MK-0608 inhibits in vitro and in vivo RNA replication of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus (IPNV) is an important pathogen that is threatening the worldwide salmon and trout industry. But there is no therapeutic drug available for now. In this study, we demonstrate that MK-0608 is highly efficient against IPNV and low cytotoxic, with a 50 % effective concentration (EC50) of 0.20 µM and selectivity index (SI) of about 268. Time of addition assay illustrated that MK-0608 targeted the early stage of IPNV life cycle. Furthermore, we found that MK-0608 blocked IPNV attachment on the premise of sufficient pre-incubation time but MK-0608 did not influence viral internalization and release. MK-0608 could inhibit IPNV genome synthesis, and combination with ribavirin enhanced the inhibition effect, which might be functional via binding to IPNV RNA dependent RNA polymerase (RdRp), which was predicted by using molecular docking methods. In vivo test showed that IPNV was extremely suppressed in the rainbow trout (Oncorhynchus mykiss) with one single dose of MK-0608, and the higher dosage of 50 mg/kg could cause 3 log decrease of IPNV loads in fish tissues.

Atlantic salmon type I interferon genes revisited.

Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.

In-vitro immunomodulatory responses and antiviral activities of antimicrobial peptide octominin against fish pathogenic viruses.

Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 µg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 µg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 µg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 µg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.

Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48 h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and + 38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

Characterization of the activity of 2'-C- methylcytidine against infectious pancreatic necrosis virus replication.

Infectious pancreatic necrosis virus (IPNV) is the pathogen of infectious pancreatic necrosis (IPN), which can cause high mortality in salmonids, harm the healthy development of salmon-trout aquaculture, and lead to huge economic losses. However, in China, there is currently neither a commercially available vaccine to prevent IPNV infection nor antiviral drugs to treat IPNV infection. The genome of IPNV consists of two segments of dsRNA named A and B. Segment B encodes the RNA-dependent RNA-polymerase (RdRp) VP1 which is essential for viral RNA replication and is therefore considered an important target for the development of antiviral drugs. In this study, we investigate whether 2'-C-methylcytidine (2CMC), a nucleoside analog which target viral polymerases, has an inhibitory effect on IPNV both in vitro and in vivo. The results show that 2CMC inhibits IPNV infection by inhibiting viral RNA replication rather than viral internalization or attachment. In vivo experiment results showed that 2CMC could inhibit viral RNA replication and reduce viral load in rainbow trout (Oncorhynchus mykiss). In our study, we have revealed that 2CMC has a potent inhibitory effect against IPNV infection. Our data suggest that 2CMC is an attractive anti-IPNV drug candidate which will be highly valuable for the development of potential therapeutics for IPNV.

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV), belonging to the genus Aquabirnavirus within the family Birnaviridae, causes huge economic loss to the global salmonid industry every year. Recently, outbreaks of disease caused by genogroup I IPNV were found in many rainbow trout (Oncorhynchus mykiss) farms worldwide. An inactivated vaccine was prepared using a genogroup I IPNV isolate with an optimized procedure as incubation with ß-propanolactone (BPL) at the final concentration of 0.5% at room temperature for 48 h. The inactivated vaccine was used to immunize rainbow trout, and the protection efficiency was evaluated by viral loads determination, immune-related genes quantification, and neutralizing antibody tests. The viral loads in immunized rainbow trout were significantly decreased and the strongest antiviral effect was observed on 30 days post-immunization (d.p.i). The expression of innate immune-related genes IFN-1, and Mx-1 genes were significantly up-regulated on 3, 7, and 15 d.p.i (p < 0.05), and adaptive immune-related genes CD4, CD8, and IgM genes were significantly up-regulated on 15 and 30 d.p.i (p < 0.05). Neutralizing antibodies were firstly detected on 30 d.p.i and the highest titer was observed on 45 d.p.i, which began to decrease on 60 d.p.i, but was still significantly higher than that in negative control fish. The results indicated that the vaccine prepared in this study could stimulate the non-specific and specific immune response and provide significant immune protection to the vaccinated rainbow trout.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss).

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-ß1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-ß1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus.

