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The demand for aquaculture is increasing, but production is declining due to high feed costs and disease outbreaks. Viral hemorrhagic septicemia (VHS) is a viral disease that seriously affects seawater and freshwater fish in aquaculture, including the olive flounder (Paralichthys olivaceus), a major aquaculture fish in Korea. However, very few vaccines are currently available for viral hemorrhagic septicemia virus (VHSV). The nutrient-rich microalga Chlorella vulgaris has been used as a feed additive in aquaculture and as a host for the industrial production of recombinant VHSV glycoprotein as an oral vaccine. In this study, VHSV glycoprotein was cloned with a salt-inducible promoter, and high levels of expression up to 41.1 mg/g wet C. vulgaris, representing 27.4% of total extracted soluble protein, were achieved by growing the transformed C. vulgaris for 5 days in the presence of 250 mM NaCl. The production of a neutralizing antibody was detected in the serum of fish given feed containing 9% VHSV glycoprotein-expressing C. vulgaris. Furthermore, relative survival rates of 100% and 81.9% were achieved following challenges of these fish with VHSV at 106 and 107 pfu/fish, respectively, indicating that C. vulgaris could be used as a platform for the production of recombinant proteins for use as oral vaccines in the control of viral diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3—which did not express NV protein—rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60–70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The introduction of reverse genetic technology to generate recombinant VHSVs (rVHSVs) has contributed to the uncovering of functional roles of viral genes and to the development of attenuated prophylactic vaccines. In this study, to assess the possible use of rVHSVs as a tool of combined vaccines, we newly rescued rVHSVs that harbor viral envelop-studded eGFP (rVHSV-A-SGT) or nucleoprotein-fused eGFP (rVHSV-A-NLG), and the ability of these rVHSVs to induce adaptive humoral immunity in olive flounder (Paralichthys olivaceus) was compared with that of rVHSV-A-eGFP that expresses eGFP as a soluble form in the cytoplasm of infected cells. The results showed that antibodies against eGFP were efficiently induced by the immunization of olive flounder with rVHSV-A-SGT and rVHSV-A-NLG, while rVHSV-A-eGFP was poor in the ability to induce antibody response against eGFP. These results suggest that the display of heterologous antigens on VHSV envelop is a good way to develop efficient combined vaccines and the fusion of foreign antigen with N protein can also be a way to enhance immunogenicity of a foreign antigen. The present recombinant VHSVs - rVHSV-A-SGT and rVHSV-A-NLG – not only express foreign antigens in host cell cytoplasm but also display antigens in or on the virus particles. Further researches on the availability of recombinant VHSVs as combined vaccines against multiple fish pathogens are needed. • The possible use of rVHSVs expressing antigen on or in viral particles was assessed. • rVHSV expressing viral envelop-studded eGFP (rVHSV-A-SGT) was rescued. • Another rVHSV expressing nucleoprotein-fused eGFP (rVHSV-A-NLG) was rescued. • Antibodies against eGFP were efficiently induced by rVHSV-A-SGT and rVHSV-A-NLG. • Display of antigens on VHSV envelop would be a good way to develop combined vaccines. [ABSTRACT FROM AUTHOR]

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. • Reverse genetic methods for the rescue of recombinant VHSVs are described. • The possible use of attenuated live VHSV and single-cycle VHSV was described. • Recombinant VHSVs as a delivery vehicle for foreign antigens are described. [ABSTRACT FROM AUTHOR]

