Body weight and water temperature shape VHSV infection dynamics and shedding in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia virus (VHSV) causes major losses in cold-season aquaculture; however, the joint effects of fish body weight and water temperature on within-host dynamics and the shedding potential remain poorly quantified. We experimentally challenged olive flounder (Paralichthys olivaceus) of three weight groups (10 g, 100 g, and 200 g) reared at 8 °C, 13 °C, and 18 °C with VHSV and quantified spleen viral loads using RT-qPCR and CPE-positivity to classify infection severity. Small fish (10 g group) rapidly reached Severe infection at 8-13 °C (7.5-8.1 log10 copies mg⁻¹; CPE 100 %), whereas at 18 °C, they showed a transient rise (∼6.3 log10 copies mg⁻¹) at days post-challenge (dpc) 3, followed by a decline to Light infection by dpc 14. Infections in medium-sized fish (100 g group) increased more slowly but were classified as having Severe infection by dpc 7-14 at 8-13 °C, and decreased to Light infection by dpc 14 at 18 °C. Large fish (200 g group) were classified as having Light-to-Moderate infections early, progressed to Severe infection by dpc 14 at 8 °C, remained largely Moderate at 13 °C, and had Light infection with no CPE by dpc 7-14 at 18 °C. Across weight groups, rearing at approximately 18 °C consistently mitigated infection, whereas 8-13 °C sustained high viral loads and CPE-positivity. Fish and water viral loads were correlated (r = 0.59) and inversely related to fish weight group (r = -0.48/-0.25), while each 1 log10 copies mg⁻¹ increase in spleen viral load increased the odds of shedding by 3.4-fold (odds ratio (OR) = 3.4) and, among shedders, predicted higher water titers (β = 0.41). These effects were evident in the 10 g and 100 g weight groups, but not in the 200 g weight group. Collectively, these results indicate that colder water and lower fish weight markedly amplified VHSV burden and likely environmental shedding risk, supporting temperature- and weight-aware risk assessment and targeted surveillance that prioritizes juvenile cohorts and colder periods for monitoring and control.