Viral Hemorrhagic Septicemia Research Articles

Poly (I:C)-Potentiated Vaccination Enhances T Cell Response in Olive Flounder (Paralichthys olivaceus) Providing Protection against Viral Hemorrhagic Septicemia Virus (VHSV).

Viral hemorrhagic septicemia (VHS), caused by viral hemorrhagic septicemia virus (VHSV), is a viral disease affecting teleosts, and is the major cause of virus-related deaths in olive flounder (Paralichthys olivaceus). Research has focused on ways to control VHS, and recently, the use of polyinosinic-polycytidylic acid poly (I:C)-potentiated vaccination has been investigated, whereby fish are injected with poly (I:C) and then with live pathogenic virus, resulting in a significant decrease in VHSV-related mortality. T cell responses were investigated in the present study after vaccinating olive flounder with poly (I:C)-potentiated vaccination to understand the ability of poly (I:C) to induce T cell immunity. Stimulation of T cell responses with the poly (I:C)-potentiated vaccination was confirmed by examining levels of CD3+ T cells, CD4-1+ T cells and CD4-2+ T cells. Higher levels of CD4-2+ T cells were found in vaccinated fish than CD4-1+ T cells, believed to result from a synergistic effect between poly (I:C) administration and pathogenic VHSV immunization. More importantly, the role of CD4-2+ T cells in the antiviral response was clearly evident. The results of this study suggest that the outstanding protection obtained with the poly (I:C)-potentiated vaccination is due to the robust immune response initiated by the CD4-2+ T cells.

Virological screening of wild and farmed fish species: Application of innovative Taqman multiplex real-time PCR protocols

Global demand for seafood is growing and fish stock maintenance becomes a pressing issue, making aquaculture production of capital importance in terms of fish resources sustainability. Therefore, fish disease outbreaks are a significant constraint to fisheries trade and aquaculture production, affecting economic development worldwide. In this study, a virological screening in wild and farmed fish populations was performed by two TaqMan multiplex real-time PCR assays developed to identify/quantify, in a single run, four viruses responsible for major economic losses in fisheries, namely: infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) and viral nervous necrosis virus (VNNV). A total of 323 fish were assessed, being the majority from four species from the Atlantic coast: gilthead seabream and seabass, two extensively farmed species in aquaculture and captured in the wild, and sardine and Atlantic horse mackerel, two of the most consumed fish species in Portugal. The genomes of IPNV (genogroup II) and VHSV (genogroup Ie) were detected in one pool of gilthead seabream and one pool of Atlantic horse mackerel, confirming the importance of molecular tools for viral screening, especially in asymptomatic hosts, since viral infections may impart major economic losses to the fish industry.

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV).

Simple SummaryIn the present report will be the first of a series of PCR-based macroarrays for the detection, typing, and quantification of several viruses of importance in fish aquaculture. The way of preparing the array—under patent process—differs from macroarrays reported by other researchers because it ensures its reliability after storage times up to 1 year at −25 °C. In this study, we show the results of the evaluation of a macroarray designed for the viral hemorrhagic septicemia virus (VHSV), of great importance for fish farmers worldwide, and the same procedure of validation is ongoing with macroarrays for other fish viruses.The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5–50 TCID50/mL), analytical specificity (intra and intergroup), efficiency (E = 100.0–101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R2 < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the ‘binary multiplex RT-qPCR system (bmRT-qPCR)’ previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at −25 °C for three months and up to one year before their use, without significant loss of efficiency.

Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting

Several reverse genetics systems for viral haemorrhagic septicaemia virus (VHSV) have been developed over the last decade. These systems have been based on genotype Ia, IVa and IVb isolates and have used the fish cell line EPC, which is less susceptible to some VHSV isolates belonging to genotype I and genotypes II and III. While developing a reverse genetics system in our laboratories for VHSV genotype Ib, we realized that the isolate in interest (SE SVA 1033 9C) did not grow in EPC cells and it was necessary to adapt the reverse genetics protocols to the BF-2 fish cell line. This cell line is very sensitive to high temperatures and is therefore not compatible with the original protocols based on the use of recombinant vaccinia virus (vTF7-3) as a provider of the T7 RNA polymerase (T7-RNAP) to the system, which includes incubation periods at 37 °C. Transfection efficiency was assessed in BF-2 cells using a reporter plasmid and it showed to be highest when using Lipofectamine™ 3000 compared to other transfection reagents. A luciferase assay was performed to determine the optimal activity of T7-RNAP in BF-2 cells with different amounts of vTF7-3. We successfully recovered recombinant VHSV (rVHSV) in BF-2 cells by reducing the incubation time at 37 °C after transfection to both 3 and 6 h. Another strategy we attempted successfully was to transfect mammalian BHK-21 cells, which are routinely used to propagate vTF7-3, and after the 37 °C incubation period, a BF-2 cell suspension was added hypothesizing that the virions formed in the transfected mammalian cells would infect the subsequently added fish cells at 15 °C incubation over the following days. We have successfully recovered rVHSV from both BHK-21 with a BF-2 cells suspension as well as a new protocol for VHSV reverse genetics in BF-2 cells has been established.

Replication of heterologous glycoprotein-expressing chimeric recombinant snakehead rhabdoviruses (rSHRVs) and viral hemorrhagic septicemia viruses (rVHSVs) at different temperatures

Water temperature is an important environmental factor for the outbreaks of fish rhabdovirus diseases. In the present study, to know the role of piscine rhabdoviral glycoproteins in the determination of replication temperature, several chimeric snakehead rhabdoviruses (SHRVs) and viral hemorrhagic septicemia viruses (VHSVs) expressing heterologous glycoproteins (rSHRV-Gvhsv, SHRV expressing VHSV G protein; rSHRV-Gsvcv, SHRV expressing spring viremia of carp virus G protein; rVHSV-Gshrv, VHSV expressing SHRV G protein; rVHSV-Gsvcv, VHSV expressing SVCV G protein) were generated using reverse genetics, and their replication characteristics at different temperatures were investigated. Furthermore, based on SHRV minigenome containing a reporter gene, the role of VHSV N, P, and L proteins in the determination of VHSV’s low-temperature replication was investigated. In Epithelioma papulosum cyprini (EPC) cells, rSHRV-Gvhsv could replicate only at low temperatures (15 and 20 °C) but not at 25 and 28 °C, while rSHRV-Gsvcv could replicate both low and high temperatures, indicating that VHSV G protein is a critical factor that determines the limit of replication-possible temperatures in VHSV. The range of replication-possible temperature of chimeric VHSVs (rVHSV-Gshrv and rVHSV-Gsvcv) was not different from that of rVHSV-wild (replicated only at 15 and 20 °C) in spite of having the G protein of high temperature-replicating viruses, suggesting that not only G protein but also other viral protein(s) would be involved in the determination of replication-possible temperature limit in VHSV. Cells harboring SHRV minigenome that expressing eGFP as a reporter protein were co-transfected with heterologous combinations of helper plasmids of SHRV and VHSV, through which we could exclude VHSV N and P proteins for the low-temperature replication of VHSV, because cells harboring SHRV minigenome showed fluorescence at high temperatures when VHSV N or P protein encoding plasmids were supplied. However, no fluorescence was observed in cells co-transfected with plasmids encoding SHRV N, SHRV P and VHSV L protein at all tested temperatures, suggesting that the combination of SHRV N, P and VHSV L proteins could not form a functional ribonucleoprotein (RNP) complex. Although we could not directly demonstrate the involvement of VHSV L protein in the temperature limit of VHSV replication, it is highly probable that not only VHSV G protein but also VHSV L protein may participate in the determination of VHSV replication temperature.

Molecular Epidemiology of Novirhabdoviruses Emerging in Iranian Trout Farms.

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.

Widespread Seropositivity to Viral Hemorrhagic Septicemia Virus (VHSV) in Four Species of Inland Sport Fishes in Wisconsin.

