Search

Introduction: Fish nucleated red blood cells (RBCs), also known as erythrocytes, play a crucial role in maintaining immune system balance by modulating protein expression in response to various stimuli, including viral attack. This study explores the intriguing behavior of rainbow trout RBCs when faced with the viral hemorrhagic septicemia virus (VHSV), focusing on the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Methods: Rainbow trout RBCs were Ficoll-purified and exposed to ultraviolet (UV)-inactivated VHSV or live VHSV at different multiplicities of infection (MOIs). Using cryo-soft X-ray tomography (cryo-SXT), we uncovered structural and cellular modifications in RBCs exposed to UV-inactivated VHSV. Moreover, RBCs were treated with 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, to investigate its effect on viral replication. Quantitative real-time PCR was also used to analyze the expression of genes related to the UPR and other related cellular pathways. Results and discussion: Beyond their antiviral response, RBCs undergo notable intracellular changes to combat the virus. Cryo-SXT highlighted a significant increase in the ER volume. This increase is associated with ER stress and the activation of the UPR pathway. Interestingly, VHSV replication levels augmented in RBCs under ER-stress inhibition by 4-PBA treatment, suggesting that rainbow trout RBCs tune up ER stress to control viral replication. Therefore, our findings suggested the induction of ER stress and subsequent activation UPR signaling in the antiviral response of RBCs to VHSV. The results open a new line of investigation to uncover additional mechanisms that may become novel cellular targets for the development of RBC-targeted antiviral strategies. [ABSTRACT FROM AUTHOR]

A newly isolated rhabdovirus-like agent, named SHRV-In, obtained from snakehead fish in India was further characterized in this study. The virus demonstrated growth in various cell lines including those derived from snakehead and seabass species. It exhibited sensitivity to acidic conditions (pH 3.0) and exposure to 56 °C. Five structural proteins of 20 to 68 kDa were found in the purified virus. Experimental infection trials conducted on catfish (Pangasius pangasius) resulted in mortality 72 h post-infection, whereas no mortality was observed in rohu (Labeo rohita) and common carp (Cyprinus carpio) fingerlings. Molecular characterization of the virus involved sequencing of the N, P, M, and G gene fragments after PCR amplification using specific primers. Sequence analysis of all four viral genes showed high similarity with the previously isolated snakehead rhabdovirus (SHRV) from Thailand. Phylogenetic analysis and gene sequence alignment placed the SHRV-In isolate within the Novirhabdovirus group, which includes viruses like VHSV (viral hemorrhagic septicemia virus), IHNV (infectious hematopoietic necrosis virus), and SHRV. However, our repeated trials failed to amplify the NV gene in this isolate SHRV-In. [ABSTRACT FROM AUTHOR]

Bacterial and viral infections cause a huge burden to healthcare settings worldwide, and mortality rates associated with infectious microorganisms have remained high in recent decades. Despite tremendous efforts and resources worldwide to explore diagnostic biomarkers, rapid and easily assayed indicators for the diagnosis of bacterial and viral infections remain a challenge. B7 homolog 3 (B7-H3), a member of the B7 family of immunoregulatory proteins, is overexpressed in patients with septicemia, meningitis, pneumonia, and hepatitis. Therefore, B7-H3 could be used as a potential clinical indicator and therapeutic target for bacterial and viral infections caused by H. pylori, S. pneumoniae, M. pneumoniae, hepatitis B virus (HBV), viral hemorrhagic septicemia virus (VHSV), respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV). Moreover, the interplay between infectious microorganisms and B7-H3 and exploration of the functional roles of the B7-H3 molecule could aid in the development of novel strategies for disease diagnosis and immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques.

Among salmonids, brook charr, Salvelinus fontinalis has been gaining popularity in aquaculture. However, major limitations associated with opportunistic diseases that result from unstable egg and fry‐rearing conditions are experienced under intensive rearing conditions. Great losses in brook charr culture result from viral (e.g., infectious pancreatic necrosis, viral hemorrhagic septicemia virus), fungal (e.g., saprolegniosis), and bacterial (e.g., furunculosis, cold water disease, columnaris) diseases. Unfortunately, there is no effective treatment for viral diseases for brook charr, and the chemical disinfectants and antibiotics commonly used against bacterial and fungal infections are reaching their limits (pathogen resistance, microbial community disruption), making alternative and more sustainable rearing approaches essential to bolster aquaculture. In this regard, recent studies show that many microbial species (bacteria, archaea, viruses, eukaryotes‐like protozoa, and unicellular fungi) are essential from the earliest life stages for the proper development and growth of fish. Based on ecological theory and work on marine fish, it has been hypothesized that current aquaculture practices favor r‐strategic, opportunistic microbes that are detrimental to fish health and development. Consequently, to ensure the success and sustainability of brook charr farming, a new aquaculture paradigm involving recirculating aquaculture systems (RAS) that select a K‐enriched, specialized microbial community as a preventive measure against those microbial diseases is a very promising avenue. In this review, after introducing brook charr biology, the role of microbes in brook charr rearing and the general problems of disease and low fry survival due to an imbalance in the microbial community will be discussed. Various curative and prophylactic treatment methods currently used to control these diseases and their impact on microbial ecology will be covered, and we will conclude with the rationale of microbial ecology management and its effectiveness in promoting fry survival through disease prevention. To our knowledge, this is the first literature review addressing the promises of managing microbial ecology in a sustainable way in the brook char industry. [ABSTRACT FROM AUTHOR]

The South Korean aquaculture industry has incurred considerable production losses due to various infectious diseases. Artificially synthesized polyinosinic–polycytidylic acid (poly I:C), structurally similar to double-stranded RNA (dsRNA) and cytidine–phosphate–guanosine oligodeoxynucleotides, can enhance immune responses and protect against diseases. Here, we investigated dsRNA molecules with different cytidine–phosphate–guanosine (CpG) motifs (dsRNA-CpGMix) as fused agents to treat Miamiensis avidus and viral hemorrhagic septicemia virus (VHSV) infection in olive flounders. We further investigated the efficacy of specific sequence motifs in dsRNA in modulating immunostimulatory effects. Fish treated with poly I:C or dsRNA-CpGMix exhibited higher survival rates than the control group. Olive flounder leukocytes stimulated with poly I:C or dsRNA-CpGMix showed increased scuticocidal activity in the presence of inactivated immune sera. dsRNA with CpG motif sequences induced higher resistance against M. avidus and VHSV infections than dsRNA without CpG motif sequences, and the dsRNA-CpGMix group showed upregulated ISG15 or Mx compared to the dsRNA-GFP group. Thus, dsRNA containing CpG motifs can be used as effective immunostimulants to enhance resistance against viral and parasitic diseases in olive flounder. The specific sequences of the CpG motifs in dsRNA may be important for enhancing immune responses and resistance against M. avidus and VHSV infections in olive flounders. [ABSTRACT FROM AUTHOR]