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. KEY POINTS: • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

Determination of VP2 sequence-based virulence motifs and phylogenetic analysis of domestic Turkish IPNV isolates.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish and is one of the most severe economic diseases in aquaculture. In Turkey, an increase in infectious pancreatic necrosis virus (IPNV) outbreaks in freshwater rainbow trout have been reported in recent years. This study aimed to analyze the VP2 gene from recent IPNV isolates from Turkey to determine whether there are epidemiological links between IPNV isolates from rainbow trout (Oncorhynchus mykiss; 62) and sea bass (Dicentrarchus labrax; 1), wild turbot (Scophthalmus maximus; 1) and the environment in order to investigate potential wild and farmed fish interactions. In this study, 62 Turkish IPNV isolates collected over 10 years (2005-2014) from rainbow trout, sea bass and turbot were genotypically characterized. The phylogenetic analysis indicated that Turkish IPNV isolates are closely related to strains from Denmark, Iran and Spain and that all Turkish IPNV isolates belong to genogroup V, serotype A2 (Sp strain). Furthermore, low genetic diversity was found among the Turkish isolates (identity, 95.5%-100% nucleotides and 97.8%-100% amino acids). The result of the analysis of the amino acid residues found at positions 217, 221 and 247 (proline, threonine and alanine, respectively) could be associated with virulence.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon.

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Inactivated infectious pancreatic necrosis virus (IPNV) vaccine and E.coli-expressed recombinant IPNV-VP2 subunit vaccine afford protection against IPNV challenge in rainbow trout.

Infectious pancreatic necrosis (IPN) is a highly contagious disease causing high mortality in juvenile trouts. Since there is no effective way to treatment against IPNV, early diagnosis and prevention play an important role in combating the disease. The different types of IPNV vaccines (inactive, live, recombinant, DNA, etc) have been produced from local isolates and have been used in developed countries. In Turkey, there is no commercial licensed vaccines against IPNV. Due to this reason, IPNV vaccine is needed in Turkey. The production of recombinant VP2 subunit vaccine (IPNV-VP2) and inactivated whole particle virus vaccine (IPNV-WPV) were attempted from selected isolate belong to sp serotype. For this purpose; the virus was produced in RTG-2 cell line and RT-PCR amplification was performed by using primers with restriction enzymes. The whole VP2 gene was cloned into a plasmid vector and VP2 was expressed by using E. coli expression system. A trial was conducted to determine the immunity ability of IPNV-VP2 and IPNV-WPV in rainbow trout. According to the SN50 assay, the IPNV-WPV stimulates immune response faster than the IPNV-VP2 vaccine. Besides, the relative percent of Survive (RPS) was detected as 79% in fish vaccinated with IPNV-WPV and 70% in fish vaccinated with IPNV-VP2. Thus, we can say that the recombinant vaccine of IPNV-VP2 is almost protected against IPNV infection as well as the inactive vaccine.

Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.

Viral Infection Drives the Regulation of Feeding Behavior Related Genes in Salmo salar.

The feeding behavior in fish is a complex activity that relies on the ability of the brain to integrate multiple signals to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Upon stress cues including viral infection or mediators such as the proinflammatory cytokines, prostaglandins, and cortisol, both Pomc and Npy/Agrp neurons from the hypothalamus are stimulated, thus triggering a response that controls both energy storage and expenditure. However, how appetite modulators or neuro-immune cues link pathogenesis and energy homeostasis in fish remains poorly understood. Here, we provide the first evidence of a molecular linkage between inflammation and food intake in Salmon salar. We show that in vivo viral challenge with infectious pancreatic necrosis virus (IPNV) impacts food consumption by activating anorexic genes such as mc4r, crf, and pomcb and 5-HT in the brain of S. salar. At the molecular level, viral infection induces an overall reduction in lipid content in the liver, favoring the production of AA and EPA associated with the increment of elovl2 gene. In addition, infection upregulates leptin signaling and inhibits insulin signaling. These changes are accompanied by a robust inflammatory response represented by the increment of Il-1b, Il-6, Tnfa, and Pge2 as well as an increased cortisol level in vivo. Thus, we propose a model in which hypothalamic neurons respond to inflammatory cytokines and stress-related molecules and interact with appetite induction/inhibition. These findings provide evidence of crosstalk between pathogenesis-driven inflammation and hypothalamic-pituitary-adrenocortical axes in stress-induced food intake behavior in fish.

Evaluation of the Antiviral Activity against Infectious Pancreatic Necrosis Virus (IPNV) of a Copper (I) Homoleptic Complex with a Coumarin as Ligand.

