Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).

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.

Herbal active small molecule as an immunomodulator for potential application on resistance of common carp against SVCV infection.

Herbal immunomodulators are an important part of prevention and control on viral diseases in aquaculture because of their propensity to improve immunity in fish. The present study was conducted to evaluate the immunomodulatory effect and antiviral activity of a synthesized derivative (serial number: LML1022) against spring viremia of carp virus (SVCV) infection in vitro and in vivo. The antiviral data suggested that LML1022 at 100 µM significantly inhibited the virus replication in epithelioma papulosum cyprini (EPC) cells, and may completely inhibit the infectivity of SVCV virion particles to fish cells by affecting the viral internalization. The results in the related stability of water environments also demonstrated that LML1022 had an inhibitory half-life of 2.3 d at 15 °C, which would facilitate rapid degradation of LML1022 in aquaculture application. For in vivo study, the survival rate of SVCV-infected common carp was increased 30% at least under continuous oral injection of LML1022 at 2.0 mg/kg for 7 d treatment. Additionally, pretreatment of LML1022 on fish prior to SVCV infection also obviously reduced the viral loads in vivo as well as an improved survival rate, showing that LML1022 was potential as an immunomodulator. As an immune response, LML1022 significantly upregulated the immune-related gene expression including IFN-γ2b, IFN-I, ISG15 and Mx1, indicating that its dietary administration may improve the resistance of common carp against SVCV infection.

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

Specificity of DNA Vaccines against the Genogroup J and U Infectious Hematopoietic Necrosis Virus Strains Prevalent in China.

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In addition to the common genogroup J IHNV, genogroup U has been newly discovered in China. However, there is no effective DNA vaccine to fight against this emerging genogroup U IHNV in China. In this study, DNA vaccines encoding the IHNV viral glycoprotein (G) gene of the GS2014 (genogroup J) and BjLL (genogroup U) strains isolated from northern China were successfully developed, which were identified by restriction analysis and IFA. The expression of the Mx-1 gene and G gene in the spleens and muscles of the injection site as well as the titers of the serum antibodies were measured to evaluate the vaccine efficacy by RT-qPCR and ELISA. We found that DNA vaccine immunization could activate Mx1 gene expression and upregulate G gene expression, and the mRNA levels of the Mx1 gene in the muscles were significantly higher than those in the spleens. Notably, DNA vaccine immunization might not promote the serum antibody in fish at the early stage of immunization. Furthermore, the efficacy of the constructed vaccines was tested in intra- and cross-genogroup challenges by a viral challenge in vivo. It seemed that the DNA vaccines were able to provide great immune protection against IHNV infection. In addition, the genogroup J IHNV-G DNA vaccine showed better immune efficacy than the genogroup U IHNV-G or divalent vaccine, which could provide cross-immune protection against the genogroup U IHNV challenge. Therefore, this is the first study to construct an IHNV DNA vaccine using the G gene from an emerging genogroup U IHNV strain in China. The results provide great insight into the advances of new prophylactic strategies to fight both the genogroup J and U IHNV in China.

Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

FIP200 restricts RNA virus infection by facilitating RIG-I activation.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation.

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Impact of Vaccination and Pathogen Exposure Dosage on Shedding Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) in Rainbow Trout.

Vaccine efficacy in preventing clinical disease has been well characterized. However, vaccine impacts on transmission under diverse field conditions, such as variable pathogen exposure dosages, are not fully understood. We evaluated the impacts of vaccination on disease-induced host mortality and shedding of infectious hematopoietic necrosis virus (IHNV) in Rainbow Trout Oncorhynchus mykiss. Fish, in up to three different genetic lines, were exposed to different dosages of IHNV to simulate field variability. Mortality and viral shedding of each individual fish were quantified over the course of infection. As the exposure dosage increased, mortality, number of fish shedding virus, daily virus quantity shed, and total amount of virus shed also increased. Vaccination significantly reduced mortality but had a much smaller impact on shedding, such that vaccinated fish still shed significant amounts of virus, particularly at higher viral exposure dosages. These studies demonstrate that the consideration of pathogen exposure dosage and transmission are critical for robust inference of vaccine efficacy.