Abstract Vaccines based on viral replicon particles would be advantageous to induce immune responses compared to inactivated viruses in that they can infect host cells (only once) and can produce viral proteins in the infected cells like live viruses. Furthermore, as viral replicon particles are replication-defective, they are safer than live attenuated viruses. Previously, we had rescued viral hemorrhagic septicemia virus (VHSV) replicon particles lacking full ORF of G gene (rVHSV-ΔG). In the present study, to enhance the immunogenicity of VHSV replicon particles, we newly generated another form of VHSV replicon particles that can produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM), and compared the protective efficacy of rVHSV-GΔTM with that of rVHSV-ΔG through immunization of olive flounder (Paralichthys olivaceus). In addition, we evaluated the safety of rVHSV-GΔTM by the analysis of effects on wild-type VHSV replication. In the vaccine experiment, olive flounder immunized with rVHSV-GΔTM showed significantly higher titers of serum neutralization activity than fish immunized with rVHSV-ΔG suggesting that the G protein that is not only spiked on the viral envelop but also secreted extracellularly can contribute to the enhancement of adaptive humoral immunity. Moreover, fish immunized with rVHSV-GΔTM showed higher survival rates than fish immunized with rVHSV-ΔG, suggesting that the amount of G protein provided to hosts is an important factor for the enhancement of vaccine efficacy against VHSV disease. In a safety aspect, rVHSV-GΔTM could not replicate in infected cells, and significantly inhibited the replication of wild-type VHSV when co-infected, suggesting that rVHSV-GΔTM would not worsen disease progression caused by wild-type VHSV infection. Highlights • Replicon particles of VHSV that can secret G protein (rVHSV-GΔTM) were generated. • Protective efficacy of rVHSV-GΔTM in olive flounder was higher than that of rVHSV-ΔG. • RVHSV-GΔTM induced significantly higher serum neutralization activity than rVHSV-ΔG. • RVHSV-GΔTM significantly inhibited the replication of wild-type VHSV when co-infected. [ABSTRACT FROM AUTHOR]

Abstract Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV. Highlights • IRF9 gene knockout EPC cells were produced using a CRISPR/Cas9 vector. • Knockout of IRF9 gene decreased the expression of ISG15 and Mx1 genes. • Decrease of type I IFN response allowed VHSV to replicate efficiently. • The present IRF9 knockout EPC cells can be used to get higher titered VHSV. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1β, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection. [ABSTRACT FROM AUTHOR]

The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12–36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3′ end of cRNA might be lower than that to the 3′ end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication. • Dynamics of 3 VHSV RNA strands in EPC cells was analyzed using a strand-specific RT-qPCR. • Viral mRNA transcription and cRNA copy number were significantly higher at 20 °C than at 15 °C. • Changes in RNA copy numbers were small but significantly higher in IRF-9 knockout cells. • The copy number of cRNA never exceeded the copy number of vRNA at all assay times. [ABSTRACT FROM AUTHOR]

Transcription factors related to the activation of type I interferons (IFNs) and nuclear factor-kappa B (NF-κB) are known to be critical in innate immune responses. Interferon regulatory factors (IRFs) are a family of transcription factors. IRF-3 is known to act as the primary regulator in type I IFN signaling in response to viral infections, and the upregulation of IRF5 by virus infection has been reported in various fish species. One of the ways to know the functional role of certain genes is the production of target gene(s) knockout cells or organisms. In the present study, we produced either IRF3 or IRF5 gene knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 system, and investigated the effect of IRF3 gene and IRF5 gene knockout on polyinosinic:polycytidylic acid (ploly (I:C))-mediated and viral hemorrhagic septicemia virus (VHSV) infection-mediated type I IFN response and NF-κB activation. Both IRF3 knockout and IRF5 knockout EPC cells showed severely decreased type I IFN responses measured by ISRE activity and the expression of Mx1 and ISG15 genes when stimulated with poly (I:C), while the decreased level of type I IFN responses was not high as by poly (I:C) stimulation when infected with VHSV. Different from type I IFN response, NF-κB activities in IRF3 and IRF5 knockout cells were not highly different between poly (I:C) stimulated cells and VHSV-infected cells. Further studies are needed to elucidate pathways responsible for the type I IFN responses and NF-κB activation by VHSV infection. • Either IRF3 or IRF5 gene of EPC cells were knocked out using a CRISPR/Cas9 system. • Both knockout cells showed severely decreased type I IFN responses by poly (I:C). • Decrease of type I IFN responses in both knockout cells were not high by VHSV infection. • NF-κB activities in both knockout cells to poly (I:C) and VHSV were not highly different. [ABSTRACT FROM AUTHOR]