Olive flounder were immersed with viral hemorrhagic septicemia virus (VHSV) and reared for 30 days at 17℃ mount resistance against the virus, but the standard for the rearing period remains unclear. Here, we investigated VHSV dynamics and localization in internal organs (heart, spleen, and kidney) after VHSV immersion treatment and rearing at a suboptimal temperature (17℃), followed by rechallenge at an optimal temperature (10℃), and compared the protective efficacy between rearing periods of 5 days and 30 days at 17℃. Juvenile olive flounder reared for 5 days at 17℃ after VHSV immersion treatment acquired resistance against the VHSV infection, as evidenced by their 100% survival. The observed VHSV N gene mRNA in the kidney of flounder (5-day rearing) gradually increased and peaked at 15 days post re-challenge (dpr), although increased until 5 dpr and thereafter decreased in the kidney of flounder (30-day rearing). VHSV mRNA signal in flounder (5-day rearing) was observed at 15 dpr, whereas it was detected at 5 dpr in flounder (30-day rearing) by RNA-ISH (in-situ hybridization). The protection provided by live VHSV vaccination at suboptimal temperatures might not be mediated by humoral immunity, with evidence of lower IgM mRNA expression in the vaccinated group. These observations provide a better understanding of the rearing period needed for immunizing fish against the virus at suboptimal temperatures. [ABSTRACT FROM AUTHOR]

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins. [ABSTRACT FROM AUTHOR]

The rainbow trout (Oncorhynchus mykiss) is the most important produced species in freshwater within the European Union, usually reared in intensive farming systems. This species is highly susceptible to viral hemorrhagic septicemia (VHS), a severe systemic disease widespread globally throughout the world. Viral hemorrhagic septicemia virus (VHSV) is the etiological agent and, recently, three classes of VHSV virulence (high, moderate, and low) have been proposed based on the mortality rates, which are strictly dependent on the viral strain. The molecular mechanisms that regulate VHSV virulence and the stimulated gene responses in the host during infection are not completely unveiled. While some preliminary transcriptomic studies have been reported in other fish species, to date there are no publications on rainbow trout. Herein, we report the first time-course RNA sequencing analysis on rainbow trout juveniles experimentally infected with high and low VHSV pathogenic Italian strains. Transcriptome analysis was performed on head kidney samples collected at different time points (1, 2, and 5 days post infection). A large set of notable genes were found to be differentially expressed (DEGs) in all the challenged groups (e.s. trim63a, acod1, cox-2, skia, hipk1, cx35.4, ins, mtnr1a, tlr3, tlr7, mda5, lgp2). Moreover, the number of DEGs progressively increased especially during time with a greater amount found in the group infected with the high VHSV virulent strain. The gene ontology (GO) enrichment analysis highlighted that functions related to inflammation were modulated in rainbow trout during the first days of VHSV infection, regardless of the pathogenicity of the strain. While some functions showed slight differences in enrichments between the two infected groups, others appeared more exclusively modulated in the group challenged with the highly pathogenic strain. [ABSTRACT FROM AUTHOR]

VHS is an extremely epizootic virus that causes high morbidity in cold-water fish. Nearly 100 species of fish in the northern hemisphere are affected by VHS disease, which has significant economic consequences for the aquaculture industry. It is crucial to understand how this virus affects fish. The spread of virus depends on environmental factors and the age of fish species. As the temperature rises, the virus's spread increases. In general, salmonid fish are particularly susceptible to VHSV and juvenile fish are also more sensitive to it. The virus is transmitted from infected water to various host species. Various methods of diagnosing and preventing diseases are presented. This review provides a comprehensive overview of VHS disease, including the species affected, diagnosis and detection of the virus, morphology, phylogeny and pathogenicity. [ABSTRACT FROM AUTHOR]

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines – viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated. [ABSTRACT FROM AUTHOR]

The demand for aquaculture is increasing, but production is declining due to high feed costs and disease outbreaks. Viral hemorrhagic septicemia (VHS) is a viral disease that seriously affects seawater and freshwater fish in aquaculture, including the olive flounder (Paralichthys olivaceus), a major aquaculture fish in Korea. However, very few vaccines are currently available for viral hemorrhagic septicemia virus (VHSV). The nutrient-rich microalga Chlorella vulgaris has been used as a feed additive in aquaculture and as a host for the industrial production of recombinant VHSV glycoprotein as an oral vaccine. In this study, VHSV glycoprotein was cloned with a salt-inducible promoter, and high levels of expression up to 41.1 mg/g wet C. vulgaris, representing 27.4% of total extracted soluble protein, were achieved by growing the transformed C. vulgaris for 5 days in the presence of 250 mM NaCl. The production of a neutralizing antibody was detected in the serum of fish given feed containing 9% VHSV glycoprotein-expressing C. vulgaris. Furthermore, relative survival rates of 100% and 81.9% were achieved following challenges of these fish with VHSV at 106 and 107 pfu/fish, respectively, indicating that C. vulgaris could be used as a platform for the production of recombinant proteins for use as oral vaccines in the control of viral diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: Fish-derived primary cells are highly valuable for investigating cell–virus interactions, virus isolation, diagnosis, and vaccine development because of their rapid growth rate and high susceptibility to viruses. We established a new cell line, the dwarf gourami fin (DGF), from the caudal fin of the dwarf gourami (Trichogaster lalius). We analyzed the optimal growth conditions, including media, temperature, and fetal bovine serum concentration, modal chromosome number, and transfection efficiency. The susceptibility of DGF cells to different viruses, including red sea bream iridovirus, infectious spleen and kidney necrosis virus, viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus was evaluated. The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis. [ABSTRACT FROM AUTHOR]