Evaluation on the antiviral effect of a hydroxycoumarin against infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis (IHN) caused by the viral pathogen infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of salmonid species, resulting in significant economic impact. The previous study showed a hydroxycoumarin derivative 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) significantly inhibited spring viraemia of carp virus (SVCV) infection, suggesting that D5 may be useful as a potential anti-IHNV agent. In this study, D5 at the concentration of up to 10 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate of >90%, maintained mitochondrial membrane potential (ΔΨm) levels, and decreased IHNV-induced apoptosis in virus-infected cells. As the consequence of protection on mitochondria, D5 enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain the antioxidant-oxidant balance of IHNV-infected EPC cells. For in vivo study, D5 via intraperitoneal injection exhibited an anti-IHNV effect in the virus-infected fish by substantially enhancing the survival rate. Meanwhile, up-regulation of six interferon (IFN) related gene expressions demonstrated that D5 may activate IFN-related expressions for inhibiting IHNV replication during the early stage of viral infection, which is beneficial for the continuous antiviral action on controlling low viral loads in rainbow trout juvenile. Thus, D5 effective regulated IHNV-induced undesirable conditions to be an excellent potential therapeutic agent against IHNV infection.

Application of antigen presenting cell-targeted nanovaccine delivery system in rhabdovirus disease prophylactics using fish as a model organism.

BACKGROUND: Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease. MATERIALS AND METHODS: In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention. RESULTS: Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group. CONCLUSION: This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

N-linked glycosylation sites in G protein of infectious hematopoietic necrosis virus (IHNV) affect its virulence and immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis in salmonid fish, resulting in substantial economic losses to the aquaculture industry worldwide. The G protein, which harbors the major antigenic determinants of IHNV, is an envelope glycoprotein that plays an important role in both pathogenicity and immunogenicity of IHNV. Previous studies have demonstrated that changes to viral glycosylation sites may affect replication and immunogenicity, but little is known about the specific contributions of G protein glycosylation to IHNV replication and pathogenicity. In this study, we predicted four N-linked glycosylation sites at position 56, 379, 401, and 438 Asp (N) in G protein, and using a reverse genetics system developed in our laboratory, constructed nine recombinant viruses with single, triple, or quadruple glycosylation site disruptions using alanine substitutions in the following combinations: rIHNV-N56A, rIHNV-N379A, rIHNV-N401A, rIHNV-N438A, rIHNV-N56A-N379A-N401A, rIHNV-N56A-N379A-N438A, rIHNV-N56A-N401A-N438A, rIHNV-N379A-N401A-N438A, and rIHNV-N56A-N379A-N401A-N438A. Our results confirmed that all four asparagines are sites of N-linked glycosylation, and Western blot confirmed that mutation of each predicted N-glycosylation sited impaired glycosylation. Among the nine recombinant IHNVs, replication levels decreased significantly in vitro and in vivo in the triple and quadruple mutants that combined mutation of asparagines 401 and 438, indicating the importance of glycosylation at these sites for efficient replication. Moreover, juvenile rainbow trout mortality after challenge by each of the nine mutants showed that, while eight mutants suffered almost 100% cumulative mortality over 30 days, the mutant with a single alanine substitution at position 438 resulted in cumulative mortality of less than 50% over 30 days. This mutant also elicited specific anti-IHNV IgM production earlier than other mutants, suggesting that glycosylation of asparagine 438 may be important for viral immune escape. In conclusion, our study reveals the effect of G protein glycosylation on the pathogenicity and immunogenicity of IHNV and provides a foundation for developing a live-attenuated vaccine.

Development of a live vector vaccine against infectious hematopoietic necrosis virus in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) leads to serious disease and economic losses in the salmonid aquaculture industry. The present study aimed to develop an effective and efficient vaccine to protect rainbow trout (Oncorhynchus mykiss) against IHNV infection. Administered via the immersion route, a live vector vaccine containing the regions of the IHNV glycoprotein (G) induced immune responses in rainbow trout. Use of the immersion route induced more-efficient mucosal immunity than intramuscular injection vaccination. IHNV G gene expression was detected in the spleens of rainbow trout at 3, 7 and 15 days post-vaccination (dpv). The G gene expression continuously decreased between 3 and 15 dpv. In addition, the expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were observed at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) gradually increased. When experimental fish were challenged with IHNV by immersion, significant differences in cumulative percentage mortality were observed in the vaccinated fish and the unvaccinated (empty-plasmid-vaccinated) fish. The relative survival rate was 92% and 6% in the vaccinated group and empty-plasmid group, respectively. Serum antibody levels gradually increased in the vaccinated fish, unlike in the unvaccinated fish, after 7 dpv. Our results suggest there was a significant increase in fish immune responses and resistance to infection with IHNV following administration of the live vector vaccine. Therefore, this live vector vaccine is a promising vaccine that may be utilized to protect rainbow trout against IHNV.

