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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]