Viral Hemorrhagic Septicemia Research Articles

Temperature-dependent immune response of olive flounder (Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV).

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 533bp 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.

Phylogenetic analysis and duplex RT-PCR detection of viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus) from Korea

Viral hemorrhagic septicemia is a serious disease that can assume epidemic proportions in cultivated olive flounders in Korea. The causal agent of this disease is viral hemorrhagic septicemia virus (VHSV), and it leads to vast economic losses in the olive flounder aquaculture industry. Therefore, the rapid and accurate detection of VHSV is paramount. At present, the manual of the World Organization for Animal Health's Office International des Epizooties (OIE) for pathogen detection is being followed for VHSV detection in Korea. However, in that manual, the primers for VHSV detection are based on the European VHSV genotype Ia. In this study, we identified 5 VHSV strains from olive flounders in Korea, and the nucleotide and amino acid sequences of VHSV genes (N, P, M, and G) were compared to investigate the genetic variation of VHSV genotypes. As expected, VHSV isolates from Korea were highly related to genotype IVa, clearly differing from the 3 European genotypes. In addition, the N gene showed low genetic variation and therefore might be considered a useful marker for VHSV detection, regardless of genotype. In contrast, the other VHSV genes (P, M, and G) had different nucleotides within genotypes. They might be suitable for designing specific primers for distinguishing between various VHSV genotypes. Duplex RT-PCR using the newly designed primers successfully detected all VHSV isolates and validated the genotype (I and IV) without sequence analysis. According to the results, all the VHSV isolates were successfully detected, and their genotypes were validated by duplex RT-PCR using the newly designed primers. Collectively, our findings suggest that duplex PCR is a convenient and appropriate method for the diagnosis of VHSV isolates in Korean aquaculture systems.

Evaluation of blue mussel Mytilus edulis as vector for viral hemorrhagic septicemia virus (VHSV)

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.

Efficacy of algal Ecklonia cava extract against viral hemorrhagic septicemia virus (VHSV)

The inhibition efficacy of an extract from Ecklonia cava (E. cava) was studied to determine whether the extract and compounds exhibited inhibitory activity against VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Based on its low toxicity and effective concentration, the E. cava extract (Ext) and compounds (eckol and phlorofucofuroeckol A) were selected for further analysis. In the plaque reduction assay, simultaneous co-exposure of VHSV to Ext, eckol and phlorofucofuroeckol A showed a higher level of inhibition than the pre- and post-exposure groups. The antiviral activity in the FHM cell line was time-dependent and increased with the exposure time with the virus and Ext or the compounds. In the in vivo experiments, different Ext concentrations were orally administered to the olive flounder. In trial I, the relative percent survival (RPS) following oral administration of 500 and 50 μg/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 μg/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 μg/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 μg/g/day) significantly induced inflammatory cytokine responses (IL-1β, IL-6 and IFN-γ) at 1 and 2 days post-oral administration (dpa). Additionally, IFN-α/β (7–12 dpa), ISG15 (2, 7 and 10 dpa) and Mx (7–12 dpa) were significantly activated in the olive flounder. In conclusion, we demonstrated an inhibitory ability of the E. cava extract and compounds against VHSV in the FHM cell line. Moreover, oral administration of the E. cava extract to the olive flounder enhanced antiviral immune responses and the efficacy of protection against VHSV, resulting in an anti-viral status in the olive flounder.

Identification and characterization of molluscan caveolin-1 ortholog from Haliotis discus discus: Possible involvement in embryogenesis and host defense mechanism against pathogenic stress

Caveolins are principal membrane proteins of caveolae that play a central role in signal transduction, substrate transport, and membrane trafficking in various cell types. Numerous studies have reported the crucial role of caveolin-1 (CAV1) in response to invading microbes; yet, very little is known about molluscan CAV1. In this study, we identified and characterized CAV1 ortholog from the disk abalone, Haliotis discus discus (HdCAV1). The cDNA sequence of HdCAV1 is 826 bp long and encodes a 127-amino acid polypeptide. Characteristic caveolin superfamily domain (Glu3 – Lys126) and two possible transmembrane domains (Cys48 - Tyr67 and Ile103 - Phe120) were identified in the HdCAV1 protein. Homology analysis revealed that HdCAV1 shared higher identity (>47%) with molluscans, but lower identity with other species. Phylogenetic tree constructed by the neighbor-joining (NJ) method revealed a distinct evolutionary pathway for molluscans. Transcriptional analysis by SYBR Green qPCR showed the highest expression of HdCAV1 mRNA in late veliger stage, as compared to that in other embryonic developmental stages of disk abalone. In adult animals, gill tissue showed highest HdCAV1 transcript levels under normal physiological condition. Stimulations with two bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), viral hemorrhagic septicemia virus, and two pathogen-associated molecular patterns (LPS and poly I:C) significantly modulated the expression of HdCAV1 transcripts. Collectively, these data suggest that CAV1 plays an important role in embryogenesis and host immune defense in disk abalone.