The aquatic infectious pancreatic necrosis virus (IPNV) causes a severe disease in farmed salmonid fish that generates great economic losses in the aquaculture industry. In the search for new tools to control the disease, in this paper we show the results obtained from the evaluation of the antiviral effect of [Cu(NN1)2](ClO4) Cu(I) complex, synthesized in our laboratory, where the NN1 ligand is a synthetic derivate of the natural compound coumarin. This complex demonstrated antiviral activity against IPNV at 5.0 and 15.0 µg/mL causing a decrease viral load 99.0% and 99.5%, respectively. The Molecular Docking studies carried out showed that the copper complex would interact with the VP2 protein, specifically in the S domain, altering the process of entry of the virus into the host cell.

Autophagy induced by infectious pancreatic necrosis virus promotes its multiplication in the Chinook salmon embryo cell line CHSE-214.

Infectious pancreatic necrosis virus (IPNV) is a common pathogen that causes huge economic losses for the salmonid aquaculture industry. Autophagy plays an important regulatory role in the invasion of pathogenic microorganisms. In this study, we explored the relationship between IPNV infection and autophagy in Chinook salmon embryo (CHSE-214) cells using standard methods. Transmission electron microscopy showed that IPNV infection produced typical structures of autophagosomes in CHSE-214 cells. Transformation of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II protein, a marker of autophagy, was observed in IPNV-infected cells using confocal fluorescence microscopy and western blot analysis. Western blotting also showed that expression of the autophagy substrate p62 was significantly decreased in IPNV-infected cells. The influence of autophagy on IPNV multiplication was further clarified with cell culture experiments using autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine. Rapamycin promoted IPNV multiplication at both the nucleic acid and protein levels, which led to higher IPNV yields; 3-methyladenine treatment had the opposite effect. This study has demonstrated that IPNV can induce autophagy, and that autophagy promotes the multiplication of IPNV in CHSE-214 cells.

Establishment of a brain cell line (SaB-1) from gilthead seabream and its application to fish virology.

Gilthead seabream (Sparus aurata) is among the most important cultured fish species in the Mediterranean area and pathogen diseases one of the bottlenecks to the aquaculture sector. For this reason, generation of laboratory tools for diagnostic and research applications would be beneficial to improve the seabream aquaculture. In this sense, we aimed to generate a seabream cell line for biological studies. Thus, we have obtained a brain-derived cell line (SaB-1) that is continuously growing for more than 4 years. Cellular characterization of the SaB-1 cells shows that they express both neural and glial cell markers, suggesting they are neural-stem cells, have a neuron-like morphology and show a rapid growth in culture. We evaluated their susceptibility to the main fish viruses: nervous necrosis virus (NNV), spring viremia carp virus (SVCV), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). SaB-1 cells are susceptible to all the tested viruses. In addition, the transcription of genes related to the type I interferon (IFN) is greatly up-regulated by the NNV infection whilst the viral infection with SVCV, IPNV or VHSV failed to do so. These data demonstrate that the seabream SaB-1 cell line is continuous, stable and could be useful, at least, for fish virology and immunity applications.

Infectious pancreatic necrosis virus inhibits infectious hematopoietic necrosis virus at the early stage of infection in a time dependent manner during Co-infection in Chinook salmon embryo cell lines.

Salmonids can be co-infected by infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) under natural or experimental conditions. To reveal the influence of IPNV on IHNV in co-infections, CHSE-214 cells were inoculated with IPNV at different time intervals prior to or after IHNV infection. Propagation of IHNV was determined by an immunofluorescence antibody test, real-time quantitative polymerase chain reaction, flow cytometry, and virus titration. The results showed that when cells were inoculated with IPNV prior to IHNV, IHNV multiplication was inhibited. This inhibitory effect became stronger with increasing time intervals (P < 0.05). When cells were inoculated with IPNV after IHNV, the inhibitory effect became weaker with increasing time intervals (P < 0.05), and no significant inhibition was observed at 12 h (P > 0.05) compared with the single IHNV infection group. The findings suggest that IHNV is inhibited at the early stage of infection by IPNV and in a time dependent manner during co-infection. Furthermore, the effect of IPNV on IHNV entry and expression of IHNV entry-related genes clathrin, dynamin-2, adaptor protein 2, and vacuolar protein sorting 35 were also determined. The results showed that IPNV did not affect the amount of IHNV entering the cells. However, the expression levels of clathrin and dynamin-2 were significantly lower in co-infection than those in single IHNV infection, which suggests that IPNV likely inhibits IHNV by affecting IHNV invasion via downregulating IHNV entry-related genes clathrin and dynamin-2.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.