Viral Hemorrhagic Septicemia Research Articles

Interleukin-8 (IL-8) Expression in the Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus (VHSV) Challenge.

Interleukin-8 (IL-8) is an inflammatory cytokine that plays an important role in the inflammatory response through the activation of neutrophil cells. The expression of IL-8 was investigated in early developmental stages of the olive flounder and in tissues of 8-month-old individuals. The expression of IL-8 increased after the initiation of the immune system rather than at the early stage of development, and high expression was observed in the gills and spleen, the organs associated with immunity and metabolism. In addition, IL-8 expression after infection by viral hemorrhagic septicemia virus significantly increased in the fin, gill, muscles, and spleen. These results suggest that IL-8 is closely related to inflammation and immune regulation in the immune response of the olive flounder and may be used as a basis for studies on the immune systems of other fish.

The aquatic animal pandemic crisis.

The growth of aquaculture over the past 50 years has been accompanied by the emergence of aquatic animal diseases, many of which have spread to become pandemic in countries or continents. An analysis of 400 emerging disease events in aquatic animals that were logged by the Centre for Environment, Fisheries and Aquaculture Science between 2002 and 2017 revealed that more than half were caused by viruses. However, in molluscs, most events were parasitic. Categorising these events indicated that the key processes underpinning emergence were the movement of live animals and host switching. Profiles of key pathogens further illustrate the importance of wild aquatic animals as the source of new infections in farmed animals. It is also clear that the spread of new diseases through the largescale movement of aquatic animals for farming, for food and for the ornamental trade has allowed many to achieve pandemic status. Many viral pathogens of fish (e.g. infectious salmon anaemia, viral haemorrhagic septicaemia) and shrimp (e.g. white spot syndrome virus) affect a large proportion of the global production of key susceptible species. Wild aquatic animal populations have also been severely affected by pandemic diseases, best exemplified by Batrachochytrium dendrobatidis, a fungal infection of amphibians, whose emergence and spread were driven by the movement of animals for the ornamental trade. Batrachochytrium dendrobatidis is now widespread in the tropics and subtropics and has caused local extinctions of susceptible amphibian hosts. Given the rising demand for seafood, aquacultural production will continue to grow and diseases will continue to emerge. Some will inevitably achieve pandemic status, having significant impacts on production and trade, unless there are considerable changes in global monitoring and the response to aquatic animal diseases.

The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout.

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.

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China.

Fish rhabdoviruses are a family of viruses responsible for large-scale fish die-offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT-PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT-PCR. Electron microscopy showed the presence of bullet-shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole-genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen-free largemouth bass juveniles. VHSVLB2018-infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%-93.3% by 7days post-infection. VHSVLB2018 was re-isolated from dead fish and confirmed by RT-PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.

Establishment and characterization of a fin tissue cell line derived from silver pomfret, Pampus argenteus.

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT-PCR and viral titre assays. In contrast, PaF cells were resistant to red-spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune-related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen-silver pomfret interaction.

Identification of the optimal insertion site for expression of a foreign gene in an infectious hematopoietic necrosis virus vector.

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.

Potential Role of Rainbow Trout Erythrocytes as Mediators in the Immune Response Induced by a DNA Vaccine in Fish.

In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV (GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor signaling pathway, the autophagy pathway, and the activation of the innate immune response, among others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the future design of novel vaccine strategies.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

Molecular characterization and expression analysis of rockfish (Sebastes schlegelii) viperin, and its ability to enervate RNA virus transcription and replication in vitro

Viperin, also known as RSAD2 (Radical S-adenosyl methionine domain containing 2), is an interferon-induced endoplasmic reticulum-associated antiviral protein. Previous studies have shown that viperin levels are elevated in the presence of viral RNA, but it has rarely been characterized in marine organisms. This study was designed to functionally characterize rockfish viperin (SsVip), to examine the effects of different immune stimulants on its expression, and to determine its subcellular localization. SsVip is a 349 amino acid protein with a predicted molecular mass of 40.24 kDa. It contains an S-adenosyl l-methionine binding conserved domain with a CNYKCGFC sequence. Unchallenged tissue expression analysis using quantitative real time PCR (qPCR) revealed SsVip expression to be the highest in the blood, followed by the spleen. When challenged with poly I:C, SsVip was upregulated by approximately 60-fold in the blood after 24 h, and approximately 50-fold in the spleen after 12 h. Notable upregulation was detected throughout the poly I:C challenge experiment in both tissues. Significant expression of SsVip was detected in the blood following Streptococcus iniae and lipopolysaccharide challenge, and viral hemorrhagic septicemia virus (VHSV) gene transcription was significantly downregulated during SsVip overexpression. Furthermore, cell viability assay and virus titer quantification with the presence of SsVip revealed a significant reduction in virus replication. As with previously identified viperin counterparts, SsVip was localized in the endoplasmic reticulum. Our findings show that SsVip is an antiviral protein crucial to innate immune defense.

