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Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHμ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5′ RACE-cDNA sequencing approach to characterize modifications of the IgHμ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as ‘sashimi’. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene–aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10–17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule — IM vaccination for small-sized fish and IP vaccination for table-sized fish.

Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.

Hirame rhabdovirus (HIRRV), a member of the genus Novirhabdovirus, causes morbidity and mortality in farmed olive flounder (Paralichthys olivaceus). As no information is available on the role of the NV gene of HIRRV, we produced a recombinant HIRRV with the NV gene deleted (rHIRRV-ΔNV) using reverse genetic technology and investigated whether the NV gene knockout affected HIRRV replication and the type I interferon response of the host cell. The rescue of rHIRRV-ΔNV was successful only when IRF9-gene-knockout Epithelioma papulosum cyprini (ΔIRF9-EPC) cells were used, suggesting that the NV protein of HIRRV might be involved in inhibition of the type I interferon response of the host cell. This conclusion was also supported by the significantly higher level of Mx gene induction in EPC cells infected with rHIRRV-ΔNV than in cells infected with recombinant HIRRV without the deletion. When cells were coinfected with rHIRRV-ΔNV and either wild-type HIRRV or wild-type viral hemorrhagic septicemia virus (VHSV), there was a decrease in the growth rate of not only wild-type HIRRV but also wild-type VHSV in a concentration-dependent manner. Further studies are required to investigate the role of HIRRV NV in virulence and its possible importance for the development of attenuated vaccines.

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Currently, no effective vaccines, Food and Drug Administration-approved inhibitors, or other therapeutic intervention options are available against VHSV. α-Lipoic Acid (LA), a potent antioxidant, has been proposed to have antiviral effects against different viruses. In this study, LA (CC50 = 472.6 μmol/L) was repurposed to exhibit antiviral activity against VHSV. In fathead minnow cells, LA significantly increased the cell viability post-VHSV infection (EC50 = 42.7 μmol/L), and exerted a dose-dependent inhibitory effect on VHSV induced-plaque, cytopathic effects, and VHSV glycoprotein expression. The time-of-addition assay suggested that the antiviral activity of LA occurred at viral replication stage. Survival assay revealed that LA could significantly upregulated the survival rate of VHSV-infected largemouth bass in both co-injection (38.095% vs. 1.887%, P

To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.

Water temperature is an important factor for immune responses in poikilothermic fish. Especially, it has been known that adaptive immunity is more sensitive to temperature than innate immunity in fish. The optimal temperature for olive flounder (Paralichthys olivaceus) culture is known between 20 and 25 °C, and there are several papers reporting the low or no effectiveness of inactivated vaccines in olive flounder kept at low water temperatures. Previously, we had reported that a vaccine based on single-cycle viral hemorrhagic septicemia virus (VHSV) that was modified to produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM) induced significantly higher survival rates in olive flounder than a vaccine of rVHSV-ΔG that had no G gene in the genome. In the present study, we evaluated the availability of rVHSV-GΔTM as a protective vaccine that can be used in olive flounder at low water temperature periods. Olive flounder fingerlings were divided into 6 groups: group 1 and 2 were kept at 14 °C, group 3 and 4 were kept at 20 °C, and group 5 and 6 were kept at 14 °C for 1 week and then shifted to 20 °C. Fish in groups 1, 3, and 5 were intramuscularly (i.m.) immunized with 8.5 × 104 PFU/fish of rVHSV-GΔTM, and fish in groups of 2, 4, and 6 were i.m. Injected with L-15 alone. In the challenge test, the survival rates of fish immunized with rVHSV-GΔTM were significantly higher than those of control group fish that were injected with L-15 alone. Among three vaccination groups (group 1, 3, and 5), group 1 showed no mortality. The cumulative mortalities of group 3 and group 5 were both 25%. While fish in control groups (group 2, 4, and 6) showed 90–100% mortalities. The qPCR genome copy number of rVHSV-GΔTM in the kidney of fish immunized at 14 °C was clearly higher than that in fish immunized at 20 °C, which suggests that higher amount of secretory viral glycoprotein would be produced in fish vaccinated at 14 °C than at 20 °C. Olive flounder immunized with rVHSV-GΔTM at 14 °C showed the serum neutralization activity as high as fish immunized at 20 °C, suggesting that the humoral immune response of olive flounder was effectively induced at lower water temperature. These results suggest that VHSV vaccines based on single-cycle viruses can be used as prophylactic vaccines even at low water temperature period.

