Viral hemorrhagic septicemia virus (VHSV) was isolated from farmed olive flounder (Paralichthys olivaceus). The viral N-gene was amplified by reverse transcriptase PCR, cloned, and sequenced for phylogenetic analysis to identify the genotype (I-IV). Virus isolates were cultured on Epithelioma papulosum cyprini cell line and, after completion of the cytopathic effects, the supernatant was collected and used to challenge virus-free flounder. The infected founder were reared in 16°C, 21°C, or 25°C, and compared to an unchallenged control. Virus titration was measured in the head kidneys, spleens, livers, brains, muscles, and gills of challenged fish using real-time quantification of the VHSV G-gene. Phylogenetic analysis confirmed that the isolates were VHSV Genogroup IV. The VHSV-challenged fish in the 16°C group showed 100% mortality with significantly increased expression of viral G-gene mRNA in the spleen, compared to fish reared in other temperatures and the control fish, suggesting that fish reared in 16°C are more susceptible to VHSV infection