Transcriptome and proteome analyses of red blood cells from rainbow trout challenged with viral haemorrhagic septicaemia virus

Chromium and tungsten contents were varied to produce three different 125 ksi grade casing steels, and their sulfide stress cracking (SSC) behaviors were evaluated using double cantilever beam testing. The effects of chromium and tungsten on the SSC resistance were clarified by microstructure analysis and hydrogen permeation test. Reducing the chromium content and adding tungsten significantly increased the number of irreversible traps and decreased the proportion of the Σ3 boundaries and high-angle grain boundaries. Moreover, the addition of tungsten led to the reduction of hydrogen permeability and diffusivity, resulting in the enhancement of SSC resistance.

In vitro rainbow trout transcriptome reveals immune evasion associated with higher virulence of viral haemorrhagic septicaemia virus

In vitro rainbow trout transcriptome reveals immune evasion associated with higher virulence of viral haemorrhagic septicaemia virus

Nanoencapsulation of inactivated-viral vaccine using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in olive flounder (Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection

Viral haemorrhagic septicaemia virus (VHSV), a (−) ssRNA virus belonging to the genus Novirhabdovirus of rhabdoviridae family, is the aetiological agent of viral haemorrhagic septicaemia (VHS) disease which causes huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. Previously, we developed an inactivated vaccine viz., formalin-inactivated VHSV mixed with squalene as adjuvant which was effective in conferring protective immunity (58–76% relative percentage survival) against VHSV but the mode of administration was intraperitoneal injection which is not feasible for small sized fingerling fish. To overcome this limitation, we presently focused on replacing the injection route of vaccine delivery by oral and immersion routes. In this context, we encapsulated the inactivated VHSV vaccine with chitosan nanoparticles (CNPs-IV) by water-in-oil (W/O) emulsification method. After encapsulation, two sets of in vivo vaccination trials were conducted viz., preliminary trial-I and final trial-II. In preliminary trial-I, olive flounder fingerlings (10.5 ± 1.7 g) were vaccinated with CNPs-IV by different delivery strategies involving oral and immersion routes (single/booster dose) followed by challenge with VHSV (1 × 106 TCID50 virus/fish) to evaluate an effective method amongst different applied delivery strategies. Subsequently, a final trial-II was conducted to better understand the immune mechanism behind the efficacy of the employed delivery strategy and also to further improvise the delivery mechanism with prime-boost (primary immersion and oral boosting) combination in order to improve the transient anti-VHSV response in the host. Evaluation of RPS analysis in trial-I revealed higher RPS of 46.7% and 53.3% in the CNPs-IV (immersion) and CNPs-IV (immersion/immersion) groups, respectively compared to 0% RPS in the CNPs-IV (oral) group and 20% RPS in the CNPs-IV (oral/oral) group when calculated against 100% cumulative mortality percentage in the NVC (non-vaccinated challenged) control group, whereas, in the trial-II, RPS of 60% and 66.6% were obtained for CNPs-IV (immersion/immersion) and CNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, CNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to control, in both the systemic (kidney) and mucosal (skin and intestine) immune compartments of the host post immunization as well as post challenge. To conclude, mucosal immunization with CNPs-IV vaccine can orchestrate an effective immunization strategy in organizing a coordinative immune response against VHSV in olive flounder thereby exhibiting higher protective efficacy to the host with minimum stress.

Differences of Viral Hemorrhagic Septicemia Virus Loads among Organs of Dead and Surviving Olive Flounder Infected by Intramuscular Injection and Immersion Challenge.

Viral hemorrhagic septicemia virus (VHSV) is an important viral pathogen in the culture of Olive Flounder Paralichthys olivaceus. Based on cumulative mortality, the virulence of VHSV was found to be highly different depending on challenge routes and exposure doses (using tissue culture infectious dose with 50% endpoint [TCID50]). Olive Flounder were injected with VHSV at 102.5 , 104.5 , 106.5 , and 108.5 TCID50/100μL/fish. A second group of fish was immersed at 103.5 , 105.5 , and 107.5 TCID50/mL at 10°C for 1h in this study. The cumulative mortality was observed at 15d postinfection. Immersion challenge at 103.5 TCID50/mL caused no mortality, while intramuscular injection challenge resulted in high levels of mortality with all VHSV exposure doses. Overall, Olive Flounder was susceptible to VHSV, with cumulative mortality of 90% or 100% in fish intramuscularly injected with high or low doses of VHSV. The cumulative mortality was 40% and 70% at 105.5 and 107.5 TCID50/mL, respectively, in the immersion challenge group. The VHSV titration and copy numbers were estimated by TCID50 and quantitative reverse transcription PCR methods. From dead Olive Flounder, VHSV titration was consistently detected in all tested organs, ranging from 105 to 109 TCID50/mL. The VHSV titration was under the detection limit from surviving Olive Flounder, but the VHSV N gene was detected.

