Red Sea Bream Iridovirus Stability in Freeze–Thaw Cycles: Quantitative Assays of Infectious Particles

Red sea bream iridovirus is a serious threat to farmed fish, but little is known about how repeated freezing and thawing affect its stability. This study investigated the effects of repeated freeze-thaw cycles on RSIV infectivity by comparing quantitative polymerase chain reaction (qPCR), viability qPCR (vqPCR), and 50% tissue culture infectious dose (TCID₅₀) assays. While qPCR detected high amounts of viral DNA after multiple cycles, both viability qPCR and TCID₅₀ revealed a significant loss of infectivity unless serum was present. Correlation analysis showed a high degree of agreement between vqPCR and TCID₅₀, indicating their high compatibility for assessing viral infectivity. However, the correlations between qPCR and vqPCR, as well as between qPCR and TCID₅₀, were significantly lower, suggesting that qPCR alone may overestimate viral infectivity by detecting non-infectious viral DNA. These results demonstrate the critical role of serum in preserving RSIV infectivity and highlight the superior accuracy of vqPCR and TCID₅₀ in assessing viral infectivity compared with qPCR. This study emphasizes the importance of serum in storage media and suggests that combining vqPCR with TCID₅₀ is a more reliable measure of RSIV infectivity than qPCR alone.