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Olive flounder were immersed with viral hemorrhagic septicemia virus (VHSV) and reared for 30 days at 17℃ mount resistance against the virus, but the standard for the rearing period remains unclear. Here, we investigated VHSV dynamics and localization in internal organs (heart, spleen, and kidney) after VHSV immersion treatment and rearing at a suboptimal temperature (17℃), followed by rechallenge at an optimal temperature (10℃), and compared the protective efficacy between rearing periods of 5 days and 30 days at 17℃. Juvenile olive flounder reared for 5 days at 17℃ after VHSV immersion treatment acquired resistance against the VHSV infection, as evidenced by their 100% survival. The observed VHSV N gene mRNA in the kidney of flounder (5-day rearing) gradually increased and peaked at 15 days post re-challenge (dpr), although increased until 5 dpr and thereafter decreased in the kidney of flounder (30-day rearing). VHSV mRNA signal in flounder (5-day rearing) was observed at 15 dpr, whereas it was detected at 5 dpr in flounder (30-day rearing) by RNA-ISH (in-situ hybridization). The protection provided by live VHSV vaccination at suboptimal temperatures might not be mediated by humoral immunity, with evidence of lower IgM mRNA expression in the vaccinated group. These observations provide a better understanding of the rearing period needed for immunizing fish against the virus at suboptimal temperatures. [ABSTRACT FROM AUTHOR]

• At a temperature of 15 °C, resembling natural conditions, the minimum infection dose for the establishment of infection through horizontal transmission is determined to be 103.4 TCID 50 /ml or higher. • The number of infected fish increased with higher infection concentrations, and no significant difference in viral load in kidneys was observed among the infected fish. • Significant differences in weight and length were observed among the farms; however, no correlation was found between the number of infected fish and either weight or length. Viral hemorrhagic septicemia virus (VHSV) affects over 80 fish species, leading to viral hemorrhagic septicemia (VHS). Horizontal VHSV transmission is widely studied, with researchers utilizing various doses to establish infection models. Infected hosts shed the virus into the environment, elevating the risk of transmission to naïve fish within the same system. This study aimed to ascertain the minimum infective dose of VHSV in olive flounder (Paralichthys olivaceus). In olive flounder, the detection of VHSV within the kidney exhibited the highest infection rate on the third day among days 1, 3 and 5. Doses of 103.0 to 104.7 TCID 50 /ml were administered to juvenile olive flounder across three farms. Results showed resistance to infection below 103.4 TCID 50 /ml at 15 °C. While infection frequency varied by concentration, higher concentrations correlated with more infections. Nonetheless, viral copy numbers did not differ significantly among infected fish at varying concentrations. This study underscores the need for early VHSV management and contributes essential data for pathogenicity assessment and foundational knowledge. [ABSTRACT FROM AUTHOR]