Oral vaccines are highly demanded by aquaculture sector that requires alternatives to injectable vaccines, involving fish handling, stress-related immunosuppression and mortalities. However, most previous attempts to obtain effective oral vaccines have failed due to a restricted tolerance mechanisms in intestine, whose mucosa is at the frontline of antigen encounter and has to balance the equilibrium between tolerance and immunity in a microbe-rich environment. Thus, the search for oral adjuvants that could augment immune responses triggered by antigens allowing them to circumvent intestinal tolerance is of great relevance. The present work focuses on the adjuvant potential of the Escherichia coli LT(R192G/L211A) toxoid (dmLT). To undertake an initial screening of the potential that dmLT has as an oral adjuvant in rainbow trout (Oncorhynchus mykiss), we have analyzed its transcriptional effects alone or in combination with Aeromonas salmonicida subsp. salmonicida or viral hemorrhagic septicemia virus (VHSV) on rainbow trout intestinal epithelial cell line RTgutGC and gut explants. Our results show that although dmLT provoked no significant effects by itself, it increased the transcription of pro-inflammatory cytokines and antimicrobial genes induced by the bacteria. In contrast, when combined with VHSV, dmLT only increased the transcription of Mx and the intracellular adhesion molecule 1 (ICAM1). Therefore, the protocol designed is an effective method to initially evaluate the effects of potential oral adjuvants, and points to dmLT as an effective adjuvant for oral antibacterial vaccines.

Background Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and are thusly notifiable diseases with the World Organization for Animal Health. This study assessed the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores the abilities of specific IHNV genes to interfere with the interferon pathway in heterogenous teleost cell-lines. Methods Optimized protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (-IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 hours post transfection (hpt) with luciferase constructs driven by a modified β-Actin promoter (pCAG). Their modulation of the innate antiviral immune response was characterized 72 hpt, using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation, upon MAVS co-transfection. Results M was generally confirmed as the strongest constitutive transcriptional suppressor while IHNV P, but not VHSV P, augmented constitutive transcription in fibroblastic cell types. Cell-specific effects were observed for viral G gene, with VHSV G exhibiting suppression of basal transcription in EPC and BF-2 but not in trout cells; while IHNV G was stimulatory in RTG-2, but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV NV compared to VHSV NV. The innate antiviral immune response, focusing on the IFN pathway, was silenced by IHNV M in all cell lines tested. IHNV N showed a dose-dependent suppression of type I IFN, but with minor effects on MX-1. IHNV P and G played minor IFN-inhibitory roles, consistent and dose-dependent only for G in rainbow trout cells. IHNV NV mediated a consistent stimulatory effect on either Type I IFN or MX-1, but much less pronounced in RTgill-W1. Conclusions This study extends our understanding of Novirhabdoviruses-host interaction, showing differential innate immune responses in heterogenous cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.

Viral hemorrhagic septicemia (VHS) is a serious viral disease that infects the olive flounder in South Korea. The Korean aquaculture industry experienced an economic loss caused by the high infectivity and mortality. This study aimed to evaluate the infection density of VHSV in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm and injected subcutaneously with 106 PFU/fish. Each 15 fish were sampled at 0, 3, and 7 days post challenge (dpc), respectively, to perform quantitative analysis of VHSV using SYBR-green based real-time PCR in various tissues including spleen, liver, head-kidney, body-kidney, muscle, esophagus, stomach, intestine, gill, and brain. Organs infected with VHSV were obtained after 3 and 7 days. Each organs were examined for viral infection using real-time PCR. The data obtained from this experiment revealed copy numbers higher than 10 copies per 100 ng cDNA in the spleen (15.26 ± 3.11 copies/100 ng of cDNA), muscle (11.24 ± 2.25 copies), and gill (14.23 ± 6.26 copies), but lower in liver, head-kidney, body-kidney, esophagus, brain and stomach. The present study, together with previous data, demonstrated that the gill, spleen, and muscle are the major target organs of VHSV in olive flounder. Therefore, central monitoring of spleen, gill and muscle should be considered and might be necessary if anti-VHSV treatment is to be successful in infected olive flounder.

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.