Objective: Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in freshwater and saltwater fish species. The susceptibility of endangered Pallid Sturgeon Scaphirhynchus albus to VHSV genotype IVb (VHSV‐IVb) infection was investigated. Methods: An in vitro assessment using two Pallid Sturgeon cell lines derived from skin and spleen tissue and in vivo evaluation of juvenile Pallid Sturgeon after exposure to VHSV‐IVb were performed. Result: Plaque assay and RT‐PCR results confirmed VHSV‐IVb replication in Pallid Sturgeon cell lines. Sturgeon were also susceptible to VHSV‐IVb infection after immersion and injection exposures during laboratory experiments. However, after widespread mortality occurred in all treatment groups, including negative control fish, it was determined that the Pallid Sturgeon stock fish were infected with Missouri River sturgeon iridovirus (MRSIV) prior to experimental challenge. Nevertheless, mortalities were equal or higher among VHSV‐exposed fish than among negative controls (MRSIV infected), and histopathological assessments indicated reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in fish from the VHSV treatment. Conclusion: These results indicate that Pallid Sturgeon is a susceptible host for VHSV‐IVb, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Research comparing susceptibility of specific pathogen‐free and MRSIV‐infected fish to VHSV‐IVb is needed to accurately assess the vulnerability of Pallid Sturgeon to VHSV‐IVb. Impact statementThis first investigation of Pallid Sturgeon susceptibility to VHSV indicated they could be infected with the highly lethal VHSV‐IVb strain; however, because the tested fish were already infected with another disease‐causing virus the level of susceptibility of Pallid Sturgeon to VHSV‐IVb alone needs to be studied further. [ABSTRACT FROM AUTHOR]

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: Environmental DNA (eDNA) has attracted attention as a monitoring tool in the aquaculture industry to detect aquatic viral diseases in seawater. To be able to use the low concentration of viruses in seawater, an additional concentration process needs to be employed. In this study, the iron flocculation method was applied to concentrate the viral hemorrhagic septicemia virus (VHSV), and the recovery rate was estimated to determine the suitability of two re-suspension buffers, oxalic and ascorbic acid. It was determined that, while both of these buffers could be efficient for recovering viral genome copy, the oxalic acid buffer was more suitable for preserving viral infectivity than the ascorbic acid buffer. Thus, the iron flocculation method using an oxalic acid buffer could be efficient for evaluating actual viral transmission and is expected to predict the occurrence of diseases in the environment. Iron flocculation is widely used to concentrate viruses in water, followed by Fe-virus flocculate formation, collection, and elution. In the elution stage, an oxalic or ascorbic acid re-suspension buffer dissolved iron hydroxide. After the concentration of viral hemorrhagic septicemia virus (VHSV) in seawater (1 × 101 to 1 × 105 viral genome copies or plaque-forming unit (PFU)/mL), the recovery yield of the viral genome using quantitative real-time PCR (qRT-PCR) and viral infectivity using the plaque assay were investigated to evaluate the validity of the two re-suspension buffers to concentrate VHSV. The mean viral genome recovery yield with oxalic and ascorbic acid was 71.2 ± 12.3% and 81.4 ± 9.5%, respectively. The mean viral infective recovery yields based on the PFU were significantly different between the two buffers at 23.8 ± 22.7% (oxalic acid) and 4.4 ± 2.7% (ascorbic acid). Notably, although oxalic acid maintains viral infectivity over 60% at a viral concentration above 105 PFU/mL, the infective VHSVs were not sufficiently recovered at a low viral concentration (102 PFU/mL, <10%). To support this result, concentrated VHSV was inoculated in Epithelioma papulosum cyprini (EPC) cells to confirm cell viability, viral gene expression, and extracellular viral titer. All results demonstrated that oxalic acid buffer was superior to ascorbic acid buffer in preserving viral infectivity. [ABSTRACT FROM AUTHOR]

Virus infection activates integrated stress response (ISR) and stress granule (SG) formation and viruses counteract by interfering with SG assembly, suggesting an important role in antiviral defense. The infection of fish cells by Viral Hemorrhagic Septicemia Virus (VHSV), activates the innate immune recognition pathway and the production of type I interferon (IFN). However, the mechanisms by which VHSV interacts with ISR pathway regulating SG formation is poorly understood. Here, we demonstrate that fish cells respond to heat shock, oxidative stress and VHSV infection by forming SG that localized key SG marker, Ras GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1). We show that PKR-like endoplasmic reticulum kinase (PERK), but not (dsRNA)-dependent protein kinase (PKR), is required for VHSV-induced SG formation. Furthermore, in VHSV Ia infected cells, PERK activity is required for IFN production, antiviral signaling and viral replication. SG formation required active virus replication as individual VHSV Ia proteins or inactive virus did not induce SG. Cells lacking G3BP1 produced increased IFN, antiviral genes and viral mRNA, however viral protein synthesis and viral titers were reduced. We show a critical role of the activation of ISR pathway and SG formation highlighting a novel role of G3BP1 in regulating VHSV protein translation and replication. [ABSTRACT FROM AUTHOR]