Serological assays were conducted for anti-viral hemorrhagic septicemia virus (VHSV) antibodies in four species of fish in Wisconsin (Bluegill Lepomis macrochirus, Brown Trout Salmo trutta, Northern Pike Esox lucius, and Walleye Sander vitreus) to examine spatial and temporal distributions of exposure. Sera were tested for non-neutralizing anti-nucleocapsid antibodies to VHSV by blocking enzyme-linked immunosorbent assay (ELISA). Results (percent inhibition [%I]) were analyzed for differences among species, across geographic distance, and among water management units. Positive fish occurred in 37 of 46 inland water bodies tested, including in water bodies far from reported outbreak events. 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. Spatial patterns of seropositivity and changes in %I between sampling years were also analyzed. These analyses explore how serology might be used to understand VHSV distribution and dynamics and ultimately to inform fisheries management.

Molecular characterization and expression analysis of Japanese flounder (Paralichthys olivaceus) chemokine receptor CXCR2 in comparison with CXCR1

Chemokines are categorized into five families; one of the families is the CXC chemokines, which are critical in the pro-inflammatory process. CXC chemokines transmit signals and mediate a cell's biological activities by binding to cell surface receptors known as chemokine receptors (CXCRs). In this study, the CXCR2 from Japanese flounder (Paralichthys olivaceus) (JfCXCR2) was identified and characterized at the molecular level. The JfCXCR2 gene has a 1077 bp open reading frame that encodes a protein of 359 amino acid residues with seven transmembrane domains. Phylogenetic analysis of JfCXCR2 revealed that it belonged to the fish CXCR2 subfamily. Furthermore, JfCXCR2 was compared with the previously identified Japanese flounder CXCR1 (JfCXCR1). The expression analysis of uninfected Japanese flounder showed that JfCXCR1 and JfCXCR2 were expressed in all the tissues and organs tested but mainly in immune-related organs, including the kidney and spleen. Infection by Streptococcus iniae significantly increased the level of JfCXCR1 and JfCXCR2 mRNA in the kidney at days 1 and 3 post-infection. On the other hand, VHSV (viral hemorrhagic septicemia virus) and Edwardsiella tarda infection significantly increased JfCXCR2 mRNA levels in the kidney at days 3 and 6 post-infection, respectively. Conversely, JfCXCR1 expression was not significantly changed by either E. tarda or VHSV infection. Additionally, the peripheral blood leukocytes (PBLs) stimulated by recombinant proteins rCXCL8_L1a and rCXCL8_L1b were found to have significantly increased levels of JfCXCR1 and JfCXCR2 mRNA. Interestingly, even higher levels of JfCXCR1 and JfCXCR2 expression were observed in PBLs stimulated with rCXCL8_L1a and rCXCL8_L1b than in PBLs stimulated with either recombinant protein. These data suggest that bacterial infections may activate JfCXCR1. By contrast, JfCXCR2 may be activated by both bacterial and viral infection to mediate the immune response. These data can contribute to further understanding the functions of CXCR1 and CXCR2 in the fish immune system.

Antiviral Actions of 25-Hydroxycholesterol in Fish Vary With the Virus-Host Combination.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

Development and validation of a lateral flow immunochromatographic assay for specific detection of viral hemorrhagic septicemia virus (VHSV, genotype IVa) in olive flounder (Paralichthys olivaceus)

A lateral flow immunochromatographic assay (LFIA) for the detection of viral hemorrhagic septicemia virus (VHSV, genotype IVa) was developed using seven anti-VHSV monoclonal antibodies (mAbs). The mAb 15E10 was not only used to bio-conjugate the VHSV antigen with colloidal gold nanoparticles but also as a capture antibody on the test line. The detection limit of the LFIA with VHSV isolates (genotype I-IV) was in the range of 105.05 to 105.55 TCID50/100 μL without any cross-reactivity with other fish viruses. Field-level validation of the assay using the heart of VHSV-infected and non-infected olive flounders (Paralichthys olivaceus) showed a specificity of 100% and sensitivity of 93.9% with a diagnostic effectiveness of 97.3%. The LFIA facilitates rapid detection of VHSV (genotype IVa) in olive flounders in farming facilities.