Viral hemorrhagic septicemia virus (VHSV) genotype IVa is an endemic pathogen to the marine waters of British Columbia, with numerous marine fishes being susceptible to infection and disease, including Atlantic salmon Salmo salar reared in open net-pen aquaculture. The susceptibility of Atlantic salmon and sockeye salmon Oncorhynchus nerka to VHSV-IVa infection was evaluated using exposure routes including injection, static immersion, and cohabitation with diseased Pacific herring Clupea pallasii. Exposed fish were monitored for mortality and external pathology, mortalities were tested by virus isolation assay, and live fish were regularly sampled and screened for infection. Among injected sockeye, VHSV was detected in 1 mortality (n = 195) and 2 sub-sampled fish (n = 30), whereas sockeye exposed by immersion and cohabitation did not experience mortality nor was systemic infection indicated by tissue screening. Injection and cohabitation exposure routes confirmed the susceptibility of Atlantic salmon to VHSV. Neither sockeye nor Atlantic salmon surviving the cohabitation served as a reservoir of VHSV, but Pacific herring did. The results suggest that VHSV-IVa poses low risk to sockeye salmon under natural routes of exposure.

Highlights • A quantitative LAMP method for identification of EEDV has been developed. • Analytical sensitivity of the qLAMP is as low as 78 pg extracted DNA from tissue. • The method is highly specific for EEDV. • The EEDV qLAMP method was evaluated against the qPCR method., Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 μM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Viperin is known to exhibit activity against RNA viral infection. Viral hemorrhagic septicemia virus (VHSV) is a negative-sense single-stranded RNA virus that causes severe loss in aquaculture species. Susceptible species include redlip mullets (Liza haematocheila), which has become an economically important euryhaline mugilid species in offshore aquaculture along the west coast of Korea. Although interferon-stimulated genes are suspected to act against VHSV, specific pathways or mechanisms of these antiviral actions in redlip mullets have not yet been established. In silico studies of the mullet viperin (Lhrsad2) revealed an S-adenosyl methionine binding conserved domain containing the 77CNYKCGFC84 sequence. In the tissue distribution, the highest levels of lhrsad2 expression were observed in the blood. When injected with poly(I:C), an approximately 17-fold upregulation (compared to the control) of viperin was detected in the blood after 24 h. Furthermore, non-viral immune stimuli, including Lactococcus garvieae (L. garvieae) and lipopolysaccharide (LPS), that were injected into redlip mullets were not found to induce considerable levels of viperin expression. Subcellular analysis revealed that Lhrsad2 localized to the endoplasmic reticulum (ER). To investigate Lhrsad2's antiviral effects against VHSV, cells overexpressing lhrsad2 were infected with VHSV, and then the viral titer and viral gene expression were analyzed. Both assays revealed the potential of Lhrsad2 to significantly reduce VHSV transcription and replication. In brief, the current study illustrates the remarkable ability of viperin to weaken VHSV in redlip mullet.

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV–S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared “public” clones. We previously reported a strong public IgHμ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.

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., Author summary Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes huge economic losses to the fish culture industry throughout the world. Virulence among naturally occurring VHSV strains varies widely. However, little is known about the viral factors that determine VHSV virulence. Here, we identify a naturally-occurring, single-amino-acid substitution in the VHSV P protein that enhances VHSV virulence in flounder. This amino acid substitution in the P protein was detected only in highly virulent VHSV strains, and it enhances viral RNA synthesis and inhibits the interferon response of host cells early after virus infection. Recombinant VHSV containing this amino acid substitution caused increased mortality in flounder compared with the wild type. This is the first study to identify a naturally occurring amino acid substitution in VHSV that determines its virulence in flounder. We expect that our result can be applied to other fish species, and this finding will provide new opportunities to generate an effective VHSV vaccine.

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.

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.