Genomic prediction utilizes relationships between phenotypes and thousands of genetic markers dispersed across the genome of a species under selection to estimate an individual's breeding value. Viral hemorrhagic septicemia (VHS) is a devastating disease that causes high mortality in the olive flounder (Paralichthys olivaceus). Selection of VHS resistant individuals using traditional pedigree-based approaches is slow and ineffective; therefore, we investigated the potential of genomic selection to identify superior VHS-resistant individuals. In this study, various statistical models and algorithms, including the multilayer perceptron (MLP) and convolutional neural network (CNN), were compared for their prediction accuracy of breeding values. Other models assessed include pedigree-based best linear unbiased prediction (PBLUP), genomic best linear unbiased prediction (GBLUP), Bayesian A (BA), Bayesian B (BB), Bayesian C (BC), Bayesian Lasso (BL), Bayesian ridge regression (BRR), elastic net (EN), ridge regression (RR), and random forest (RF). These models were assessed for their ability to predict VHS resistance based on genomic data obtained from high-quality 70 K single nucleotide polymorphism (SNP) Affymetrix® Axiom® myDesign™ Genotyping Array from 865 animals. Furthermore, we investigated the population structure of the selected flounder population using a genomic relatedness matrix, PCA analysis, kinship coefficients, and multidimensional scaling. The results revealed that RF had the highest prediction accuracy for the three VHS-resistance traits (binary survival, days to death, and time of death), followed by BRR and GBLUP. PBLUP exhibits the lowest accuracy for these traits. Machine learning (ML) models, such as RF, outperformed traditional PBLUP and GBLUP, showing the greatest improvement over other Bayesian methods (BA, BB, and BC). The optimal parameters were determined for the best models, including specific marker sizes and sample size recommendations. The selected models and their parameters significantly improved the prediction of VHS resistance, demonstrating the potential of genomic selection to outperform the traditional pedigree-based methods. Our findings indicate that genomic selection can be more effective using ML models than conventional approaches in predicting VHS resistance and offers a means to enhance the genetic resistance of olive flounder in aquaculture to this disease. • Identifying disease resistance traits are necessary for breeding programs to create better animals. • A 70 K high density SNP array was used in the genomic prediction for VHSV traits. • The prediction accuracy ranged from 0.53 to 0.69 (lowest for PBLUP and highest for RF and CNN) • Machine learning models outperformed traditional PBLUP, GBLUP, and Bayesian models. • Increasing marker and population numbers also increase the genomic prediction ability. [ABSTRACT FROM AUTHOR]

In this study, the Peroxiredoxin-5 (PRDX5) gene from starry flounder (Platichthys stellatus , PsPRDX5) was identified through next-generation sequencing (NGS) and verified via cloning and sequencing. The predicted amino acid sequence, derived from the cDNA, was confirmed by multiple sequence alignment and phylogenetic analysis, establishing its homology with related sequences. PsPRDX5 mRNA was expressed across all examined tissues in healthy starry flounder, with distinct tissue-specific expression profiles. Following artificial infection with viral hemorrhagic septicemia virus (VHSV) and Streptococcus parauberis PH0710, significant alterations in PsPRDX5 mRNA expression were observed. VHSV infection resulted in a pronounced downregulation of PsPRDX5 in most major tissues, except for a significant upregulation in the intestine at 7 days post-infection. Conversely, infection with S. parauberis PH0710 led to a significant upregulation of PsPRDX5 in nearly all tissues. These results suggest that the differential expression of PsPRDX5 in response to VHSV and S. parauberis PH0710 reflects pathogen-specific immune and antioxidant defense mechanisms, highlighting the role of PsPRDX5 in the immune response of starry flounder to both viral and bacterial infections. • PRDX5 was identified from starry flounder and its expression was detected in all tissues examined. • Tissue- and gene-specific expression levels of PRDX5 mRNA were confirmed in healthy starry flounder. • PsPRDX5 mRNA expression was significantly regulated by S. parauberis PH0710 and VHSV infections. [ABSTRACT FROM AUTHOR]

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii ; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi , AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H 2 O 2 -treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii. • The membrane-associated Ring-CH5 (MARCH5) from Amphiprion clarkii (AcMARCH5) was identified and characterized. • Temporal expression of AcMARCH5 in blood, spleen and head kidney is drastically modulated by various immune stimuli. • AcMARCH5 overexpression suppressed antiviral gene expression and promoted VHSV viral replication. • AcMARCH5 overexpression reduced pro-inflammatory cytokine production in Poly I:C stimulated macrophages. • AcMARCH5 showed an anti-apoptotic property during H 2 O 2 induced oxidative stress. [ABSTRACT FROM AUTHOR]

Viral hemorrhagic septicemia virus (VHSV) is a pathogen that causes hemorrhagic septicemia in olive flounder at water temperatures below 15 °C, leading to symptoms such as abdominal swelling due to ascites and muscle hemorrhaging, and in severe cases, mortality. In this study, we investigated the proliferation of NCCs, the transcriptional analysis of CD4 and CD8, and the expression of pro-inflammatory cytokines (IL-1β, IL6, TNFα) and the cytokines (IL12, IL15, IFN-1β, IFNγ) involved in cytotoxic cell activation in the kidney of olive flounder during VHSV infection at suboptimal temperature (17 °C) and following a shift to optimal temperature (10 °C). Following viral infection, the population of NCCs and CD8 gene expression steadily increased. Meanwhile, IL12, IL15, and IFN-1β levels exhibited an early surge, reaching their peak within 1–2 days post-challenge at 17 °C. Notably, in olive flounder that were re-challenged with VHSV at 10 °C after an initial challenge at 17 °C, there was a rapid and robust expansion of NCCs and increased CD4 and CD8 gene expression, demonstrating the mounting of immune memory by NCCs and an efficient adaptive immune response from CD4+ and CD8+ T cells against VHSV infection. • Olive flounder initially exposed to VHSV at 17 °C acquired resistance to VHSV upon secondary exposure at 10 °C. • Pro-inflammatory cytokines (IL-1β, IL-6) were upregulated in kidney of olive flounder reinfected with VHSV at 10 °C, then regulated after 2 days. • In olive flounder secondarily infected with VHSV at 10 °C, NCCRP-1+ cells (NCCs) appear to have been activated by IL15. • Rapid activation of NCCs and CD8+ T cells in olive flounder upon VHSV reinfection likely suppressed viral replication. [ABSTRACT FROM AUTHOR]

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay. Replacing GDNV with GDNQ showed no significant difference in luciferase activity. Further characterization using reverse genetically engineered recombinant viruses revealed that rVHSV-L GDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells than rVHSV-wild. Olive flounder infected with rVHSV-L GDNQ experienced higher early-stage mortality but lower overall mortality than those infected with rVHSV-wild. These findings suggest that while the GDNQ motif may positively influence VHSV replication speed, it may not confer an overall advantage for the ultimate viral pathogenicity. • Change of GDNV motif of VHSV L protein to GVNV resulted in completely abolishing polymerase activity. • Alteration of GDNV motif to GVNQ reduced polymerase activity but was not significant. • rVHSV-L GDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells. • rVHSV-L GDNQ induced a faster mortality rate than rVHSV-wild during early infection stages. • Final cumulative mortalities by rVHSV-L GDNQ were lower than those by rVHSV-wild. [ABSTRACT FROM AUTHOR]