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines – viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated. [ABSTRACT FROM AUTHOR]

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are potent immunostimulators in vertebrates. Based on the backbone structure and the oligonucleotides sequences, CpG-ODNs can be classified into three different classes (A, B, and C) that induce class-specific immune responses. Plasmids with stimulatory CpG motifs in their backbone also show immunostimulatory effects, and a plasmid-based delivery of CpG motifs can be an alternative approach to expensive CpG-ODNs. In this study, we constructed a plasmid (pL-CpGmix) that harbors multiple copies of CpG-ODN 2216 (class A), 1668 (class B), and 2395 (class C) motifs, and evaluated the potential of the constructed plasmid to protect of olive flounder ( Paralichthys olivaceus ) from viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish were administered pL-CpGmix, pL-GFP that harbored a green fluorescent protein (GFP) gene, empty vector, or phosphate buffered saline (PBS) alone. On 3 days post-administration, fish were challenged with VHSV or M. avidus . Fish administered pL-CpGmix showed the lowest cumulative mortality (60%) against a VHSV challenge, which was significant compared to that of the control groups (empty vector and PBS). In the M. avidus challenge experiment, fish in the group administered pCpG-mix showed the highest survival rate and serum scuticocidal activity, and those were statistically significant compared to those of control groups. Although fish administered pL-GFP showed lower survival rates than fish administered pL-CpGmix, the GFP-containing vector induced higher survival rates against M. avidus and VHSV infections and higher serum scuticocidal activity compared to the control groups, suggesting that GFP sequence might have an immunostimulating property. The present results showed that the plasmid harboring different classes of CpG motifs enhanced resistance of olive flounder against both M. avidus and VHSV infections. [ABSTRACT FROM AUTHOR]

Abstract: The role of viral hemorrhagic septicemia virus (VHSV) NV gene in nuclear factor-κB (NF-κB) activation was investigated. Epithelioma papulosum cyprini (EPC) cells pre-treated with tumor necrosis factor (TNF)-α showed a strong resistance against VHSV infection, but cells treated with TNF-α after VHSV infection showed no resistance, suggesting that immediate early TNF-α-mediated responses inhibit VHSV replication. Activation of NF-κB is a key step in TNF-α-mediated immunomodulatory pathways. In this study, activation of NF-κB by TNF-α exposure was inhibited in EPC cells harboring NV gene expressing vectors, indicating that the NV gene of VHSV can suppress TNF-α-mediated NF-κB activation. Furthermore, the NV gene knock-out recombinant VHSV (rVHSV-ΔNV-EGFP) induced significantly higher NF-κB activity in EPC cells than wild-type VHSV, suggesting that VHSV adopted a strategy to suppress early activation of NF-κB in host cells through and NV gene. [Copyright &y& Elsevier]

Abstract: To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish. [Copyright &y& Elsevier]

Abstract: Recently, we have reported the rescue of a recombinant viral hemorrhagic septicemia virus (VHSV) that has enhanced green fluorescent protein (EGFP) gene ORF instead of NV gene ORF (rVHSV-ΔNV-EGFP), and demonstrated that intramuscular injection or oral administration of the recombinant VHSV induced high protection against wild-type VHSV challenge in olive flounder (Paralichthys olivaceus). In the present study, we have firstly generated two types of recombinant VHSV chimeras; one has hirame rhabdovirus (HIRRV) G gene instead of VHSV G gene (rVHSV-HG-ΔNV-EGFP), the other harbors both HIRRV G and VHSV G genes (rVHSV-HG-VG-ΔNV-EGFP), and analyzed their ability to induce serum neutralization activity against VHSV and HIRRV. The neutralization activity against each virus (HIRRV and VHSV) was dependent on the presence of the corresponding G gene in the genome, supporting that G protein is the major antigen to induce neutralizing antibodies against rhabdoviruses. Induction of high neutralization activity against both VHSV and HIRRV by immunization with rVHSV-HG-VG-ΔNV-EGFP suggests that recombinant VHSVs might be expected to be used as combined vaccines in fish. [Copyright &y& Elsevier]