Galectins are well-known β-galactoside-binding proteins, which play vital roles in innate immune responses of both vertebrates and invertebrates. However, knowledge regarding invertebrate galectins is still in its infancy. With the intention of filling the knowledge gap, here we identified a quadruple domain-containing galectin from marine invertebrate disk abalone, Haliotis discus discus (AbGalec), and characterized it. AbGalec consisted of four distinct carbohydrate-recognition domains (CRDs) and lacked a signal peptide. Expression analysis revealed AbGalec to be ubiquitously expressed in all the examined early embryonic stages of abalone, with highest expression in the 16-cell stage, suggesting the importance of AbGalec in early developmental processes. Tissue distribution analysis revealed the highest expression of AbGalec in abalone mantle, followed by that in gills and hemocytes. Immune challenge experiments revealed significant upregulation of AbGalec at 24 h and 48 h post injection (p.i.) with bacterial and viral components. These results suggested the possible involvement of AbGalec in host defense mechanisms. Polyinosinic: polycytidylic acid (Poly I:C) and viral hemorrhagic septicemia virus (VHSV) injections were capable of inducing AbGalec transcript expression more prominently than bacterial stimulants, thus providing evidence for its role in viral infections. We determined the virus-neutralizing ability of a quadruple domain-containing galectin for the first time, by analyzing the downregulation of VHSV transcripts during the overexpression of AbGalec. Significant downregulation of VHSV transcripts was observed after 24 h and 48 h of post infection. Collectively, our findings reveal the potent antiviral responses of molluscan quadruple domain-containing galectin, AbGalec, along with its involvement in innate immunity.

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

Determining the origin of recurrent outbreaks of fish diseases occurring on fish farms is essential for disease prevention and control measures. In this study, we investigated the potential reservoir role of wild fish species living near salmonid farms which were regularly found to be positive for viral hemorrhagic septicemia virus (VHSV). In addition to VHSV, infectious hematopoietic necrosis virus (IHNV) was also isolated from several pike Esox lucius samples collected from a pond near the salmonid farms of interest. All isolates of VHSV and IHNV analyzed had 100% identical partial glycoprotein gene sequences. VHSV pike strain OO128-25 belonged to the Ia genotype and shared 99.1 to 99.5% nucleotide identity with strains recently isolated from the farms. IHNV pike strain OO121-8, European genotype, appeared to be different from strains from France characterized since the first isolation in 1987. Isolates representative of both viral species were highly virulent in rainbow trout Oncorhynchus mykiss. OO128-25 induced 65% mortality in pike fingerlings, whereas only weak mortality was observed with OO121-8, despite characteristic symptoms in infected fish. High levels of specific antibodies to VHSV and IHNV were detected in adult pike in the absence of clinical signs. Infection of rainbow trout in contact with experimentally VHSV- or IHNV-infected pike fingerlings indicates possible horizontal transmission. These results suggest that pike could act as a reservoir for VHSV and IHNV in the wild, providing additional evidence to explain viral persistence and resurgence in certain areas.

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2&alpha, (p-eIF2&alpha, ), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2&alpha, During VHSV IVb infection, eIF2&alpha, phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2&alpha, Similarly, IVb NV null VHSV infection induced less p-eIF2&alpha, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2&alpha, pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion‐dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10−5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV.

RNAlater is a commonly used transport and storage solution for samples collected for fish health investigations, particularly those potentially involving viruses. However, the infectivity of fish viruses after storage in RNAlater have not been determined. Nevertheless, knowledge of pathogen infectivity of preserved samples is crucial for ensuring safe transport and storage protocols. Therefore, the infectivity of three fish RNA viruses in RNAlater was examined at four temperatures: −80 °C, 4 °C, room temperature (RT, approximately 22 °C) and 37 °C. The viruses were viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and chum salmon reovirus (CSV). Overall, three consistent outcomes were observed. First, all three viruses remained infectious in RNAlater at RT or lower. High log titres of these viruses remained over 30 d of storage in either RNAlater or PBS. Second, RNAlater delayed the thermal inactivation of these viruses when compared to PBS at 37 °C. For VHSV, the titre remained high in RNAlater after one day of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For IPNV, the titre remained high in RNAlater after 30 d of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For CSV, the titre was slightly higher in RNAlater than PBS at 37 °C over 7 d, and by day 30, only samples stored in RNAlater proved infectious at titres above the detection threshold. Third, RNAlater delayed the inactivation of these viruses when they were stored together with head kidney homogenates. For VHSV, infectious virus was recovered from samples stored at 4 °C in RNAlater by day 7 of incubation, whereas it was inactivated to below threshold in PBS over the same period. For both IPNV and CSV, infectious virus was recovered from samples stored at 37 °C in RNAlater for 7 d, but not so in PBS. In summary, fish viruses can remain infectious and are even temporarily protected from inactivation while in RNAlater. This makes RNAlater a potentially useful solution for the transport of fish viruses. At the same time, precautionary measures must be taken when transporting potentially infectious samples in RNAlater.