Although the viral hemorrhagic septicemia virus (VHSV) is a significant aquatic disease, its shedding dynamics at different water temperatures remain insufficiently studied. In this study, we investigated VHSV infection and shedding patterns in infected olive flounder (Paralichthys olivaceus) at different water temperatures. Briefly, fish infected with VHSV were divided into two groups: (1) maintained at 8 °C, 15 °C, and 22 °C, and (2) exposed to gradually increasing (from 15 °C to 22 °C in 1 °C increments per day) and decreasing (from 22 °C to 15 °C in 1 °C decrements per day) temperatures. The spleens of five randomly selected fish and 1 L of rearing water were sampled to quantify VHSV using a previously developed quantitative polymerase chain reaction (qPCR) assay. The VHSV decay rate was assessed at 8 °C, 15 °C, and 22 °C for 14 days, and 50% tissue culture infectious dose (TCID 50) viral titers were measured. Viral loads in fish spleens increased at 8 °C and decreased at 22 °C over time. At low water temperatures, VHSV loads in spleen were higher and had a strong positive correlation with viral shedding. Notably, only fish infected with VHSV at >106 copies per spleen mg−1 seemed to shed enough virus for detection. Viral loads in fish were significantly higher in the increasing temperature group than in the decreasing temperature group, indicating that the temperature at the time of infection was an essential factor in the development of the disease. When the water temperature rose above 18 °C in the increasing temperature group, viral loads rapidly decreased, and VHSV was not detected in the rearing water. Extended virus survival, especially in colder temperatures, highlights the risk of environmental persistence and potential waterborne transmission. Our results suggest a higher likelihood of horizontal VHSV transmission in fish when the viral load exceeds 106 copies and water temperature is below 18 °C. • VHSV loads were higher at lower temperatures and exhibited a strong positive association with viral shedding. • VHSV was more pathogenic in olive flounder at increasing temperatures than at decreasing temperatures. • Horizontal VHSV transmission in olive flounder is likely at viral load > 106 copies per spleen and water temperature < 18 °C. [ABSTRACT FROM AUTHOR]

Viral vector-based vaccines are promising candidates that deliver antigens of interest into hosts. Particularly, a single-cycle virus possesses the immunogenicity of a live virus vaccine and safety of an inactivated virus vaccine because they do not produce progeny virion after infection. The rVHSV-GΔTM single-cycle, which removes the transmembrane region and C-terminal cytoplasmic region of the G gene from the KJ2008 isolate, virus demonstrates its superiority as a vaccine in controlling the viral hemorrhagic septicemia virus (VHSV) in olive flounder, although research for establishing its suitability is still lacking. Therefore, we established optimal usage through various concentrations and booster immunizations in olive flounder and confirmed the cross-protective effect against KJ2008 and GCVP-02 belonging to different subclades of the glycoprotein genotype IVa. The olive flounder were intramuscular prime or boost immunized from 1 × 102 pfu/fish to 1 × 104 pfu/fish of rVHSV-GΔTM. After four weeks of immunization, each group was challenged with KJ2008 and GCVP-02 isolates. In the prime immunization group, protective efficacy against the GCVP-02 isolate was relatively low compared with the KJ2008 isolate, but the boost immunization group showed improved protective efficacy in both isolates. Our findings suggest that the rVHSV-GΔTM single-cycle virus vaccine based on the KJ2008 isolate can be effective in controlling VHSV in the fish culture industry because it showed a protective efficacy even against other isolates and high protective efficacy upon boosting. • A single-cycle virus possesses the immunogenicity of a live virus vaccine and the safety of an inactivated virus vaccine. • The rVHSV-GΔTM single-cycle virus induced cross-protection against different VHSV genotype IVa isolates (KJ2008 and GCVP-02). • The booster immunization demonstrated improvement in notable protective efficacy compared to the prime immunization. • The rVHSV-GΔTM single-cycle virus vaccine, based on the KJ2008 isolate, can effectively control VHSV in the fish culture industry. [ABSTRACT FROM AUTHOR]

Introduction: Viral hemorrhagic septicemia virus (VHSV) is the most lethal pathogen in aquaculture, infecting more than 140 fish species in marine, estuarine, and freshwater environments. Viral hemorrhagic septicemia virus is an enveloped RNA virus that belongs to the family Rhabdoviridae and the genus Novirhabdovirus. The current study is designed to infer the worldwide Viral hemorrhagic septicemia virus isolates' genetic diversity and evolutionary dynamics based on G-gene sequences. Methods: The complete G-gene sequences of viral hemorrhagic septicemia virus were retrieved from the public repositories with known timing and geography details. Pairwise statistical analysis was performed using Arlequin. The Bayesian model-based approach implemented in STRUCTURE software was used to investigate the population genetic structure, and the phylogenetic tree was constructed using MEGA X and IQ-TREE. The natural selection analysis was assessed using different statistical approaches, including IFEL, MEME, and SLAC. Results and Discussion: The global Viral hemorrhagic septicemia virus samples are stratified into five genetically distinct subpopulations. The STRUCTURE analysis unveiled spatial clustering of genotype Ia into two distinct clusters at K = 3. However, at K = 5, the genotype Ia samples, deposited from Denmark, showed temporal distribution into two groups. The analyses unveiled that the genotype Ia samples stratified into subpopulations possibly based on spatiotemporal distribution. Several viral hemorrhagic septicemia virus samples are characterized as genetically admixed or recombinant. In addition, differential or subpopulation cluster-specific natural selection signatures were identified across the G-gene codon sites among the viral hemorrhagic septicemia virus isolates. Evidence of low recombination events elucidates that genetic mutations and positive selection events have possibly driven the observed genetic stratification of viral hemorrhagic septicemia virus samples. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a powerful innate immune mechanism to knock down translation of specific proteins whose machinery is conserved from plants to mammals. The template used to determine which mRNA's translation is inhibited is dsRNA, whose origin can range from viruses (long dsRNA, ∼100–1000s bp) to host (micro(mi)RNA, ∼20mers). While miRNA-mediated RNAi is well described in vertebrates, the ability of long dsRNA to guide RNAi-mediated translation inhibition in vertebrates is controversial. Indeed, as long dsRNA is so effective at inducing type I interferons (IFNs), and IFNs down-regulate RNAi machinery, it is believed that IFN-competent cells are not capable of using long dsRNA for RNAi. In the present study the ability of long, sequence specific dsRNA to knock down both host protein expression and viral replication is investigated in IFN-competent rainbow trout cells. Before exploring RNAi effects, the optimal dsRNA concentration that would funnel into RNAi without triggering the IFN response was determined. After which, the ability of sequence specific long dsRNA to target knockdown via RNAi was evaluated in: (1) uninfected host cells using inducible luciferase gene expression and (2) host cells infected with chum salmon reovirus (CSV), frog virus 3 (FV3) or viral hemorrhagic septicemia virus genotype IVa (VHSV-IVa). Induced expression studies utilized RTG-P1, a luciferase reporter cell line, and dsRNA containing luciferase sequence (dsRNA-Luc) or a mis-matched sequence (dsRNA-GFP), and subsequent luminescence intensity was measured. Anti-CSV studies used dsRNA-CSVseg7 and dsRNA-CSVseg10 to target CSV segment 7 and CSV segment 10 respectively. Inhibition of virus replication was measured by viral titration and RT-qPCR. Taking advantage of the fact that long dsRNA can accommodate more sequences than miRNAs, the antiviral capability of dsRNA molecules containing both CSV segment 7 and segment 10 simultaneously was also measured. Target sequence appears important, as dsRNA-FV3MCP did not knock down FV3 titres, and while dsRNA-VHSV-N knocked down VHSV-IVa, dsRNA-VHSV-G and dsRNA-VHSV-M did not. This is the first study in fish to provide evidence that sequence specific long dsRNA induces potent gene expression silencing and antiviral responses in vitro via an RNAi-like mechanism instead of an IFN-dependent response. • A long dsRNA IFN induction threshold was determined for RTG-P1 and RTG-2. • Sequence-specific long dsRNA induced potent gene expression silencing in RTG-P1 and antiviral responses in RTG-2. • Mis-matched sequence long dsRNA failed to mediate gene and viral knockdown. • This suggests that the observed knockdown is an RNAi-like mechanism instead of an IFN-dependent response. [ABSTRACT FROM AUTHOR]

