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Viral hemorrhagic septicemia is a serious disease that can assume epidemic proportions in cultivated olive flounders in Korea. The causal agent of this disease is viral hemorrhagic septicemia virus (VHSV), and it leads to vast economic losses in the olive flounder aquaculture industry. Therefore, the rapid and accurate detection of VHSV is paramount. At present, the manual of the World Organization for Animal Health's Office International des Epizooties (OIE) for pathogen detection is being followed for VHSV detection in Korea. However, in that manual, the primers for VHSV detection are based on the European VHSV genotype Ia. In this study, we identified 5 VHSV strains from olive flounders in Korea, and the nucleotide and amino acid sequences of VHSV genes (N, P, M, and G) were compared to investigate the genetic variation of VHSV genotypes. As expected, VHSV isolates from Korea were highly related to genotype IVa, clearly differing from the 3 European genotypes. In addition, the N gene showed low genetic variation and therefore might be considered a useful marker for VHSV detection, regardless of genotype. In contrast, the other VHSV genes (P, M, and G) had different nucleotides within genotypes. They might be suitable for designing specific primers for distinguishing between various VHSV genotypes. Duplex RT-PCR using the newly designed primers successfully detected all VHSV isolates and validated the genotype (I and IV) without sequence analysis. According to the results, all the VHSV isolates were successfully detected, and their genotypes were validated by duplex RT-PCR using the newly designed primers. Collectively, our findings suggest that duplex PCR is a convenient and appropriate method for the diagnosis of VHSV isolates in Korean aquaculture systems. [ABSTRACT FROM AUTHOR]

Olive flounder ( Paralichthys olivaceus ) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10 −5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV. [ABSTRACT FROM AUTHOR]