Oral administration of recombinant Lactococcus lactis expressing largemouth bass (Micropterus salmoides) IFNa3 protein enhances immune response against largemouth bass virus (LMBV) infection.

Largemouth bass virus (LMBV) is a highly pathogenic pathogen that often causes high mortality of affected largemouth bass and significant financial losses. Type I interferon as an effective and broad spectrum tool has been successfully used for therapeutic or prophylactic treatment some viral infections. However, the implementation of immunotherapies based on interferon administration to combat LMBV infections has not been reported. And Lactic Acid Bacteria (LAB) are a powerful vehicle for expressing cytokines or immunostimulant peptides at the gastrointestinal level after oral administration. In this study, Lactococcus lactis (L. lactis) expression system with lactose as a screening marker was utilized to express the Micropterus salmoides interferon a3 (IFNa3) protein and orally administered to largemouth bass. The genetically engineered strain pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 was successfully constructed, and its potential to elicit immune protection response by oral administration was evaluated. After orally administration, the recombinant L. lactis was detected in guts of experimental fish and remained detectable for 72 h. Additionally, IFNa3 was able to enhance the test fish's immune response, as determined by the relatively increased mRNA relative expression of immune-related genes in the liver, spleen, and kidney tissues, including IFN-γ, TNF-α, IL-1ß, IL-8, IgM and IgT. Following LMBV challenge, the experiment group of pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 exhibited a 70 % survival rate, while survival rate were 15 % in the PBS control group, 45 % in the pNZ8149/L. lactis NZ3900 group. Furthermore, the viral load in the surviving fish was significantly lower than that of the control groups. These findings suggest that oral administration of recombinant L. lactis producing IFNa3 induces largemouth bass immune responses at a systemic level to effective prevent and combat of LMBV infection.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

Infectious hematopoietic necrosis virus truncated G protein effect on survival, immune response, and disease resistance in rainbow trout.

The major antigenic protein of infectious hematopoietic necrosis virus (IHNV) is the surface glycoprotein G, which contains neutralizing epitopes that induce the production of immune neutralizing antibodies. In this study, the IHNV G gene sequence was truncated according to bioinformatics principles and then recombinantly expressed via an E. coli expression system. We then assessed the specific antibody immunoglobin M (IgM) levels of rainbow trout immunized with recombinant truncated G protein (emulsified with Freund's incomplete adjuvant), and showed that antibody IgM levels of immunized fish were significantly higher than in the control group (p < 0.01). The mRNA expression levels of interferon 1 (IFN1) and interleukin-8 (IL-8) were also up-regulated significantly (p < 0.01) in head kidneys and spleens of rainbow trout immunized with recombinant truncated G protein. Also, after challenge with wild-type IHNV HLJ-09 virus on Day 28, rainbow trout immunized with recombinant truncated G protein showed cumulative survival rates of 60%. These results indicate that the truncated G protein of IHNV expressed by the E. coli prokaryotic expression system can be used as a candidate immunogen for an IHNV subunit vaccine, which lays a theoretical foundation for the study of further potential IHNV subunit vaccines.

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.

EvaGreen-based real-time PCR assay for sensitive detection of salmonid alphavirus.

Salmonid alphaviruses (SAVs), which include the etiological agents of salmon pancreas disease (PD) and sleeping disease (SD), are significant viral pathogens of European salmonid aquaculture, resulting in substantial economic losses to the salmonid-farming industry. Even though many countries including China have not reported the presence of SAV infections, these countries may be seriously threatened by these diseases as the salmon fish import trade increases. Thus, it is indeed necessary to develop efficient detection methods for the diagnosis and prevention of SAV infection. Real-time PCR assays have been increasingly used in viral detection, and in many cases scientists prefer dye-based real-time PCR assays for their high sensitivity and low cost. In this study, we developed a novel, sensitive, low-cost detection method, EvaGreen-based real-time PCR assay for the detection of SAV. This assay exhibited high specificity for SAV1, SAV2, and SAV5 and was able to detect SAV at concentrations as low as 1.5 × 101 copies, making them more sensitive than the approved conventional RT-PCR method (detection limit, 1.5 × 106 copies). Assessment of infected fish samples showed that the sensitivity of EvaGreen-based assay was higher than previously developed SYBR Green assay (227 assay). Thus, we report that the EvaGreen real-time PCR assays is an economical alternative diagnostic method for the rapid detection of SAV1, SAV2, and SAV5 infection, providing improved technical support for the clinical diagnosis and epidemiological investigation of SAV.