Nucleated Teleost Erythrocytes Play an Nk-Lysin- and Autophagy-Dependent Role in Antiviral Immunity.

With the exception of mammals, vertebrate erythrocytes are nucleated. Nevertheless, these cells are usually considered as mere carriers of hemoglobin. In this work, however, we describe for the first time an unrecognized role of teleost red blood cells (RBCs). We found that Nk-lysin (Nkl), an antimicrobial peptide produced by NK-cells and cytotoxic T-lymphocytes, was also expressed in flatfish turbot (Scophthalmus maximus) erythrocytes. Although the antiviral role of Nkl remains to be elucidated, we found a positive correlation between the transcription of nkl and the resistance to an infection with Rhabdovirus in a teleost fish. Surprisingly, Nkl was found to be present in the autophagolysosomes of erythrocytes, and therefore this higher resistance provided by Nkl could be related to autophagy. The organelles of RBCs are degraded through autophagy during the maturation process of these cells. In this work, we observed that the blockage of autophagy increased the replication of viral hemorrhagic septicemia virus in nucleated teleost erythrocytes, which suggests that this mechanism may also be a key process in the defense against viruses in these cells. Nkl, which possesses membrane-perturbing ability and was affected by this modulation of RBC autophagy, could also participate in this process. For the first time, autophagy has been described not only as a life cycle event during the maturation of erythrocytes but also as a pivotal antiviral mechanism in nucleated erythrocytes. These results suggest a role of erythrocytes and Nkl in the antiviral immunity of fish and other vertebrates with nucleated RBCs.

Molecular identification and functional analysis of two variants of myeloid differentiation factor 88 (MyD88) from disk abalone (Haliotis discus discus)

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein of the Toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and is involved in a diverse array of inflammatory responses via NF-κB activation. In the present study, two MyD88 variants were identified from disk abalone (Haliotis discus discus) and designated AbMyD88-2 and AbMyD88-X. The deduced AbMyD88-2 and AbMyD88-X comprised 433 and 354 amino acids with predicted molecular masses of 48.85 kDa and 40.17 kDa, respectively. AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features including an N-terminal death domain (DD) and C-terminal toll interleukin 1 receptor (TIR) domain similar to those in mammals. Expression analysis of AbMyD88-2 and AbMyD88-X mRNA at different early embryonic developmental stages of abalone by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably higher values at the 16-cell (AbMyD88-2) and morula stages (AbMyD88-X). In unchallenged disk abalones, AbMyD88-2 was highly expressed in muscles, while AbMyD88-X mRNA was predominantly transcribed in hemocytes. Moreover, AbMyD88-2 and AbMyD88-X mRNA were differentially modulated in abalone hemocytes after a challenge with live bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and pathogen-associated molecular patterns (lipopolysaccharides and Poly I:C). Overexpression of AbMyD88-2 and AbMyD88-X in HEK293T cells induced the activation of the NF-κB promoter. AbMyD88-2 and AbMyD88-X involvement in inflammatory responses was characterized by their overexpression in RAW264.7 murine macrophage cells. These results revealed comparatively higher NO (Nitric oxide) production, induction of inflammatory mediator genes (iNOS and COX2), and proinflammatory genes (IL1β, IL6 and TNFα) expression in abalone MyD88s-overexpressing cells than in mock control in the presence or absence of LPS stimulation. Altogether, these results suggest that existence of a MyD88-dependent like signaling pathway in disk abalone and that both AbMyD88-2 and AbMyD88-X might be involved in innate immune and inflammatory responses.

The theoretical reliability of PCR-based fish viral diagnostic methods is critically affected when they are applied to fish populations with low prevalence and virus loads

The reliability of polymerase chain reaction (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 wild fish 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 Cádiz. To evaluate the reliability of different diagnostics test used, we have re-analysed 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, the predictive negative value, and the positive and negative likelihood ratio (LR+ and LR- ). For viral nervous necrosis virus, a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral haemorrhagic septicaemia virus, the highest values were provided by RT-PCR supplemented by dot-blot hybridization. In the case of infectious pancreatic necrosis virus, none of the procedures yielded 100% for any parameter. 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. 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.