Abstract: The objective of this study was to investigate the effects of different classes of CpG oligodeoxynucleotides (CpG-ODNs) on resistance of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish administered CpG-ODN 2216 (A-class ODN) showed the highest resistance against VHSV infection and the strongest expression of Mx and ISG15 genes. Fish administered CpG-ODN 2395 (C-class ODN) showed intermediate resistance against VHSV between ODN 1668 and ODN 2216. Whereas, olive flounder intraperitoneally (i.p.) injected with CpG-ODN 1668 (B-class ODN) showed no enhanced resistance against VHSV challenge and no significant increase in expression of Mx and ISG15 genes. In contrast, in the M. avidus challenge experiment, fish injected with CpG-ODN 1668 showed the highest survival rate and the strongest scuticocidal activity of serum. Similar to the results in the VHSV infection experiment, administration of CpG-ODN 2395 elicited secondly higher survival rate against M. avidus challenge. Although fish administered CpG-ODN 2216 eventually showed 100% mortality by M. avidus challenge, the cumulative mortality until 2weeks post-challenge was similar to the group of fish injected with CpG-ODN 1668. The present results suggest that CpG-ODNs belonging to different classes have different abilities to increase resistance against different pathogens in olive flounder. Therefore, selection of a CpG-ODN appropriate to the characteristics of a certain pathogen is crucial to induce immune responses that are optimal for defense against the pathogen. [Copyright &y& Elsevier]

Viral hemorrhagic septicemia virus (VHSV) has been a main cause of morbidity and mortality in cultured olive flounder (Paralichthys olivaceus) in Korea. The aim of the present study was to construct recombinant baculovirus-based VHSV G protein delivery tools for the immunization of olive flounder. We rescued recombinant A. californica multiple nucleopolyhedrovirus (AcMNPV) i) displaying VHSV G protein on the viral envelop, ii) harboring a CMV promoter-driven VHSV G protein gene cassette, or iii) having a bicistronic construct equipped with both envelope-spiked G protein and a CMV promoter driven G protein cassette. In the immunization experiment, groups of olive flounder immunized with recombinant baculoviruses carrying VHSV G protein on the envelope showed significantly higher survival rates than groups of fish in the control group (medium only) and the control recombinant baculovirus (expressing green florescent protein) immunized group, suggesting the possible use of recombinant baculoviruses carrying foreign antigens on the envelope for the development of fish vaccines. The survival rate of fish immunized with a recombinant baculovirus having a CMV-driven VHSV G protein expressing cassette was higher than that of fish immunized with a baculovirus having a reporter protein-expressing cassette, but was much lower compared to previously reported results of VHSV G protein-based DNA vaccines, suggesting that the delivery efficiency of the present baculovirus-mediated VHSV G protein gene into olive flounder cell's nucleus might not be high compared to plasmid-based DNA vaccines. Although the group of fish immunized with the recombinant baculovirus equipped with both envelope-spiked G protein and a CMV promoter driven G protein cassette showed the highest survival rate against VHSV challenge, further elaborate studies are needed to uncover the synergic effect of the two forms of VHSV G protein delivered by recombinant baculoviruses. • Recombinant baculoviruses harboring or expressing VHSV G protein were rescued. • Baculovirus with VHSV G protein on envelope induced higher protection in olive flounder. • Baculovirus with CMV promoter-driven VHSV G protein induced a weak protection. • Baculovirus can be used as a delivery vehicle of antigens or genes for fish vaccination. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Production of functional miR-30e by a CMV promoter-driven vector was verified. • Down-regulation of reporters containing 3′-UTR of NFκbiαb by miR-30e was demonstrated. • A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) was rescued. • Expression of miR-30e by rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. • RVHSV-A-miR30e ncreased NF-κB activity and type I interferon induced genes expression. [ABSTRACT FROM AUTHOR]

