report detailed new methods for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells. [ABSTRACT FROM AUTHOR]