Novirhabdoviruses belong to the Rhabdoviridae family of RNA viruses. All of the four members are pathogenic for bony fish. Particularly, Infectious hematopoietic necrosis virus (IHNV) and Viral hemorrhagic septicemia virus (VHSV) often cause mass animal deaths and huge economic losses, representing major obstacles to fish farming industry worldwide. The interactions between fish and novirhabdoviruses are becoming better understood. In this review, we will present our current knowledge of fish innate immunity, particularly type I interferon (IFN-I) response, against novirhabdoviral infection, and the evasion strategies exploited by novirhabdoviruses. Members of Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) appear to be involved in novirhabdovirus surveillance. NF-κB activation and IFN-I induction are primarily triggered for antiviral defense. Autophagy can also be induced by viral glycoprotein (G). Although sensitive to IFN-I, novirhabdoviruses have nucleoprotein (N), matrix protein (M), and non-virion protein (NV) to interfere with host signal transduction and gene expression steps toward antiviral state establishment. Moreover, novirhabdoviruses may exploit some microRNAs for immunosuppression.

The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that infects farmed fish. In this study, we measured the replication of VHSV increased steadily at 9, 24, 72, and 120 h after infection and progression of necrosis was observed at 72 hpi. We performed transcriptomic and metabolomics profiling of kidney tissues collected at each infection time using Illumina HiSeq2000 and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and 72 metabolites were selected to identify the mechanisms of infection and they were integrated using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the immune system was activated by VHSV infection. A decrease in levels of amino acids such as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated analysis of transcriptome and metabolome using KEGG pathway database revealed four types of responses in the kidneys of olive flounder to VHSV infection. Among these, the mechanisms related to the immune system and protein synthesis were activated, whereas ATP synthesis and the antioxidant system activity were suppressed. This is the first study describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The results suggest that the suppression of ATP synthesis and antioxidant systems, such as glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation of the immune system could result in the inflammation of kidney tissue in VHSV-infected olive flounder.

Olive flounder (Paralichthys olivaceus) is one of the most economically important aquaculture fish. However, its production is often affected by various diseases, especially viral hemorrhagic septicemia virus (VHSV) that cause serious economic losses. In this study, we sequenced the whole transcriptome of the P. olivaceus using Illumina RNA-sEq. De novo assembly of control and virus-infected cDNA libraries of head kidney at 13 and 20 °C was accomplished with 2,007,532,438 raw reads, resulting in 244,578 unigenes with an average length of 533 bp and found 65,535 candidate coding unigenes with homology to other species by BLAST analysis. DEG analysis among control and virus-infected head kidney samples of 13 and 20 °C revealed that 1290 up-regulated and 162 down-regulated genes (p ≤ 0.01), linked to metabolism, virulence factors, adhesion and immune-response. We constructed an expressed gene catalog for the P. olivaceus to serve as a resource for marine environmental genomic and immuno-genetic/genomic studies focused on uncovering the molecular mechanisms underlying the responses of P. olivaceus to VHSV under different temperature.

When viral diseases occur in aquaculture farms, the virus released into the seawater from infected animals can re-infect other susceptible species or accumulate in filter-feeding organisms. We conducted a viral hemorrhagic septicemia virus (VHSV) survivability analysis of blue mussel Mytilus edulis digestive enzymes, viral depuration, and infectivity tests via in vitro and in vivo inoculation to evaluate the infectious state. VHSV particles were not completely digested within 24 h in vitro and were maintained for 7 d in the mussel digestive gland. Mussels cohabitating with naturally VHSV-infected olive flounder Paralichthys olivaceus could accumulate the viral particles. Although the viral particles in the gill as the entrance of filter-feeding organisms are infectious, the presence of these particles in the digestive gland were not able to induce cytopathic effects in vitro. Viral particles detected by RT-PCR from bivalve mollusks (Pacific oyster Crassostrea gigas and mussel) from the field did not produce cytopathic effects in cell culture and did not replicate after intraperitoneal injection into olive flounder. Therefore, VHSV particles in blue mussel might be in a non-infectious stage and the possibilities of VHSV transmission to fish under field conditions are scarce.