Abstract Rhabdoviral G protein-based DNA vaccines have been recognized as a useful way to protect cultured fish from rhabdoviral diseases. In Korea, viral hemorrhagic septicemia virus (VHSV) genotype IVa has been the primary culprit of high mortalities of cultured olive flounder (Paralichthys olivaceus). In this study, we inserted a miR-155-expressing cassette into the VHSV's G protein-based DNA vaccine, and analyzed the effects of miR-155 on the antiviral activity and on the vaccine efficacy in olive flounder. Olive flounder fingerlings were intramuscularly (i.m.) immunized with 10 μg/fish (1st experiment) or 1 μg/fish (2nd experiment) of DNA vaccine plasmids. However, there were no significant differences in mortalities and serum neutralization titers between fish immunized with 1 μg and 10 μg plasmids/fish, suggesting that i.m. injection with 1 μg plasmids/fish would be enough to induce effective adaptive immune responses in olive flounder fingerlings. In survival rates, as fish immunized with just G protein expressing plasmids showed no or too low mortalities, the adjuvant effect of miR-155 was not discernible. Also, in the serum neutralization activities, although G gene or G gene plus miR-155 expressing DNA vaccines induced significantly higher activities than control vaccines (PBS and vacant vector), no significant differences were found between G gene alone and G gene plus miR-155 expressing DNA vaccines. In the serum virucidal activity, fish immunized with G gene plus miR-155 expressing DNA vaccine showed significantly higher activity against hirame rhabdovirus (HIRRV) at 3 days post-immunization (d.p.i.) compared to other groups, suggesting that miR-155 produced from the vector can enhance innate immune responses in olive flounder. The significantly enhanced serum virucidal activities against VHSV especially at 28 d.p.i. in the groups immunized with G gene alone and G gene plus miR-155 expressing DNA vaccines reflect the increased antibodies against G protein, which could activate the classical complement pathway and subsequent viral inactivation. As the available information on the DNA vaccines in olive flounder is not sufficient, more diverse researches on the protective efficacy of DNA vaccines are needed to make more practical use of DNA vaccines in olive flounder farms. Highlights • The potential of miR-155 as a molecular adjuvant for VHSV DNA vaccine was assessed. • Adjuvant effect of miR-155 was not discernible due to too low mortalities in immunized groups. • Significantly higher serum virucidal activity against HIRRV was induced by pFC-vG-miR155. • Immunization with 1 μg plasmids/fish was enough to induce effective adaptive immune responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vaccines based on inactivated or attenuated viruses can be a way to prevent viral hemorrhagic septicemia virus (VHSV) disease, and the efficiency of viral production is a critical factor that can determine the practical use of developed vaccines in aquaculture farms. To know the effects of epithelioma papulosum cyprini (EPC) cells over-subculture on VHSV replication, the VHSV titer produced from high-passage EPC cells (subcultured more than 200 times in our laboratory) was compared to the titer produced from low-passage EPC cells (subcultured 5–15 times). Furthermore, to know whether immune factors are involved in VHSV titers, differences not only in the expression of Mx1 and ISG15 genes but also in the apoptosis progression by VHSV infection between high- and low-passage EPC cells were analyzed. The VHSV titers from high-passage EPC cells were significantly higher than titers from low-passage EPC cells, suggesting that the changed properties of EPC cells by over-subculture were favorable for VHSV proliferation. The DNA laddering of high-passage EPC cells by VHSV infection took a longer time than that of low-passage EPC cells, suggesting that over-subculture might delay apoptosis in VHSV infected EPC cells, and the delay of apoptosis by over-subculture can be thought as one of the factors that increased VHSV titers in high-passage EPC cells. The increased folds of Mx1 and ISG15 genes in high-passage EPC cells were significantly lower than those in low-passage EPC cells when exposed to either poly (I:C) or VHSV. However, the expression levels of Mx1 and ISG15 genes of high-passage EPC cells that were not stimulated with poly I:C or VHSV were almost equal to or higher than the expression levels of low-passage EPC cells that were exposed to poly (I:C) or VHSV. This result suggests that high-passage EPC cells were already in an excited state in type I interferon responses without any stimulants. The full open reading frame (ORF) sequences of Mx1 gene between high- and low-passage EPC cells were completely same. However, there were some differences in the amino acids sequences of ISG15 gene between high- and low-passage EPC cells, suggesting that ISG15-mediated pathways might be different between high- and low-passage EPC cells, which might influence on the replication of VHSV. The present results showed that the changed properties of EPC cells by over-subculture were favorable for VHSV proliferation. [ABSTRACT FROM AUTHOR]

