Isolation and Infectious Temperature Optimization of Genetically Similar VHSV Isolates in Farmed Olive Flounder, Paralichthys olivaceus

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 degrees C, 21 degrees C, or 25 degrees 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 degrees 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 degrees C are more susceptible to VHSV infection.