Aims The reliability of PCR techniques is an important issue in viral diagnosis, and it is even crucial when they must be applied for detection of viruses in asymptomatic carriers. The problems will arise when the aim is to study fish wild populations, where the viral loads and prevalence values are extremely low. We have evaluated several PCR procedures employed by two laboratories for monitoring fish captured in several oceanographic campaigns performed in the Gulf of Cadiz. Methods and Results To evaluate the reliability of different diagnostics test used, we have re-analyzed fish samples that had been previously subjected to diagnosis for a surveillance of viruses performed in 2010-2011 in wild fish populations. The following parameters were employed: the clinical sensitivity (Ss), the clinical specificity (Sp), the predictive positive value (PPV), the predictive negative value (PNV), and the positive and negative likelihood ratio (LR+ and LR-). For viral nervious necrosis virus (VNNV), a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral hemorrhagic septicemia virus (VHSV), the highest values were provided by RT-PCR supplemented by dot blot hybridization. In the case of Infectious pancreatic necrosis virus (IPNV), none of the procedures yielded 100% for any parameter. Conclusions The results obtained for viral prevalence indicate: (i) that the conservation of the samples at -80 °C did not affect to the capacity of detection of the virus in the tissues, and (ii) that the reproducibility of the diagnosis can be affected by factors including the staff experience and/or the materials employed. Finally, the use of a combination of procedures in advised to ensure the maximum reliability of the diagnosis when it is applied to asymptomatic fish populations. Significance and Impact of study This paper describes a strategy of combining diagnostic tests for the surveillance and monitoring of wild fish populations to reduce underestimation of the prevalence of viruses this type of populations. This article is protected by copyright. All rights reserved.

This study aims to express fish Viral hemorrhagic septicemia virus (VHSV) G main antigen domain by using Bac-to-bac expression system. Using bioinformatics tools, B cell epitope of VHSV G gene was predicted, and G main antigen domain was optimized. GM gene was inserted into pFastBac1 vector, then transferred recombinant plasmid into DH10Bac to get recombinant rBacmid-GM. Obtained shuttle plasmid rBacmid-GM was transfected into sf9 cells. GM expression was examined using by PCR and western-blot. Results indicated that G main antigen domain gene of VHSV was successfully cloned and sequenced which contains 1209 bp. PCR proved that shuttle plasmid rBacmid-GM was constructed correctly. SDS-PAGE electrophoresis analysis detected a band of protein about 45kD in expression product of G gene. Obtained recombinant G protein reacted with VHSV-positive serum that was substantiated by western-blot analysis. In conclusion, the main antigen domain of VHSV G was successfully expressed in the Bac-to-Bac baculovirus system.

The Novirhabdovirus viral hemorrhagic septicemia virus (VHSV) genotype IVb has caused serious fish kills and become endemic throughout the Great Lakes basin of North America. This is troublesome since there are no protective vaccines currently approved against this deadly disease even though recombinant technology has become increasingly common. Herein, we explored the production of a recombinant VHSV-IVb glycoprotein, believed to be important for virus infectivity, and determined its ability to elicit protection against challenge with the wild virus strain. A recombinant baculovirus containing a 5' 6x polyhistidine tag embedded in the VHSV-IVb G gene was used to infect the larvae of the cabbage looper Trichoplusia ni. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of affinity-purified protein yielded apparent VHSV-IVb glycoprotein at the expected molecular weight of ~65 kDa. The recombinant protein (rG) was used successfully in coating microtiter plate wells in an indirect enzyme-linked immunosorbent assay (ELISA), and positive anti-VHSV-IVb antibodies in Muskellunge Esox masquinongy were capable of binding to both the rG and purified whole VHSV-IVb, indicating epitope resemblance. In addition, the rG elicited a protective response in Muskellunge during a VHSV-IVb immersion challenge, resulting in 80% relative percent survival. Our results demonstrate that cabbage looper larvae can serve as an excellent production system for apparently conformationally correct viral glycoprotein. The incorporation of a polyhistidine tag facilitates obtaining highly purified protein in a relatively high concentration, which has potential in the development of an efficacious subunit vaccine against this deadly virus. Received September 11, 2016; accepted March 10, 2017.