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV. • Rapid neutralization assay against VHSV was developed using recombinant SHRV. • rSHRV-Gvhsv-eGFP expressing eGFP was rescued and evaluated for neutralization assay. • Neutralization assay using rSHRV-Gvhsv-eGFP was faster than that using rVHSV-A-eGFP. • Assay using rSHRV-Gvhsv-eGFP was more rapid and convenient than conventional method. [ABSTRACT FROM AUTHOR]

In the development of inactivated or attenuated viral vaccines for cultured fish, viral titers harvested from the cultured cells would be the most important factor for the determination of vaccine's cost effectiveness. In this study, we hypothesized that the lengthening of cell survival time by the inhibition of apoptosis can lead to an increase of the final titer of viral hemorrhagic septicemia virus (VHSV). To test the hypothesis, we investigated the effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on VHSV infection-mediated apoptosis in Epithelioma papulosum cyprini (EPC) cells and on the VHSV titers. VHSV infection induced the DNA laddering in EPC cells, and the progression of DNA fragmentation was in proportion to the CPE extension. The progression of DNA fragmentation in EPC cells infected with VHSV was clearly inhibited by exposure to Z-VAD(OMe)-FMK, and the inhibition was intensified according to the increase of the inhibitor concentration. These results confirmed the previous reports that the death of host cells by VHSV infection is through apoptosis. Cells infected with a recombinant VHSV, rVHSV-ΔNV-eGFP, that was generated from our previous study by replacement of the NV gene ORF with the enhanced green fluorescent protein (eGFP) gene ORF, showed earlier and more distinct DNA fragmentations compared to the cells infected with wild-type VHSV, suggesting the inhibitory role of the NV protein in VHSV-mediated apoptosis that was previously reported. The final viral titers in the supernatant isolated from Z-VAD(OMe)-FMK treated cells after showing an extensive CPE were significantly higher than the viral titers from cells infected with virus alone, indicating that the delay of apoptosis by Z-VAD(OMe)-FMK extended the survival time of EPC cells, which lengthen the time for VHSV replication in the cells. In conclusion, Z-VAD(OMe)-FMK-mediated inhibition of apoptosis significantly increased the final titers of both wild-type VHSV and rVHSV-ΔNV-eGFP, indicating that apoptosis inhibition can be a way to get higher titers of VHSV. [ABSTRACT FROM AUTHOR]

Generation of recombinant viruses lacking an essential gene for the production of infective viral particles would be a way to produce safety-enhanced live viral vaccines. The rhabdoviral envelope-spiked glycoprotein (G) plays critical roles in the attachment of viruses on the cell surface receptor and in the transfer of endocytosed viruses to the cytoplasm by fusion to the endosomal membrane. In the present study, we produced a G gene-lacking recombinant viral hemorrhagic septicemia virus (rVHSV-ΔG) that has no ability to form plaques in the cells without a trans-supply of the G protein, which suggests that rVHSV-ΔG is a single cycle virus and progenies of rVHSV-ΔG are not infectious. One of the major advantages of attenuated vaccines is the maintenance of replication ability in the host, which enforces the adaptive immune responses. However, in spite of lacking an ability to produce infective viral particles, immunization with the present rVHSV-ΔG induced significantly higher serum neutralization titers and survival rates against virulent VHSV challenge in olive flounder ( Paralichthys olivaceus ) fingerlings, indicating that the present rVHSV-ΔG has a high potential as a prophylactic vaccine. [ABSTRACT FROM AUTHOR]