Development of an electrochemical genosensor for detection of viral hemorrhagic septicemia virus (VHSV) using glycoprotein (G) gene probe

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of a highly contagious disease of both saltwater and freshwater fish around the world. VHSV, a member of viral Rhabdoviridae family, listed as a notifiable pathogen by the World Organization for Animal Health (OIE). This study was aimed to fabricate an electrochemical DNA genosensor for detection of the VHSV Glycoprotein gene. Immobilization of the DNA probe was carried out through self-assembly by thiol binding on RGO/Au-nanocomposite-modified pencil graphite electrode (PGE). The modified PGE was characterized by field emission scanning electron microscope (FESEM). The hybridization between the target sequences and the probe was analyzed by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods. Detection in these methods was performed by a decrease in the current and an increase in the charge transfer resistance (Rct) followed by target nucleic acids hybridization with biosensor probe. Genosensor sensitivity was tested by some mismatched sequences by 1, 2, and 3 bases in the methylene blue solution. The limit of detection (LOD) was at 125 pM of DNA target within the linear range of 10−4 to 10−10 M. The selectivity, stability, regeneration, and reproducibility of the fabricated electrochemical DNA biosensor were acceptable. A pilot analysis on real samples confirmed its applicability. Altogether, the genosensor can be suggested for direct detection of negative sense RNA genome of VHSV because of its fast assay, and high sensitivity.

Comparison of immune response between Escherichia coli ‐derived recombinant subunit vaccine and formol‐inactivated whole particle vaccine against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout

Comparison of immune response between <i>Escherichia coli</i> ‐derived recombinant subunit vaccine and formol‐inactivated whole particle vaccine against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

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

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

Efficacy of an optimized extract from licorice roots ( Glycyrrhiza uralensis fischer) against viral hemorrhagic septicemia virus in olive flounder ( Paralichthys olivaceus )

Viral hemorrhagic septicemia causes significant economic losses for olive flounder (Paralichthys olivaceus) farms in Korea. The purpose of this study was to optimize extraction conditions for licorice (Glycyrrhiza uralensis roots) extract using the 23 full factorial design and thereby optimizing the antiviral activity against viral hemorrhagic septicemia virus (VHSV). The optimum conditions were ethanol concentration 100%, extraction temperature 40˚C and extraction time 5 h. Optimized extract (OE) prepared under these conditions showed potent antiviral activity against VHSV with a 95.2% CPE reduction at 75 µg/ml of OE, and a predictive capacity of 94%. Fish that received 250 and 500 mg/kg body weight/day of OE as either preventive or therapeutic administration had higher relative survival rates of 25 to 62.5% (p < 0.05) compared with VHSV-infected control group. Active components of licorice, glycyrrhizin (GL) and glycyrrhetinic acid (GLA) showed antiviral activity against VHSV with 50% effective concentration values of 100.6 and 26.0 µM respectively. Besides, time course studies by plaque reduction assay showed that GLA exhibited a direct virucidal effect on VHSV or may inhibit the penetration and internalization of the virus to cells. These results suggest that OE may be used as an anti-VHSV agent in aquaculture.

Establishment of recombinase polymerase amplification technology for detecting Viral Hemorrhagic Septicemia Virus (VHSV) in cultured aquatic animals

We established a recombinase polymerase amplification (RPA) assay for the rapid detection of viral hemorrhagic septicemia virus (VHSV) using primers designed to the N gene in this species. Optimization experiments showed that the optimal amplification temperature of the RPA assay was 37 °C and that the reaction could be completed within only 15 min. Specificity tests showed that the RPA assay did not exhibit any cross-reactivity with other infective fish viruses. Sensitivity tests further showed that the detection limit of the new RPA assay was 8.3 copies/μL, indicating that this assay was more sensitive than traditional RT-PCR method. The method was verified by detecting VHSV in 1924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method met the detection requirements for aquatic animal diseases. Collectively, our findings indicate that the novel RPA assay is fast, simple, specific, sensitive, and has significant potential for the clinic, on-site testing, and a wide range of other applications.