Poly (I:C) and imiquimod induced immune responses and their effects on the survival of olive flounder (Paralichthys olivaceus) from viral haemorrhagic septicaemia

The stimulation of immune genes by polyinosinic:polycytidylic acid (poly (I:C)) and imiquimod in olive flounder (Paralichthys olivaceus) and their role in control of viral haemorrhagic septicaemia virus (VHSV) infection were examined. Poly (I:C) (100 μg/fish) treated olive flounder had very low mortality (5%) post VHSV infection, while the imiquimod treated group had 65% and 85% mortality at a dose of 100 μg/fish and 50 μg/fish, respectively. Though the imiquimod treated group had high mortality, it was lower than the untreated group, which had 90% mortality. In vivo experiments were conducted to determine effect of the two ligands on immune modulation in the head kidney of olive flounder. Poly (I:C) activated the immune genes (TLR-3, TLR-7, MDA-5, LGP-2, IRF-3, IRF-7, IL-1β type I IFN and Mx) very early, within 1 d post stimulation, faster and stronger than imiquimod. Though Mx levels were enhanced by imiquimod, the host was still susceptible to VHSV. The poly (I:C) treated group had a high immune response at the time of infection and 1 dpi, though it decreased at later stages. The imiquimod treated group and the unstimulated group had a higher immune response to VHSV compared to the poly (I:C) treated group. The nucleoprotein copies of VHSV were very low in the poly (I:C) treated group but interestingly, were high in both untreated and imiquimod treated fish. Thus, host survival from a viral infection does not only depend on the quantity of immune response but also the time of response. Although imiquimod enhanced immune gene expression in olive flounder, a delayed response could be the reason for high mortality to VHS compared with poly (I:C), which induced the immune system effectively and efficiently to protect the host.

Viral haemorrhagic septicaemia virus (VHSV Id) infections are detected more consistently using syndromic vs. active surveillance.

The eradication of viral haemorrhagic septicaemia virus (VHSV Id) from Finnish brackish-water rainbow trout Oncorhynchus mykiss farms located in the restriction zone in the Province of Åland, Baltic Sea, failed several times in the 2000s. The official surveillance programme was often unable to find VHSV-positive populations, leading to the misbelief in the fish farming industry that virus eradication could be achieved. The ability of 3 other surveillance programmes to detect infected fish populations was compared with the official programme. One programme involved syndromic surveillance based on the observation of clinical disease signs by fish farmers, while 2 programmes comprised active surveillance similar to the official programme, but included increased sampling frequencies and 2 additional tests. The syndromic surveillance concentrated on sending in samples for analysis when any sign of a possible infectious disease at water temperatures below 15°C was noticed. This programme clearly outperformed active surveillance. A real-time reverse transcriptase-polymerase chain reaction method proved to be at least as sensitive as virus isolation in cell culture in detecting acute VHSV infections. An ELISA method was used to test fish serum for antibodies against VHSV. The ELISA method may be a useful tool in VHSV eradication for screening populations during the follow-up period, before declaring an area free of infection.

Integrated profiling of global metabolomic and transcriptomic responses to viral hemorrhagic septicemia virus infection in olive flounder

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.

Movement of small-bodied fishes from Lake Michigan into Chicago Area Waterways: Insights from otolith chemistry

The Chicago Area Waterways System (CAWS), an artificial connection between Lake Michigan (LMI) and the Illinois River watershed, has served as a conduit for invasive species and is a potential pathway for viral hemorrhagic septicemia (VHS) to spread from the Great Lakes into the Illinois River drainage. Although some fishes are known to have moved from LMI into the Illinois River via the CAWS, the rate of fish passage from the lake into the CAWS is unclear. Stable carbon isotopic signatures (δ13C) in otoliths differ between lake- and CAWS-resident fish and were used as a natural tag to identify individuals that may have moved into the CAWS from LMI. The objectives of this study were to (i) estimate relative frequency of individuals with otolith δ13C indicative of prior residency in LMI among small-bodied (<150 mm total length) VHS-susceptible fishes collected from several locations in the CAWS, and (ii) to assess differences in relative frequency of probable immigrants from LMI among CAWS locations that differ in distance from the lake. Thirty-six percent of small-bodied fishes collected from the CAWS were identified as potential immigrants based on otolith δ13C. The percentage of individuals that had otolith δ13C suggestive of prior residency in LMI was higher among fish collected from the Calumet River compared to the Chicago River and North Shore Channel and decreased with increasing distance from lock and dam structures at the three entrances to the CAWS from the lake. Movement of small-bodied fishes from LMI into the CAWS appears to be common and may represent a pathway for VHS-susceptible species to transport the virus into the CAWS and potentially beyond.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

Prediction of G gene epitopes of viral hemorrhagic septicemia virus and eukaryotic expression of major antigen determinant sequence.

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.

Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-γ and its effects on type I interferon response and virulence

Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-γ that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-γ (rVHSV-A-IFNγ) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFNγ infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFNγ was evaluated by infection to olive flounder (Paralichthys olivaceus). Using a recombinant VHSV full genome vector in which the olive flounder IFN-γ ORF was inserted between N and P genes, rVHSV-A-IFNγ was successfully rescued, and the recombinant virus was grown well in EPC cells. On the other hand, the growth of rVHSV-A-IFNγ rescued from EPC cells was severely retarded when infected into hirame natural embryo (HINAE) cells that were originated from olive flounder. These results indicate that the EPC cell's IFN-γ receptor could not bind to olive flounder IFN-γ, but the species-specific binding of IFN-γ in HINAE cells induced antiviral responses. The expression of Mx1 gene in EPC cells infected with rVHSV-A-IFNγ was not greatly different from cells infected with rVHSV-Arfp (a recombinant VHSV harboring red fluorescent protein gene between N and P genes of the genome), however, in HINAE cells, rVHSV-A-IFNγ infection induced distinctively higher Mx1 gene expression compared to other recombinant viruses. These results suggest that olive flounder IFN-γ produced from rVHSV-A-IFNγ effectively enhanced type I IFN response in HINAE cells. In the present study, the lowest mortality of olive flounder fingerlings was recorded in the group of fish challenged with rVHSV-A-IFNγ, suggesting that the recombinant VHSV was attenuated by production of IFN-γ by itself. However, although rVHSV-A-IFNγ induced significantly lower mortality, the mortality still reached to 40%. Therefore, to be safely used in the aquaculture farms as prophylactic vaccines or immunostmulators, further manipulations that can guarantee safety are needed.

Influence of Temperature on the Efficacy of Homologous and Heterologous DNA Vaccines against Viral Hemorrhagic Septicemia in Pacific Herring.

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species. Received August 1, 2016; accepted March 10, 2017.

Evolutionary conservation and expression of miR-10a-3p in olive flounder and rock bream

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that mainly bind to the seed sequences located within the 3′ untranslated region (3′ UTR) of target genes. They perform an important biological function as regulators of gene expression. Different genes can be regulated by the same miRNA, whilst different miRNAs can be regulated by the same genes. Here, the evolutionary conservation and expression pattern of miR-10a-3p in olive flounder and rock bream was examined. Binding sites (AAAUUC) to seed region of the 3′ UTR of target genes were highly conserved in various species. The expression pattern of miR-10a-3p was ubiquitous in the examined tissues, whilst its expression level was decreased in gill tissues infected by viral hemorrhagic septicemia virus (VHSV) compared to the normal control. In the case of rock bream, the spleen, kidney, and liver tissues showed dominant expression levels of miR-10a-3p. Only the liver tissues in the rock bream samples infected by the iridovirus indicated a dominant miR-10a-3p expression. The gene ontology (GO) analysis of predicted target genes for miR-10a-3p revealed that multiple genes are related to binding activity, catalytic activity, cell components as well as cellular and metabolic process. Overall the results imply that the miR-10a-3p could be used as a biomarker to detect VHSV infection in olive flounder and iridovirus infection in rock bream. In addition, the data provides fundamental information for further study of the complex interaction between miR-10a-3p and gene expression.

Validation of a model for ranking aquaculture facilities for risk-based disease surveillance

A semi-quantitative model for risk ranking of aquaculture facilities in Switzerland with regard to the introduction and spread of Viral Haemorrhagic Septicaemia (VHS) and Infectious Haematopoietic Necrosis (IHN) was developed in a previous study (Diserens et al., 2013). The objective of the present study was to validate this model using data collected during field visits on aquaculture sites in four Swiss cantons compared to data collected through a questionnaire in the previous study. A discrepancy between the values obtained with the two different methods was found in 32.8% of the parameters, resulting in a significant difference (p<0.001) in the risk classification of the facilities. As data gathered exclusively by means of a questionnaire are not of sufficient quality to perform a risk-based surveillance of aquaculture facilities a combination of questionnaires and farm inspections is proposed. A web-based reporting system could be advantageous for the factors which were identified as being more likely to vary over time, in particular for factors considering fish movements, which showed a marginally significant difference in their risk scores (p≥0.1) within a six- month period. Nevertheless, the model proved to be stable over the considered period of time as no substantial fluctuations in the risk categorisation were observed (Kappa agreement of 0.77).Finally, the model proved to be suitable to deliver a reliable risk ranking of Swiss aquaculture facilities according to their risk of getting infected with or spreading of VHS and IHN, as the five facilities that tested positive for these diseases in the last ten years were ranked as medium or high risk. Moreover, because the seven fish farms that were infected with Infectious Pancreatic Necrosis (IPN) during the same period also belonged to the risk categories medium and high, the classification appeared to correlate with the occurrence of this third viral fish disease.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout.

Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.