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Whole-genome next-generation sequencing was used to investigate the local evolution of viral haemorrhagic septicaemia virus, a serious pathogen affecting economically important fish such as rainbow trout and turbot in Europe and olive flounder in Asia. Sequence analysis showed that all isolates were genotype IVa, but could be classified further into four subgroups (K1-K4). In addition, genomic regions encompassing the nucleoprotein, phosphoprotein, matrix protein and non-virion protein genes, as well as the seven non-coding regions, were relatively conserved, whereas glycoprotein and RNA-dependent RNA polymerase genes were variable in the coding region. Taken together, the data demonstrate that whole-genome next-generation sequencing may be useful for future surveillance, prevention and control strategies against viral haemorrhagic septicaemia., (© 2020 John Wiley & Sons Ltd.)

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., (Copyright © 2018. Published by Elsevier Ltd.)

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP -like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP -like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2. • The Prosaposin-like (PSAPL) was identified by NGS analysis from Platichthys stellatus. • PsPSAPL showed high homology and sequence conservation with other species PSAPL. • PsPSAPL mRNA showed significant expression change due to pathogen infection. • The antimicrobial function and in vivo safety of PsPSAPL peptides were confirmed. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor (TLR) 5 is responsible for the bacterial flagellin recognition in vertebrates. Synergistic role of TLR 5 membrane form (TLR 5M) and TLR 5 soluble form (TLR 5S) have been reported from the study on rainbow trout. This system is regarded as the unique system in teleost fish. However, systemic response of TLR 5 genes in teleost fish has not been fully understood. Hence, we cloned Japanese flounder (Paralichthys olivaseus) TLR 5M and TLR 5S genes and their expressions were analyzed. The coding region of Japanese founder TLR 5M and TLR 5S cDNA were 2670 bp and 1923 bp, encoding 889 and 640 amino acid residues, respectively. The Japanese flounder TLR 5M was composed of an extracellular leucine rich repeats (LRRs), a transmembrane and an intracellular Toll/interleukin-1 receptor (TIR) domains, whereas TLR 5S possessed only the LRR domain. TLR 5M was highly expressed in the gill, head kidney, heart and liver. TLR 5S was highly expressed in the brain, head kidney and heart. Flagellin stimulation (1 and 5 μg/ml) led to strong gene expression of TLR 5S in peripheral blood leukocytes (PBLs) and liver cells. In contrast to TLR 5S, TLR 5M was down-regulated until 3 h after flagellin stimulation in PBLs and liver cells. The flagellin stimulation also resulted in the production of the flounder IL-1β and IL-6 from the liver cells and PBLs. The gene expression of TLR 5M was highly induced in the liver, while TLR 5S gene expression was drastically increased in the intestine following challenge with Edwardsiella tarda. Increased number of TLR 5M- and 5S-expressing cell populations were detected by in situ hybridization in the lamina propria of the intestine and liver after E. tarda infection, respectively. These results imply that the expression of these TLR 5 paralogs in Japanese flounder are differently regulated in the whole body and play important roles in the immune response against bacterial pathogens. [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]

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]