Abstract: A recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) that has enhanced green fluorescent protein (EGFP) gene instead of NV gene was previously generated using reverse genetics technology. In this study, potential of the rVHSV-ΔNV-EGFP to be used as a live oral vaccine candidate was assessed. The presence of the recombinant virus in internal organs of orally administered olive flounder (Paralichthys olivaceus) was analyzed by semi-quantitative RT-PCR. Although the recombinant VHSV-specific band was detected only when the number of PCR cycle was increased to 35, the band was detected from internal organs, such as kidney, spleen, and liver of fish that were reared at either 15 °C or 20 °C till even 20 days, suggesting that a few orally administered rVHSV-ΔNV-EGFP might be transported to internal organs, and might keep weak replication ability in the organs. VHSV-neutralizing activity was induced by oral immunization of olive flounder with the NV gene knock-out recombinant VHSV not only in skin and intestinal mucus but also in serum, suggesting that mucosal and systemic adaptive immune responses were elicited by oral immunization. In challenge experiment, groups of fish immunized with 104, 105, and 2 × 105 PFU of rVHSV-ΔNV-EGFP/fish showed 25%, 50%, and 70% of relative percent survival (RPS), respectively. The RPSs were elevated to 60%, 75%, and 90% by a boost immunization in fish boost immunized with 104, 105, and 2 × 105 PFU of rVHSV-ΔNV-EGFP, respectively. The cumulative mortality of fish in the control groups was 100%. Conclusionly, the present results demonstrate that the NV gene knock-out recombinant VHSV administered orally to olive flounder can induce dose- and boosting-dependent VHSV-neutralizing antibody in mucus and serum, and can provide a high protection in olive flounder against a virulent VHSV challenge. [Copyright &y& Elsevier]

• Rescue of chimeric SHRVs and VHSVs having heterologous G protein. • SHRV minigenome to check role of VHSV N, P, L proteins in low temperature replication. • VHSV N and P proteins were not main factors for low-temperature replication of VHSV. • Not only G protein but L protein was responsible for low-temp replication of VHSV. 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. [ABSTRACT FROM AUTHOR]

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. • Replicon particles of VHSV that can express CXCL12 (rVHSV-ΔG-CXCL12) were rescued. • Supernatant from cells infected with rVHSV-CXCL12a attracted the highest number of leukocytes. • rVHSV-ΔG-CXCL12a induced higher survival than rVHSV-ΔG-CXCL12b or rVHSV-ΔG-eGFP. • Fish immunized with rVHSV-ΔG-CXCL12a showed the highest serum neutralization activity. [ABSTRACT FROM AUTHOR]

• Recombinant VHSVs genotype IVa with mutations in M protein (M-D62A E181A) were rescued. • Most rescued recombinant viruses showed non-targeted secondary mutations in M protein. • VHSV mutants having only targeted mutations showed weakened ability against antiviral responses. • Most secondary mutations compensated for the deleterious effect of the engineered mutations. The matrix (M) protein of rhabdoviruses locates between the inner line of the viral envelope and the nucleocapsids core and plays an important role in viral replication. In the present study, we aimed to rescue a mutant of VHSV genotype IVa that has artificial mutations in the M protein (M-D62A E181A). However, most rescued recombinant viruses unexpectedly showed non-targeted secondary mutations in the M protein. Therefore, this study was conducted to know whether the targeted artificial mutation can lead to specific non-targeted secondary mutations in the M protein and whether the secondary mutations are compensatory for the targeted artificial mutations. Experiments were conducted to rescue three kinds of M protein mutants (rVHSV-M-D62A, -E181A, and -D62A E181A), and rVHSV-M-E181A and rVHSV-M-D62A E181A without the secondary mutations were rescued only from IRF-9 gene-knockout EPC cells. Recombinant VHSVs having only targeted mutation(s) (rVHSV-M-D62A, -E181A, and -D62A E181A) showed slower CPE progression and retarded growth compared to rVHSV-wild. Although the sites of secondary mutations were changed in every transfection experiment to generate recombinant VHSVs, the positions of the secondary mutations were not random. Some amino acid residues in the M protein showed more frequent mutations than others, and the changed amino acid residues were always the same. EPC cells infected with rVHSV-M-D62A E181A showed significantly higher type I interferon response and NF-κB activity, and the inhibitory activity against type I interferon response and NF-κB activity in other recombinant VHSVs having secondary mutations in M gene were similar to those of rVHSV-wild. In conclusion, the present results showed that VHSV actively responded to the artificial mutation of M protein through the secondary mutations, and those secondary mutations occurred when the artificial mutations were deleterious to viral replication and protein stability. Furthermore, most secondary mutations in recombinant viruses compensated for the deleterious effect of the engineered mutations. [ABSTRACT FROM AUTHOR]