The antiviral activity of an extract mixture from Celosia cristata and Raphanus sativus was tested against viral hemorrhagic septicemia virus (VHSV). Pretreatment of EPC cells with this extract up to 72 h before VHSV infection markedly reduced the virus titer, but it had no effect when added after virus inoculation. In olive flounder that received 5 µg of extract per fish, Mx expression peaked at 48 h after treatment. In contrast, ISG15 and TLR2 expression peaked at 72 h, and that of TLR7 peaked at 48 h, followed by a slight decrease at 72 h, indicating that the antiviral activity was mediated by induction of gene expression involved in the innate immune response.

MicroRNAs are small non-coding RNAs and are involved in the regulation of wide biological processes. Viral hemorrhagic septicemia virus (VHSV) is the causative agent of viral hemorrhagic septicemia (VHS) disease causing a heavy loss in aquaculture farms. In this study, we tried to explore the effect of VHSV infection on microRNAs profile of Epithelioma papulosum cyprini (EPC) cells at different points of time (0, 3, 12, 24, and 48 h post infection). A total of 355 conserved microRNAs and 3 novel microRNAs were identified, and among them, 103 microRNAs were differentially expressed. The number of differentially expressed microRNAs was highly increased at 24 h.p.i compared to 3 h.p.i and 12 h.p.i., suggesting that EPC cells might not actively respond to VHSV infection at an early infection period, which can allow viruses to transcript and translate their genes enough to produce viral particles that can infect to another cells. Among the differentially expressed microRNAs, 2 miRNAs (miR-735 and miR-738) that were reported only in fish species were highly upregulated, and based on the target prediction, they could regulate several immune pathways. Furthermore, the present results showed the upregulation of representative immune regulating microRNAs such as miR-146a, miR-155, and miR-99. The target prediction of differentially expressed miRNAs, GO, and KEGG pathways analysis revealed that several biological processes and different pathways were affected by the viral infection. The present dynamical changing patterns of differentially expressed microRNAs in response to the progression of VHSV infection suggest that microRNA profile that was analyzed at one time point cannot provide enough information for the interpretation of the disease mechanism. Considering the wide and complex interactions between microRNAs and genes expression, the present results provide the basis for the understanding of VHSV infection-mediated cellular responses and for future investigations on the development of possible control measures.

Viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) belong to the genus Novirhabdovirus and are the causative agents of a serious disease in cultured flounder. However, infectious hematopoietic necrosis virus (IHNV), a prototype of the genus Novirhabdovirus, does not cause disease in flounder. To determine whether IHNV growth is restricted in flounder cells, we compared the growth of IHNV with that of VHSV and HIRRV in hirame natural embryo (HINAE) cells infected with novirhabdoviruses at 1 multiplicity of infection. Unexpectedly, we found that IHNV grew as well as VHSV and HIRRV. For successful growth in host cells, viruses modulate innate immune responses exerted by virus-infected cells. Our results suggest that IHNV, like VHSV and HIRRV, has evolved the ability to overcome the innate immune response of flounder cells. To determine the innate immune response genes of virus-infected HINAE cells which are commonly modulated by the three novirhabdoviruses, we infected HINAE cells with novirhabdoviruses at multiplicity of infection (MOI) 1 and performed an RNA sequencing-based transcriptome analysis at 24 h post-infection. We discovered ~12,500 unigenes altered by novirhabdovirus infection and found that many of these were involved in multiple cellular pathways. After novirhabdovirus infection, 170 genes involved in the innate immune response were differentially expressed compared to uninfected cells. Among them, 9 genes changed expression by more than 2-fold and were commonly modulated by all three novirhabdoviruses. Interferon regulatory factor 8 (IRF8), C-X-C motif chemokine receptor 1 (CXCR1), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), cholesterol 25-hydroxylase (CH25H), C-X-C motif chemokine ligand 11, duplicate 5 (CXCL11.5), and Toll-like receptor 2 (TLR2) were up-regulated, whereas C-C motif chemokine receptor 6a (CCR6a), interleukin-12a (IL12a), and Toll-like receptor 1 (TLR1) were down-regulated. These genes have been reported to be involved in antiviral responses and, thus, their modulation may be critical for the growth of novirhabdovirus in flounder cells. This is the first report to identify innate immune response genes in flounder that are commonly modulated by IHNV, VHSV, and HIRRV. These data will provide new insights into how novirhabdoviruses survive the innate immune response of flounder cells.

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout. IMPORTANCE Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.

MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence. Furthermore, the down-regulation of reporter gene containing 3'-UTR of NF-κb inhibitor α-like protein B (NFκbiαb) by miR-30e was demonstrated, suggesting that miR-30e overexpression can increase immune responses related to NF-κB activation through inhibition of IκB. A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) that had primary microRNA-30e sequence between N and P genes was rescued using the reverse genetic method, and the successful expression of miR-30e in the cells infected with rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. Cells infected with rVHSV-A-miR30e showed the increase of NF-κB activation and type I interferon induced genes expression, suggesting that rVHSV-A-miR30e can produce functional miR-30e in fish cells, and VHSV can be used as a vehicle to deliver functional microRNAs in fish.

Viral hemorrhagic septicemia (VHS) causes serious economic loss in olive flounder aquaculture industry in Korea. Water temperature is known to play a critical role in VHS disease outbreak. Here, we assessed the potential efficacy of VHSV immersion treatment in relation to resistance conferred at differential water temperatures in olive flounder. VHSV acquired resistance was compared between formalin-killed VHSV immersion treatment and live VHSV immersion treatment at three different water temperatures viz., 10 °C, 17 °C, and 20 °C. At 10 °C, cumulative mortality was around 80% in live VHSV immersed group while 30% cumulative mortality was observed in formalin-killed VHSV treated group. After 4 weeks, surviving olive flounder at 17 °C and 20 °C were challenged with VHSV at 10 °C following which the VHS outbreaks took place at host susceptible water temperature. For the pre-treated flounder at 17 °C, survival rates were 80% and 30% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Whereas, the pre-treated flounder at 20 °C showed survival rate of 75% and 20% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Our results propose the fact that live VHSV immersion using non-susceptible water temperature has the potential to protect olive flounder against VHSV infection. Moreover, the protective efficacy of live immersion treatment in a non-excited immune state without the use of an adjuvant combined with water temperature adjustment was investigated for the first time at 17 °C. Further studies should be targeted to explore the host-associated immune factors responsible for the protective effect and acquired resistance in olive flounder after live VHSV immersion treatment.

Viral hemorrhagic septicemia virus (VHSV) has been one of the major causes of mortality in a wide range of freshwater and marine fishes worldwide. Although various types of vaccines have been tried to prevent VHSV disease in cultured fishes, there are still no commercial vaccines. Reverse genetics have made it possible to change a certain regions on viral genome in accordance with the requirements of a research. Various types of VHSV mutants have been generated through the reverse genetic method, and most of them were recovered to investigate the virulence mechanisms of VHSV. In the reverse genetically generated VHSV mutants-based vaccines, high protective efficacies of attenuated VHSVs and single-cycle VHSV particles have been reported. Furthermore, the application of VHSV for the delivery tools of heterologous antigens including not only fish pathogens but also mammalian pathogens has been studied. As not much research has been conducted on VHSV mutants-based vaccines, more studies on the enhancement of immunogenicity, vaccine administration routes, safety to environments are needed for the practical use in aquaculture farms.

Viral hemorrhagic septicemia virus (VHSV) is an ongoing cause of disease and mortality in freshwater fishes across the Great Lakes region of the Midwestern United States. Antibody detection assays such as enzyme-linked immunosorbent assay (ELISA) are nonlethal serological methods that can have significantly shorter turnaround times than the current validated viral detection diagnostic methodology for VHSV: cell culture with confirmation by polymerase chain reaction (PCR). This study evaluated an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV in Northern Pike Esox lucius. Juvenile Northern Pike were experimentally infected with VHSV by intraperitoneal injection. The infected fish were monitored for 12 weeks for signs of disease, and weekly serum samples were obtained. An analysis of the survival data showed that mortality occurred significantly more quickly in inoculated fish than in control fish. Fish that were infected by injection showed a significant increase in antibody response by 2 weeks postinfection. However, variation in the rate and pattern of antibody response among the infected fish was high at any given point. The optimum window for detecting antibodies in Northern Pike is 2-12 weeks postinfection, which generally follows the median time to appearance of clinical signs (21 d postinfection). The receiver-operating characteristic curve analysis showed the ELISA to have a sensitivity of 80.5% and a specificity of 63.2% in Northern Pike, but these values can be adjusted by choosing different percent inhibition cutoffs, which may facilitate the use of the test for specific management goals. The results of this study offer insights into the disease progression and immune kinetics of VHSV, including interindividual variation, which will aid in the management of this economically important virus.