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin−/− compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin−/− fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin−/− fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections. [Display omitted] • A zebrafish knockout model (KO) for the viperin gene was constructed by CRISPR/Cas9-mediated genome editing. • Zebrafish viperin expression was analyzed by in situ hybridization and cryosectioning. • Fish morphology, virus tropism and titer were analyzed in VHSV-infected KO fish. • Interferon system and neutrophile migration were studied. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent years, with the continuous development of the mariculture industry, the scale of mariculture has gradually expanded, and the problem of mariculture diseases is prominent. Among them, viral diseases and bacterial diseases are the main concerns. Japanese flounder (Paralichthys olivaceus), as the main marine aquaculture fish, has high economic value, mainly distributed in China, Korea, and Japan. At present, the viruses that are extremely harmful to Japanese flounder are Lymphocytes disease virus (LCDV), Hirame rhabdovirus (HIRRV), Viral hemorrhagic septicemia virus (VHSV), et al., were difficult to control in the event of a large-scale epidemic, causing significant economic losses to the Japanese flounder culture industry. Fish cell lines are an important research tool with the advantages of low assay conditions, accurate control of assay results, and ease of operation, and thus have been widely used in virology research. We established a new cell line Japanese flounder spleen (JFSP) from the spleen tissue of the Japanese flounder (P. olivaceus). We explored the optimal growth conditions, chromosome morphology, and number and transfection experiment of JFSP cells in vitro. The susceptibility of JFSP cells to different viruses, including Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV) and the expression changes of immune-related genes after virus infection were detected. The established JFSP cell line has enriched the fish cell resource bank and can be used as an excellent tool for genetic manipulation, studying host-virus interactions, and the development of potential vaccines. A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases. [ABSTRACT FROM AUTHOR]

Indigenous fishery harvest goals and data from locally driven wildlife research can inform ecosystem-based fisheries management, guided by cultural values of respect for and reciprocity with wildlife. Using highly conservative species-specific thresholds (mean %I of presumptive uninfected fish +2 SDs), 4.3% of Bluegill, 13.4% of Brown Trout, 19.3% of Northern Pike, and 18.3% of Walleye tested positive for VHSV antibodies by ELISA. A 10% decrease in fishing yield allows the bear population to increase by 10%, creating an ecosystem-based fisheries management plan that supports Wuikinuxv fisheries goals, which includes maintaining a sustainable bear population density. [Extracted from the article]

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout. [ABSTRACT FROM AUTHOR]

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV. • The Bcl-2 gene was identified and cloned from Amphiprion clarkii. • AcBcl-2 was highly expressed in blood, heart, and spleen, mediating host defense mechanisms. • Transcription of AcBcl-2 was modulated upon immune challenges. • The overexpressed AcBcl-2 increased the VHSV gene transcription. [ABSTRACT FROM AUTHOR]

Advances in genomics and the gradual reduction of cost for technologies like whole-genome sequencing have provided exciting opportunities for developing modern biotechnological-based vaccines in aquaculture. This systemic review describes the prospects and challenges of implementing these high-tech vaccines in fish species. The majority of the commercial vaccines in aquaculture utilize conventional procedures for which cost of administration, protective immunity and safety issues are the major challenges. In recent years, more efficient vaccines are being developed by adopting the advances in vaccine technology. Vaccines based on surface antigens, protein/peptide/polysaccharide subunits, recombinant DNA/mRNA/plasmids, novel antigen expression and delivery systems (bacteriophage particles, virus like particles/VLPs, recombinant yeast, mucosal vaccines), novel molecular adjuvants (IL-8, IL-12, HSPs), and encapsulation polymers and polysaccharides like chitosan nanoparticles and PLGA microcapsule were successfully developed. These biotechnology-based vaccines have proved to be very efficient in field trials, but are always in the research pipeline or as patents. Only very few of them are licensed for use, that too, in high-valued fishes like salmonids. Currently, commercial aquaculture vaccines are available for Aeromonas salmonicida , Vibrio salmonicida , Yersinia ruckeri , Vibrio anguillarum , Edwardsiella ictalurid , and for certain Betanodaviruses. Nevertheless, no registered vaccines are available for other major infectious diseases/pathogens such as viral hemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNN) and certain other betanodaviruses, channel catfish virus (CCV), gill disease bacteria, mycobacteria, flavobacterium, Edwardsiella tarda , and certain streptococci. Despite the important economic losses that the pathogens cause to aquaculture worldwide, the commercialization of vaccines remains limited due to immunological pitfalls in aquatic species, large-scale vaccination issues, unregulated use of antibiotics and chemicals, gene-based vaccine regulations and commercial viability. If attempts are to be made to develop novel delivery methods, cost-effective procedures, and relaxations in DNA vaccine regulations, biotechnology-based vaccination could circumvent the emerging disease challenges in aquaculture. [ABSTRACT FROM AUTHOR]

