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Poly (I:C) showed promise as an immunoprotective agents in rock bream against rock bream iridovirus (RBIV) infection. In this study, we evaluated the time-dependent virus replication pattern and antiviral immune responses in RBIV-infected rock bream with and without poly (I:C) administration. In the poly (I:C)+virus-injected group, virus copy numbers were more than 18.9-, 24.0- and 479.2-fold lower than in the virus only injected group at 4 (4.73 × 104 and 8.95 × 105/μl, respectively), 7 (3.67 × 105 and 8.81 × 106/μl, respectively) and 10 days post infection (dpi) (1.26 × 105 and 6.02 × 107/μl, respectively). Moreover, significantly high expression levels of TLR3 (8.6- and 7.7-fold, at 4 and 7 dpi, respectively) and IL1β (3.6-fold at 2 dpi) were observed in the poly (I:C)+virus-injected group, but the expression levels were not significantly in the virus-injected group. However, IL8 and TNFα expression levels showed no statistical significance in both groups. Mx, ISG15 and PKR were significantly highly expressed from 4 to 10 dpi in the virus-injected group. Nevertheless, in the poly (I:C)+virus-injected group, Mx and ISG15 expression were significantly expressed from 2 dpi. In summary, poly (I:C) administration in rock bream induces TLR3, IL1β, Mx and ISG15-mediated immune responses, which could be a critical factor for inhibition of virus replication.Poly (I:C) inhibited virus replication in spleen, kidney, liver and blood of rock bream.TLR3 and IL1β were highly expressed in the poly (I:C)+virus-injected group.Mx and ISG15 were significantly expressed earlier (from 2 dpi) in the poly (I:C)+virus-injected group than in the virus-injected group (from 4 dpi).