Computational study of zebrafish immune-targeted microarray data for prediction of preventive drug candidates.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus reported to cause economic loss in fish farms. Because of the lack of adequate preventative treatments, the identification of multipath genes involved in VHS infection might be an alternative to explore the possibility of using drugs for the seasonal prevention of this fish disease. We propose labeling a category of drug molecules by further classification and interpretation of the Drug Gene Interaction Database using gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment scores. The study investigated disease networks of up-and down-regulated genes to find those with high interaction as substantial genes in pathways among the different disease networks. We prioritized these genes based on their relationship to those associated with VHS infection in the context of human protein-protein interaction networks and disease pathways. Among the 29 genes as potential drug targets, nine were selected as promising druggable genes (ERBB2, FGFR3, ITGA2B, MAP2K1, NGF, NTRK1, PDGFRA, SCN2B, and SERPINC1). PDGFRA is the most important druggable up-and down-regulated gene and is considered an important gene in the IMATINIB pathway. This study findings indicate a promising approach for drug target prediction for VHS treatment, which might be useful for disease therapeutics.

An expert‐based risk ranking framework for assessing potential pathogens in the live baitfish trade

As global trade of live animals expands, there is increasing need to assess the risks of invasive organisms, including pathogens, that can accompany these translocations. The movement and release of live baitfish by recreational anglers has been identified as a particularly high‐risk pathway for the spread of aquatic diseases in the United States. To provide risk‐based decision support for preventing and managing disease invasions from baitfish release, we developed a hazard identification and ranking tool to identify the pathogens that pose the highest risk to wild fish via this pathway. We created a screening protocol and semi‐quantitative stochastic risk ranking framework, combining published data with expert elicitation (n = 25) and applied the framework to identify high‐priority pathogens for the bait supply in Minnesota, USA. Normalized scores were developed for seven risk criteria (likelihood of transfer, prevalence in bait supply, likelihood of colonization, current distribution, economic impact if established, ecological impact if established and host species) to characterize a pathogen's ability to persist in the bait supply and cause impacts to wild fish species of concern. The generalist macroparasite Schizocotyle acheilognathi was identified as presenting highest overall threat, followed by the microsporidian Ovipleistophora ovariae, and viral haemorrhagic septicaemia virus. Our findings provide risk‐based decision support for managers charged with maintaining both the recreational fishing industry and sustainable, healthy natural resources. Particularly, the identification of several high‐risk but currently unregulated pathogens suggests that focusing risk management on pathogens of concern in all potential host species could reduce disease introduction risk. The ranking process, implemented here for a single state case study, provides a conceptual framework for integrating expert opinion and sparse available data that could be scaled up and applied across jurisdictions to inform risk‐based management of the live baitfish pathway.

Bacillus subtilis Inhibits Viral Hemorrhagic Septicemia Virus Infection in Olive Flounder (Paralichthys olivaceus) Intestinal Epithelial Cells

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that infects a wide range of host fish species causing high economic losses in aquaculture. Epithelial cells in mucosal organs are target sites for VHSV entry into fish. To protect fish against VHSV infection, there is a need to develop antiviral compounds able to prevent establishment of infection at portals of virus entry into fish. Bacillus subtilis is a probiotic with excellent antiviral properties, of which one of its secretions, surfactin, has been shown to inhibit viral infections in mammals. Herein, we demonstrate its ability to prevent VHSV infection in olive flounder (Paralichthys olivaceus) intestinal epithelial cells (IECs) and infection in internal organs. Our findings show inhibition of VHSV infection in IECs by B. subtilis and surfactin. In addition, our findings showed inhibition of VHSV in Epithelioma Papulosum Cyprini (EPC) cells inoculated with intestinal homogenates from the fish pretreated with B. subtilis by oral exposure, while the untreated fish had cytopathic effects (CPE) caused by VHSV infection in the intestines at 48 h after the VHSV challenge. At 96 h post-challenge, samples from the untreated fish had CPE from head kidney and spleen homogenates and no CPE were observed in the intestinal homogenates, while the B. subtilis-pretreated fish had no CPE in all organs. These findings demonstrate that inhibition of VHSV infection at portals of virus entry in the intestines culminated in prevention of infection in internal organs. In summary, our results show that B. subtilis has the potential to prevent VHSV infection in fish and that its use as a probiotic in aquaculture has the potential to serve as an antiviral therapeutic agent against different viral infections.

Molecular characterization, immune and xenobiotic responses of glutathione S-transferase omega 1 from the big-belly seahorse: Novel insights into antiviral defense

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.