Oral immunization with a recombinant Lactobacillus expressing CK6 fused with VP2 protein against IPNV in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonid fish causing high mortality with serious economic losses to salmonid aquaculture. Ideally, the method of oral immunization should prevent the infection of rainbow trout juveniles with IPNV. In the present study, genetically engineered Lactobacillus casei 393 pPG-612-VP2/L. casei 393 and pPG-612-CK6-VP2/L. casei 393 constitutively expressing VP2 protein of IPNV were constructed. The recombinant strains pPG-612-CK6-VP2/L. casei 393 and pPG-612-VP2/L. casei 393 were orally administrated to juvenile rainbow trouts, and significant titers of IgM and IgT of pPG-612-CK6-VP2/L. casei 393 were observed. The results demonstrate that the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in trouts immunized with pPG-612-CK6-VP2/L. casei 393 showed that the recombinant strain could induce a strong cellular immune response. The IL-1ß, IL-8, CK6, MHC-II, Mx, ß-defensin, and TNF-1α levels in the spleen and gut suggest that the target molecular chemokine has the ability to attract relevant immune cells to participate in the inflammatory response and enhance the function of the innate immune response. Additionally, the pPG-612-CK6-VP2/L. casei 393 induced the expression of cytokines, which have the effect of promoting inflammation to drive the differentiation of macrophages and clear target cells. After challenging with IPNV, the reduction in viral load caused by pPG-612-CK6-VP2/L. casei 393 was significantly higher than that of the other groups. Thus, the recombinant pPG-612-CK6-VP2/L. casei 393 is a promising candidate for the development of an oral vaccine against IPNV.

Recombinant infectious hematopoietic necrosis virus expressing infectious pancreatic necrosis virus VP2 protein induces immunity against both pathogens.

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Although vaccines against IHNV and IPNV have been commercialized in many countries, the prevalence of IHNV and IPNV is still widespread in modern aquaculture. In the present study, two IHNV recombinant viruses displaying IPNV VP2 protein (rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE) were generated using the RNA polymerase Ⅱ system to explore the immunogenicity of IHNV and IPNV. The recombinant IHNV viruses were stable, which was confirmed by sequencing, indirect immunofluorescence assay, western blotting, transmission electron microscopy and viral growth curve assay. IHNV and IPNV challenge showed that the recombinant viruses had high protection rates against IHNV and IPNV with approximately 65% relative percent survival rates. Rainbow trout (mean weight 20 g) vaccinated with these two recombinant viruses showed a high level of antibodies against IHNV and IPNV infection. Taken together, our findings demonstrate that rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE might be promising vaccine candidates against IHNV and IPNV.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in the modulation of NF-κB pathway during IHNV infection.

Viral infections frequently lead to the activation of host innate immune signaling pathways involved in the defense against invading pathogens. To ensure their survival, viruses have evolved sophisticated mechanisms to overcome the host immune responses. The present study demonstrated for the first time that infectious hematopoietic necrosis virus (IHNV) activated NF-κB pathway in fish cells. We further identified that the IHNV L protein could activate the NF-κB signaling pathway and that IHNV NV functioned as an inhibitor of NF-κB activation. Further results demonstrated that the NV protein blocked the degradation of the inhibitor of NF-κB (IκBα) and suppressed the SeV-induced NF-κB nuclear translocation. In conclusion, our study explored the functions of different IHNV proteins on NF-κB activation, and revealed a potential mechanism by which IHNV evades innate immune responses.