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1β1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture. • Full-length and hypervariable region-deleted flagellins from Marinobacter algicola enhanced the transcription of pro-inflammatory genes in rainbow trout cell lines. • Immunostimulation with the full-length flagellin was slightly more potent when compared to the version with the deleted hypervariable region. • VHSV infection was significantly reduced in RTgutGC cells after pre-incubation with both flagellins. • VHSV G and N gene transcription was significantly reduced after incubation with the full-length flagellin. [ABSTRACT FROM AUTHOR]

Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3—which did not express NV protein—rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60–70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity. [ABSTRACT FROM AUTHOR]

In the fish examined 18-30 h post mortem, resting spores were observed in 47 and 71 fish macroscopically and microscopically, respectively, whereas I Ichthyophonus i hyphae were observed in 43 of the fishes. A study of Northwest Atlantic mackerel from New Jersey coastal waters found that 39% of the mackerel (N = 91) contained granulomas in the kidneys, but only 5% contained identifiable I Ichthyophonus i sp. stages (Murchelano et al., 1986). Resting spores and hyphae observed microscopically were similar in size and appearance to those observed in NEA mackerel by Sproston (1944), and the histological sections showed the presence of typical I Ichthyophonus i sp. resting spores in the examined tissues (Figure 2). The presence of aseptate hyphae in combination with resting spores in NEA mackerel tissues is therefore highly indicative of infection with live I Ichthyophonus i sp. [Extracted from the article]

Treatments to control infectious diseases in aquacultured fish commonly focus on the use of antibiotics (which are ineffective against viruses) and inorganic disinfectants. Currently, antiviral treatments are not available for use in aquaculture. The use of plasmid vectors to overexpress antiviral proteins in fish has been reported, with positive results against viral infections. The aim of this work was to overexpress the antiviral proteins interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) and natural killer enhancing factor (NKEF) using plasmid vectors (pIFIT5 and pNKEF) as novel therapeutic agents against viral infections. The antiviral activity of pIFIT5 against viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) was evaluated in vitro in epithelioma papulosum cyprinid and Chinook salmon embryo cell lines. Moreover, pNKEF antiviral activity against IPNV was also evaluated. Results showed that pIFIT5 had antiviral activity against both viruses, but pNKEF did not protect against IPNV. In addition, we evaluated the use of pIFIT5 and pNKEF constructs as therapeutics in rainbow trout. Biodistribution assays demonstrated systemic distribution of the IFIT5-TFP1 and NKEF-TFP1 proteins in rainbow trout injected with the plasmids, and pIFIT5 and pNKEF triggered an immune response in the injected fish. In a pilot study, we evaluated the level of protection conferred by pIFIT5 and pNKEF against VHSV in rainbow trout, and we observed 10 % survival for pIFIT5 compared to pTFP. Together, these results suggest the potential of pIFIT5 as therapeutic agent in aquaculture. • Overexpression of proteins IFIT5 and NKEF using plasmid vectors as antivirals. • pIFIT5 exerts antiviral activity against two different kind of viruses in vitro. • pIFIT5 and pNKEF triggered an immune response in vivo. • The antiviral activity of pIFIT5 and pNKEF against VHSV was probed in rainbow trout. [ABSTRACT FROM AUTHOR]

Water temperature is a crucial factor for adaptive immune responses in fish vaccinology, wherein the immune effects of vaccines are time × temperature (degree-days) dependent i.e. the higher the water temperature, the faster the fish develop protective immunity in response to the vaccine. In contrast, at low water temperature there happened to be reduced or no protective efficacy of vaccines as reported by several studies. Previously, we developed a squalene and aluminium hydroxide (Sq + Al) – based inactivated viral hemorrhagic septicemia virus (VHSV) vaccine, which showed good promises as a potential immunoprotective agent against VHSV infection in olive flounder (Paralichthys olivaceus) with >50% relative percent survival (RPS) at 3 to 40 weeks post-vaccination. In continuation, the present study investigated the role of the ambient water temperatures (at 10, 13, 15, and 20 °C) and immunization periods (10, 20, 30, and 40 days post-vaccination) on the development of protective efficacy of the developed (Sq + Al) – based vaccine in olive flounder using three in vivo experimental trials. The results showed fish immunized at 20 °C obtained significantly high protection starting at 10 dpv (200 degree-days) with a RPS of 86.67% (trial I), 66.67% (trial II), and 90.89% (trial III). Challenge tests at 20, 30, and 40 dpv also conferred a high level of protection when the fish were immunized at 20 °C. Moreover, immunization at 15 and 13 °C showed the best results, the RPS at 20, 30, and 40 dpv were 88.07–90.96% at 15 °C and 87.89–72.84% at 13 °C for trial I and II, respectively. Interestingly, olive flounder immunized at 10 °C also exhibited 20.00–80.02% RPS at 10–40 dpv, however in relative terms the protection was lower and slower as compared to immunization at 13, 15, and 20 °C. In addition, the study also displayed significant upregulation of various immune genes and higher specific antibody titers in the fish immunized both at 10 °C as well as 20 °C (trial III). Further, the vaccine groups showed comparatively high protection when 300 degree-days were reached in the respective groups irrespective of the temperature at which immunization was carried out. Thus, the present study indicates useful guidelines for sufficient time requirement of the (Sq + Al) – based inactivated VHSV vaccine to show its full protective efficacy against the virus in olive flounder. • Adjuvanted VHSV vaccine conferred protection at 300 degree days over 10–20 °C. • It provided significantly high protection from 10 dpv at 20 °C (200 degree days). • Vaccine efficacy increased with antibody titer at suboptimal water temperature (10 °C). [ABSTRACT FROM AUTHOR]

The study of mussels (Mytilus galloprovincialis) has grown in importance in recent years due to their high economic value and resistance to pathogens. Because of the biological characteristics revealed by mussel genome sequencing, this species is a valuable research model. The high genomic variability and diversity, particularly in immune genes, may be responsible for their resistance to pathogens found in seawater and continuously filtered and internalized by them. These facts, combined with the lack of proven mussel susceptibility to viruses in comparison to other bivalves such as oysters, result in a lack of studies on mussel antiviral response. We used RNA-seq to examine the genomic response of mussel hemocytes after they were exposed to poly I:C, simulating immune cell contact with viral dsRNA. Apoptosis and the molecular axis IRFs/STING-IFI44/IRGC1 were identified as the two main pathways in charge of the response but we also found a modulation of lncRNAs. Finally, in order to obtain new information about the response of mussels to putative natural challenges, we used VHSV virus (Viral Hemorrhagic Septicemia Virus) to run some functional analysis and confirm poly I:C's activity as an immunomodulator in a VHSV waterborne stimulation. Both, poly I:C as well as an injury stimulus (filtered sea water injection) accelerated the viral clearance by hemocytes and altered the expression of several immune genes, including IL-17, IRF1 and viperin. [Display omitted] • Poly I:C acts as an antiviral immunostimulant in mussels, activating apoptosis pathways and IRFs/STING-IFI44/IRGC1. • The response is specific and different from that induced by a VHSV. • A network of interactions between lncRNAs and coding genes (IFI44, TRIM56, DHX40 and IRGC) has been identified. • Poly I:C enhances hemocytes' ability in removing viral particles. • Poly I:C leads changes in gene expression after a VHSV waterborne infection. [ABSTRACT FROM AUTHOR]

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H 2 O 2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara. • Interferon-related developmental regulator 1 (IFRD1) in the red spotted grouper was identified and characterized. • EaIFRD1 mRNA expression upregulated significantly in peripheral blood, liver, and spleen cells after the immune challenge. • EaIFRD1 upregulates the wound-healing activity in FHM cells. • EaIFRD1 increased the cell viability under oxidative stress in FHM cells and reduced NO production in macrophages. • Overexpression of EaIFRD1 activates ISGs and reduces VHSV replication. [ABSTRACT FROM AUTHOR]

Despite the growing emphasis on aquatic disease monitoring via environmental DNA (eDNA) in water samples, detecting low concentrations of viruses in aquatic environments remains challenging. Prior studies demonstrated that plankton act as mechanical vectors for several aquatic viral pathogens but not for the viral hemorrhagic septicemia virus (VHSV). In this study, we aimed to identify the potential interaction between VHSV and microplankton and further assess the effectiveness of the water concentration method using a plankton net for VHSV detection in water. Briefly, VHSV was co-cultured with Chlorella , rotifer, and microcosms generated using plankton nets with sieve sizes of 20, 40, and 70 μm. A close interaction between VHSV and rotifer and microplankton collected with the 70 μm-plankton net with an abundant zooplankton population was found, likely influenced by the filter-feeding behaviors of zooplankton. We also found that VHSV could adsorb to fish feces at concentrations 25–88 times higher than that in the equivalent volume of seawater. Subsequently, we evaluated the efficiency of a 70 μm-plankton net for VHSV concentration in rearing water containing infected fish compared to the iron flocculation method. Notably, the plankton net outperformed the iron flocculation method in terms of sensitivity (∼10 to 20-fold), possibly due to its ability to capture fish-derived organic materials, such as feces, shed from infected fish. Our overall findings advocate for the plankton net concentration approach, not only for its pronounced sensitivity but also its operational simplicity, identifying it as a promising avenue for efficient VHSV detection in aquaculture water monitoring. • This study demonstrates an association between VHSV and microplankton. • Concentration of rearing water using a plankton net outperformed iron-flocculation in terms of viral detection. • The plankton net method, with its operational simplicity, can be an effective eDNA tool for monitoring aquatic diseases. [ABSTRACT FROM AUTHOR]

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC 50 of 100 μg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 μg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1β, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed. • Umbelliferone exhibits strong direct virucidal activity against VHSV. • Umbelliferone-medicated feed prevents VHS in olive flounder. • The anti-VHSV effects occur via TNF-α, IL-1β, IFN I, and caspase 3 inhibition. • Antiviral effects were also linked to IL-8, IFN II, and ISG15 upregulation. [ABSTRACT FROM AUTHOR]

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3 -KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3- KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3 -KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3 -KO after 5 dpi, indicating irf7 's control over ifnphi3 expression. The significantly reduced expression of isg15 , viperin , mxa , and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3 -KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3 -KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes. • The irf3 mutant zebrafish was generated using CRISPR/Cas9-mediated genome editing. • The irf3 knockout zebrafish showed increased susceptibility to VHSV infection. • Irf3 depletion significantly affected the transcription of ifnphi1 and ifnphi2 upon VHSV challenge. [ABSTRACT FROM AUTHOR]

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry. • Formalin treatment (0.05 %, 16 °C, 48 h) effectively inactivated VHSV. • Vaccinated fish showed 99 % increased survival and elevated serum antibodies. • RNA sequencing unveiled inactivated VHSV vaccine could boost strong immune response. [ABSTRACT FROM AUTHOR]

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51–96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus. • EFEMP2 cDNA was characterized in Paralichthys olivaceus. • PoEFEMP2 gene was predominantly expressed in the heart, testis, ovary and muscle. • PoEFEMP2 mRNA level initially increased and then decreased in VHSV-infected HINAE cells. • VHSV G mRNA expression was upregulated in PoEFEMP2-disrupted HINAE cells. [ABSTRACT FROM AUTHOR]

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet. • Expression of PhGal8X1 increased in response to Poly I:C, LPS, and L. garvieae stimulation. • rPhGal8X1 agglutinated both Gram-positive and Gram-negative bacteria by directly binding to bacterial glycans. • PhGal8X1 restricted VHSV replication through upregulation of pro-inflammatory cytokines and chemokines. • Overexpression of PhGal8X1 exerted protective effects against H 2 O 2 -induced oxidative stress. • PhGal8X1 demonstrated regulatory effects on enzymatic antioxidant defense mechanisms in teleosts. [ABSTRACT FROM AUTHOR]