To evaluate the protective effect of viral hemorrhagic septicemia virus genotype IVa (VHSV IVa) genome-based single-cycle viruses against VHSV genotype Ia (VHSV Ia) and infectious hematopoietic necrosis virus (IHNV) in rainbow trout, three kinds of single-cycle VHSVs were rescued using reverse genetic technology: i) rVHSV-IaGΔTM-IVaG containing the transmembrane and cytoplasmic region-deleted G protein (GΔTM) of VHSV Ia instead of VHSV IVa full G gene ORF and having VHSV IVa G proteins on the envelope; ii) rVHSV-IaGΔTM-IaG containing VHSV Ia GΔTM instead of VHSV IVa full G gene ORF and having VHSV Ia G proteins on the envelope; iii) rVHSV-IaGΔTM-ihnvGΔTM-IVaG containing not only VHSV Ia GΔTM instead of full G gene but also IHNV GΔTM instead of NV gene and having VHSV IVa G proteins on the envelope. Rainbow trout immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG showed significantly higher serum antibody titers against both VHSV Ia and VHSV IVa, and showed no mortality against VHSV Ia infection, while fish in the control groups showed 100% mortalities. Fish immunized with rVHSV-IaGΔTM-ihnvGΔTM-IVaG showed significantly higher serum antibody titers against VHSV IVa, VHSV Ia, and IHNV compared to fish in the control group. Immunization with rVHSV-IaGΔTM-ihnvGΔTM-IVaG induced significantly higher protection against not only VHSV Ia but also IHNV. These results suggest that the present single-cycle rVHSV-based system can be used as a platform to produce combined vaccines that can protect fish from multiple pathogenic species. However, the mechanism of the high protection against IHNV despite comparatively low antibody titer remains to be investigated. • VHSV IVa genome-based single-cycle viruses as bivalent vaccines in rainbow trout was evaluated. • rVHSV-IaGΔTM-IVaG had Ia G ectodomain instead of IVa G in genome and IVa Gs on envelope. • rVHSV-IaGΔTM-ihnvGΔTM-IVaG had ectodomain of Ia and IHNV G, and IVa Gs on envelope. • No mortality in fish immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG against Ia challenge. • rVHSV-IaGΔTM-ihnvGΔTM-IVaG simultaneously protected against VHSV Ia and IHNV challenges. [ABSTRACT FROM AUTHOR]

To know the effect of viral hemorrhagic septicemia virus (VHSV) infection on the cellular microRNA expression profile in olive flounder ( Paralichthys olivaceus ), fish were infected with VHSV, and cellular microRNAs expression was analyzed at 0 (control), 6, 12, 24, 48 and 72 h post-infection (h.p.i.) by the high-throughput sequencing. A total of 372 mature miRNAs were identified, and, among them, 63 miRNAs were differentially expressed during VHSV infection. The differentially expressed microRNAs number was greatly increased from 24 h.p.i. compared to the number at 6 and 12 h.p.i., suggesting that the alteration of microRNAs expression by VHSV infection may be related to the progression of VHSV disease. The target prediction analysis, the GO enrichment analysis, and the KEGG pathway analysis of the predicted target genes showed that various biological pathways could be affected by VHSV infection through the down-regulation or up-regulation of host miRNAs. The present results provide a basic information on the microRNAs related to VHSV infection in olive flounder. Considering broad effects of microRNAs on various biological pathways, data in this study can be used to interpret the mechanism of VHSV pathogenesis, which, vice versa, can be used to develop control measures against VHSV. [ABSTRACT FROM AUTHOR]