Kinetics of infectious virus and viral RNA copy number in the blood of olive flounder infected with viral hemorrhagic septicemia virus (VHSV)

Viral hemorrhagic septicemia (VHS) is a cold-water disease caused by viral hemorrhagic septicemia virus (VHSV) at an optimal temperature of 9 °C–15 °C. VHSV isolation and detection have been accomplished by using a number of diagnostic methods such as cell culture and qRT-PCR. Spleen and kidney have been reported as the main target organs of VHSV-infection; however, how VHSV spreads throughout the fish body has not been clearly studied. The purpose of this study was 1) to investigate viral titer and viral RNA copy number in the blood of VHSV-infected olive flounder at 10 °C and 13 °C; 2) to compare VHSV titer and viral RNA copy numbers in blood from fish exposed to the virus by two different challenges. VHSV titer at 10 °C was higher than at 13 °C in blood samples of injection challenged group. Whereas, similar titer was observed at 10 °C and 13 °C in the blood samples of the immersion challenged group. At 10 °C, copy numbers of VHSV-N gene in blood of immersion challenged group increased slightly in comparison to injection challenged group. At 13 °C, similar patterns were observed between the injection and immersion challenged groups. Also, higher titer and copy number were observed in fish blood compared to tested organs from our previous study. Our results indicate that VHSV genome existed in fish blood at earlier time points after infection, and the blood may contribute to the spread of the virus in whole fish body. In addition, VHSV diagnosis by qRT-PCR from fish blood samples, not requiring sacrificing the host fish can be valuable to collect the kinetic information of viral infection.

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers.

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.

Investigation of round goby viral haemorrhagic septicaemia outbreak in New York

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT-qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.

IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells

Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6 h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, these results suggest a possible role of IFIT5 in the antiviral response of RBCs against VHSV.

Effect of CXCL12-expressing viral hemorrhagic septicemia virus replicon particles on leukocytes migration and vaccine efficacy in olive flounder (Paralichthys olivaceus)

Viral replicon particles are single-cycle viruses defective for function(s) needed for viral replication, which allow them to be recognized as a safer form for the vaccination of animals compared to attenuated live viruses. However, deletion of genes that are critical for the induction of protective immunity can diminish the vaccine potential of viral replicon particles. Therefore, the manipulation of viral replicon particles to produce a molecular adjuvant can be a way to increase immunogenicity of vaccines based on viral replicon particles. Chemokines are a class of chemotactic cytokines that control the migration of diverse cells of vertebrates. CXC chemokine ligand 12 (CXCL12) binds to a receptor CXCR4, and CXCL12-CXCR4 signaling plays an important role in the migration of hematopoietic cells during embryogenesis and the attraction of leukocytes. In the present study, to evaluate the possible use of CXCL12 as a molecular adjuvant for an rVHSV-ΔG vaccine and to know differences between CXCL12a and CXCL12b in the adjuvant ability, we rescued VHSV replicon particles that are expressing olive flounder CXCL12a, CXCL12b, or eGFP (rVHSV-ΔG-CXCL12a, rVHSV-ΔG-CXCL12b, or rVHSV-ΔG-eGFP), and compared the ability to attract olive flounder leucocytes and to induce protection against a VHSV challenge. In the leukocytes migration assay, supernatants collected from cells infected with rVHSV-ΔG-CXCL12a and rVHSV-ΔG-CXCL12b showed significantly higher ability to attract olive flounder leukocytes than the supernatant of cells infected with rVHSV-ΔG-eGFP. Moreover, the significantly higher number of leukocytes were attracted to rVHSV-CXCL12a supernatant compared to rVHSV-CXCL12b supernatant, suggesting that CXCL12a would be more appropriate for the induction of immunity than CXCL12b in olive flounder. In the immunization experiment, olive flounder immunized with rVHSV-ΔG-CXCL12a showed significantly higher survival rate than fish immunized with rVHSV-ΔG-CXCL12b or rVHSV-ΔG-eGFP. In addition, fish immunized with rVHSV-ΔG-CXCL12a showed the highest serum neutralization activity. These results suggest the availability of CXCL12a for a molecular adjuvant of vaccines based on VHSV replicon particles.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56–6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout

BackgroundViral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence).MethodsOverlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection.ResultsComplete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0–10% mortality).ConclusionsWhen chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.