Naturally occurring substitution in one amino acid in VHSV phosphoprotein enhances viral virulence in flounder.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.

RNA-seq transcriptome analysis in flounder cells to compare innate immune responses to low- and high-virulence viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.

Two short antimicrobial peptides derived from prosaposin-like proteins in the starry flounder (Platichthys stellatus).

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.

Current use and management of commercial fish vaccines in Korea.

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders.

Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major).

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Molecular characterization and gene expression data of liver expressed antimicrobial Peptide-2 (LEAP-2) isolated from rock bream (Oplegnathus fasciatus).

Antimicrobial peptides (AMPs) are known to play a role as a first line of defence against microbial invasion. Liver Expressed Antimicrobial Peptides-2 (LEAP-2) is one of the AMPs. LEAP-2 includes four highly conserved cysteine residues and belongs to a cysteine-rich peptides group. We identified and characterized the molecular properties of LEAP-2 in rock bream. The expression levels of rock bream LEAP-2 (RbLEAP-2) in the 12 different tissues of healthy fish and the RbLEAP-2 expression pattern after infections with Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red seabream iridovirus (RSIV) were examined. This data provide that RbLEAP-2 plays an important role in innate immunity when rock bream is infected with a pathogen.

The atypical chemokine receptor 4 in red sea bream (Pagrus major): Molecular characterization and gene expression analysis.

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection.

Data on molecular characterization and expression profiles of the NF-κB repressing factor gene in red sea bream (Pagrus major).

Nuclear factor-kappaB (NF-κB) repressing factor (NKRF) specifically inhibits the transcriptional activity of NF-κB protein. The PmNKRF cDNA is composed of 757 amino acid residues. Alignment analysis revealed that the G-patch and R3H domains are conserved in different organisms. We aimed to analyse red sea bream NKRF (PmNKRF) gene expression after infection with pathogens [Streptococcus iniae or red sea bream iridovirus (RSIV)] and in healthy individuals. In healthy individuals, PmNKRF was ubiquitously expressed in all 12 tested tissues, predominantly in the head kidney and spleen. Expression of PmNKRF was significantly up-regulated in the gills, kidney, liver and spleen after RSIV infection. After S. iniae infection, PmNKRF expression was significantly down-regulated in the gills and significantly up-regulated in the kidney, liver and spleen.

Differential expression of olive flounder (Paralichthys olivaceus) transcriptome during viral hemorrhagic septicemia virus (VHSV) infection at warmer and colder temperature.

The data presented here are related to the research article entitled "Temperature-dependent immune response of olive flounder (Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV)" [1]. In the cited article, we sequenced the whole transcriptome of the olive flounder using Illumina RNA-Seq. Differentially expressed genes (DEG) analysis of VHSV infected head kidney samples showed perturbations in gene expression. Herein we made a comparison of DEGs at early stage of VHSV infection of olive flounder (4 h post infection) in colder (13 °C) and warmer (20 °C) temperatures. The analysis of signaling pathways showed that several major immune pathways were altered. The gene ontology terms associated with the genes differentially expressed are also presented.

Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus.

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens.

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Olive flounder CD276 (B7-H3) a coinhibitory molecule for T cells: Responses during viral hemorrhagic septicemia virus (VHSV) stimulation.

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.

The first report of cathepsin A gene in a teleost (rock bream, Oplegnathus fasciatus): An investigation of immune functions upon infection with several pathogens.

We isolated and characterised a cDNA encoding the lysosomal protective protein (serine protease) cathepsin A (CTSA) from rock bream (Oplegnathus fasciatus). The full-length rock bream CTSA (RbCTSA) cDNA (1814 bp) contains an open reading frame of 1419 bp that encodes 472 amino acids. Alignment of multiple CTSA protein sequences revealed that the active site serine and histidine residues were well-conserved among the other CTSA sequences. RbCTSA is highly expressed in the peripheral blood leukocytes, kidney, spleen, liver, intestine, gill, heart, brain, stomach, and eye. RbCTSA expression was also examined in several tissues, including whole kidneys and spleens, under bacterial and viral challenge. In general, all of the examined tissues that were infected with Edwardsiella tarda, Streptococcus iniae, or red sea bream iridovirus (RSIV) exhibited significant upregulation of RbCTSA expression compared to the controls. Our results reveal that RbCTSA may be involved in the immune responses of rock bream.

The first report of CD2 associated protein gene, in a teleost (Rock bream, Oplegnathus fasciatus): An investigation of the immune response upon infection with several pathogens.

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes.

Molecular characterization of a T cell co-stimulatory receptor, CD28 of rock bream (Oplegnathus fasciatus): Transcriptional expression during bacterial and viral stimulation.