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)-GN438A expresses VP2 gene-encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross-protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV-N438A-ΔNV-EGFP and rIHNV-N438A-ΔNV-VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild-type (wt) IHNV HLJ-09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ-09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV-N438A-ΔNV-VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.

Intein-mediated expression and purification of common carp IFN-γ and its protective effect against spring viremia of carp virus.

IFN-γ is a pleiotropic cytokine with significant roles in antiviral, antitumor and immune regulation. It could be used as an immuno-enhancer to improve fish protectiveness against pathogens. In this study, the prokaryotic expression plasmid pTwin1-N-IFN-γ was constructed to express Cyprinus carpio (common carp) IFN-γ fused with a chitin binding domain (CBD) and a self-cleavable intein-tag, Synechocystis sp DnaB. The recombinant protein CBD-DnaB-IFN-γ with the molecular weight of 44.25 kD was successfully expressed in soluble form, and the rIFN-γ (approximate 18.61 kD) was further cleaved and eluted under pH = 7.0 at 25 °C. rIFN-γ could be recognized by western blotting with rabbit anti-grass carp IFN-γ polyclonal antibody. Cytotoxicity studies on EPC cells showed that only 500 ng/ml rIFN-γ had a subtle effect on cells growth and its proliferation rate was reduced to 76.2%. EPC cells incubated with 100 ng/ml rIFN-γ showed significantly higher resistance against SVCV, reducing the TCID50/ml by more than 800-fold. In vivo studies suggested that intraperitoneal injection of rIFN-γ significantly improved the survival rate of common carps compared with SVCV challenge alone. These results implied that rIFN-γ would act as an immuno-enhancer in carp aquaculture.

IFN-stimulated P2Y13 protects mice from viral infection by suppressing the cAMP/EPAC1 signaling pathway.

Among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. However, the function of P2 receptors in viral infection has been little explored. Here we demonstrated that P2Y13 and its ligand ADP play an important role in protecting hosts from viral infections. First, we demonstrate that P2Y13, as a typical interferon-stimulated gene, is induced together with extracellular ADP during viral infection. Most importantly, extracellular ADP restricts the replication of different kinds of viruses, including vesicular stomatitis virus, Newcastle disease virus, herpes simplex virus 1, and murine leukemia virus. This kind of protection is dependent on P2Y13 but not P2Y1 or P2Y12, which are also considered as receptors for ADP. Furthermore, cyclic adenosine monophosphate and EPAC1 are downregulated by extracellular ADP through the P2Y13-coupled Gi alpha subunit. Accordingly, inhibition or deletion of EPAC1 significantly eliminates ADP/P2Y13-mediated antiviral activities. Taken together, our results show that P2Y13 and ADP play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets.

In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene.

Spring viremia of carp, a fatal viral disease, is caused by the spring viremia of carp virus (SVCV) and can result in up to 70% mortalities in common carps and significant economic losses in several other cyprinid aquaculture. The present study aimed to investigate the possible control of SVCV replication in Epithelioma papulosum cyprini (EPC) cells using the RNA interference technology targeting the RNA-dependent RNA polymerase (L) gene of the SVCV that is essential for its replication. Three stealth small interfering RNA (siRNA) sequences were designed to target three different regions on the SVCV-L gene. The specific siRNAs designed were investigated individually or in combinations to inhibit the SVCV-L gene expression and the virus replication. Results showed that the most effective siRNA sequence was the siRNA-602 that specifically reduced the SVCV replication by two logs as indicated by the virus titration and quantitative real-time PCR. Results, also, showed that the minimum effective concentration of siRNA-602 was 20 nM when used to transfect the EPC cells before the virus inoculation. Results of this study clearly indicate that targeting the SVCV-L gene by RNAi can reduce the SVCV replication in vitro, that may lead to the control of SVCV in fish.

Broad-spectrum antiviral JL122 blocks infection and inhibits transmission of aquatic rhabdoviruses.

The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections.