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Abstract: Mammalian Toll-like receptor 3 (TLR3) recognizes extracellular and intracellular viral dsRNA, and then initiates signaling cascades leading to NF-κB activation and interferon (IFN) production. To understand the roles of TLR3 in the fish immune system, TLR3 gene (JfTLR3) was identified from Japanese flounder (Paralichthys olivaceus), which consisted of 4 exons and 3 introns. Its expression in peripheral blood leukocytes increased upon stimulation with poly I:C and CpG ODN 1668. Exposure to viral hemorrhagic septicemia virus increased expression of JfTLR3 in the blood, liver, head kidney and spleen. Intracellular poly I:C stimulation in JfTLR3-overexpressing YO-K cells significantly induced IFN-inducible and NF-κB-regulated genes. NF-κB activity in JfTLR3-overexpressing YO-K cells was significantly induced by intracellular poly I:C while expression of IFN-inducible genes and NF-κB reporter activity in JfTLR3-overexpressing HINAE cells increased upon stimulation by extracellular poly I:C. These results suggest that JfTLR3 plays an important role in the induction of antiviral immune response. [Copyright &y& Elsevier]

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]

The viral hemorrhagic septicemia virus (VHSV) is a viral pathogen to cause severe losses in farming industry of olive flounder. Among six-encoding VHSV proteins, NV protein has been shown to have an impact on virulence. Thirteen VHSV strains are isolated from olive flounders showing VHS symptoms and mortality in Korea, and then molecular variation of nucleotide and amino acid sequences of the NV genes were compared. Although common mutations were detected at four sites in nucleotide sequences of NV gene, there was no difference in the amino acid sequences among 13 VHSV variants. We identified substitution of one base of 34 positions within the NV ORF of 13 VHSV isolates and these point mutations resulted in changes in amino acid sequences at 6 residues of NV (8, 56, 81, 88, 117, and 119). Based on finding of NV-mediated decrease of intracellular ATP level, protein expression of six NV variants in flounder cells decreased more than control NV protein. The strongest decreasing activity on cellular ATP level was shown by No. 56 (serine → leucine) of NV variation. The variation changes the hydropathy index significantly. The results imply that we can use appearance of some amino acid variations as virulence markers in VHSV-derived mortality. Statement of relevance 1. Recently VHSV has infected > 80 kinds of fish species. The number of infection host of VHSV is rapidly increased. Among the VHSV-infected fishes, olive flounder is one of the most important fish of aquaculture in Korea, Japan, and China. In every year, we found the massive VHSV-derived mortality of olive flounder aquaculture in Korea. To overcome this VHSV pathogenesis, we need to identify high-pathogenic VHSV appearance at aquaculture in earlier time. 2. Since VHSV gene is highly mutated in time-dependent manner, the efficient vaccine for VHSV-derived disease prevention is not well achieved. Therefore, the other approaches for prevention of epidemics by VHSV should be required. Our research goal is to develop prevention of epidemics propagation of VHSV infection by high pathogenic VHSV prediction with gene variation analysis. 3. In this study, we identified several gene variations of VHSV NV protein from 13 VHSV specimens of severe VHS-showing olive flounders in Korea. The gene variations are associated with amino acid changes of VHSV NV proteins. The amino acid variants of NV protein show the inhibitory activity of fish cellular energy (ATP) generation. The cellular energy deprivation might give a big impact on fish health and mortality. The DNA mutation on specific amino acid changes is possibly applied to develop the PCR diagnosis system for early detection of high pathogenic VHSV appearance. The early pathogenic gene diagnosis of VHSV gives more efficient prevention of epidemics. [ABSTRACT FROM AUTHOR]

Abstract: Type I interferon (IFN) induces the antiviral response in innate immunity. The type I IFN gene cloned from Japanese flounder (Paralichthys olivaceus) has a length of 1189bp and consisting of 5 exons and 4 introns. In a phylogenetic tree of type I IFNs, Japanese flounder grouped with other Acanthopterygii. To gain insight into the transcriptional regulation of IFN gene, the 1.36kb 5′-upstream region including numerous canonical motifs to bind transcription factors [for example, IFN regulatory factor (IRF)] was analyzed. In HINAE cells using a luciferase reporter assay, poly I:C-responsive transcriptional activity was found in the region from −634 to −179bp. This region includes several IRF motifs. In the presence of poly I:C, overexpression of IRF3 and RLR strongly enhanced transcriptional activity. These results suggest that the transcriptional regulation of Japanese flounder type I IFN is regulated by IRF3 after triggering with dsRNA sensors. [Copyright &y& Elsevier]

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea. [ABSTRACT FROM AUTHOR]

Rockfish, Sebastes schlegelii (mean weight 14.53 ± 1.14 cm, and mean weight 38.36 ± 3.45 g) were exposed for 4 weeks (2 weeks and 4 weeks) with the different levels of ammonia in the concentrations of 0, 0.1, 0.5, 1.0 mg/L at 19 and 24 °C. The ammonia exposure induced significant alterations in antioxidant responses. The activities of SOD, CAT, and GST were considerably increased by the ammonia exposure depending on water temperature, whereas the GSH level was notably decreased after 2 and 4 weeks. In the stress indicators, the cortisol and HSP 70 were significantly elevated by the exposure to ammonia depending on water temperature. In innate immune responses, the phagocytosis and lysozyme activity were notably decreased by ammonia exposure depending on water temperature after 2 and 4 weeks. The results suggest that ammonia exposure depending on water temperature can induce the considerable alterations in antioxidant responses, stress, and immune inhibition. [ABSTRACT FROM AUTHOR]