CD28 is a co-stimulatory receptor that provides a critical second signal alongside T cell receptors for the activation of naive T cells. We characterized the CD28 gene of rock bream, which has a deduced amino acid sequence of 221 residues with an extracellular Ig-superfamily V domain, transmembrane region, and cytoplasmic tail. The conservation in domain structures and other motifs shows that it is highly likely that RbCD28 is a homologue of mammalian CD28 and may have related co-stimulatory functions. RbCD28 is constitutively expressed in most tissues that were analysed, with a relatively higher expression in teleost lymphoid organs, such as spleens, gills, trunk kidneys and skin. Unlike human CD28, RbCD28 is highly expressed in skin and gill-associated lymphoid organs. Although gills showed constitutive expression of RbCD28 in control animals, after a pathogen challenge, induction of CD28 was low, particularly in RSIV and E. tarda infection. Whereas induction of RbCD28 was observed in kidney during E. tarda and S. iniae infection, downregulation was observed during RSIV infection. In the case of the liver, E. tarda caused an initial upregulation of RbCD28. RbCD28 activation of T cells in the spleen was limited to S. iniae infection. Activation of RbCD28 observed in lymphoid organs during infection of various pathogens shows its key role as a co-stimulatory receptor of T cells.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection.

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions.

Molecular cloning and expression analysis of a new lily-type lectin in the rock bream, Oplegnathus fasciatus.

A new lily-type lectin RbLTL was identified from rock bream (Oplegnathus fasciatus) and its expression analysed. In this study, a new lily-type lectin gene (RbLTL) was cloned from rock bream using expressed sequence tag (EST) analysis. The full-length RbLTL cDNA was encoding a 117-amino acid protein. The deduced amino acid sequence of RbLTL contained all of the conserved features crucial for its fundamental structure, including B-lectin domain and three d-mannose binding sites. RbLTL mRNA was predominately expressed in the gills, with reduced expression noted in intestine tissue. Expression analysis of time series sampled fertilized eggs revealed that expression gradually increased 1, 3, 12, and 24 h: However, expression decreased at 36 h. RbLTL expression was differentially up-regulated in rock bream gills challenged with Streptococcus iniae, Edwardsiella tarda and RSIV. Our results revealed that novel rock bream lily-type lectin may be an important molecule involved in pattern recognition and pathogen elimination in the innate immunity of rock bream.

Coagulation factor II from rock bream (Oplegnathus fasciatus): First report on the molecular biological function and expression analysis in the teleost.

The rapid haemostasis of fish prevents bleeding or infection that could be caused by physical properties of the aquatic environment. Additionally, the innate immune system is the first line of defence against infection and is responsible for the recognition of pathogen-associated molecular patterns, which are important for the activation of acquired immune responses. Coagulation factor II (CFII) is an important factor in the coagulation system and is involved in recognition and interaction with various bacterial and extracellular proteins. In this study, we identified and characterised the gene encoding CFII in rock bream (Oplegnathus fasciatus) (RbCFII) and analysed its expression in various tissues after a pathogen challenge. The full-length RbCFII cDNA (2079 bp) contained an open reading frame of 1854 bp encoding 617 amino acids. Alignment analysis revealed that a gamma-carboxyglutamic acid-rich domain, two kringle domains, and a trypsin-like serine protease domain of the deduced protein were well conserved. RbCFII was ubiquitously expressed in all tissues examined but, predominantly detected in the liver and skin. RbCFII expression was dramatically up-regulated in the kidney, spleen and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. The recombinant protein RbCFII (rRbCFII) produced using an Escherichia coli expression system was able to bind all examined bacteria. Interestingly, rRbCFII has agglutination activities towards E. coli and E. tarda, while no agglutination was shown toward Vibrio ordalii and S. iniae. These findings indicate that rRbCFII performs an immunological function in the immune response, and might be involved in innate immunity as well as blood coagulation.

First description of programmed cell death10 (PDCD10) in rock bream (Oplegnathus fasciatus): Potential relations to the regulation of apoptosis by several pathogens.

In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses.

First molecular characterisation and expression analysis of a teleost thioredoxin-interacting protein (TXNIP) gene from rock bream (Oplegnathus fasciatus).

Thioredoxin-interacting protein (TXNIP) is an important regulator of glucose metabolism that functions by inhibiting cellular glucose uptake. The full-length rock bream (Oplegnathus fasciatus) TXNIP (RbTXNIP) cDNA (2499 bp) contains an open reading frame of 1188 bp encoding 396 amino acids. Furthermore, multiple alignments showed that the arrestin domain was well conserved among the other TXNIP sequences tested. RbTXNIP was predicted to contain a PxxP and PPxY motif. Phylogenetic analysis indicated that RbTXNIP is most closely related to Fugu rubripes TXNIP. RbTXNIP was expressed significantly in the RBC, intestine, and spleen. RbTXNIP mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. Generally, all tissues examined from fish infected with Streptococcus iniae, Edwardsiella tarda and red sea bream iridovirus (RSIV) showed significant downregulation in RbTXNIP expression compared to controls. However, RbTXNIP expression showed significant upregulation in the spleen and kidney after injection of recombinant rock bream TRx1 protein. These findings provide a molecular foundation for functional studies and applications in teleosts.