A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the Infectious Spleen and Kidney Necrosis Virus.

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

Protective efficiency and immune responses to single and booster doses of formalin-inactivated scale drop disease virus (SDDV) vaccine in Asian seabass (Lates calcarifer).

BACKGROUND: Scale drop disease virus (SDDV) threatens Asian seabass (Lates calcarifer) aquaculture production by causing scale drop disease (SDD) in Asian seabass. Research on the development of SDDV vaccines is missing an in-depth examination of long-term immunity and the immune reactions it provokes. This study investigated the long-term immune protection and responses elicited by an SDDV vaccine. The research evaluated the effectiveness of a formalin-inactivated SDDV vaccine (SDDV-FIV) using both prime and prime-booster vaccination strategies in Asian seabass. Three groups were used: control (unvaccinated), single-vaccination (prime only), and booster (prime and booster). SDDV-FIV was administered via intraperitoneal route, with a booster dose given 28 days post-initial vaccination. RESULTS: The immune responses in vaccinated fish (single and booster groups) showed that SDDV-FIV triggered both SDDV-specific IgM and total IgM production. SDDV-specific IgM levels were evident until 28 days post-vaccination (dpv) in the single vaccination group, while an elevated antibody response was maintained in the booster group until 70 dpv. The expression of immune-related genes (dcst, mhc2a1, cd4, ighm, cd8, il8, ifng, and mx) in the head kidney and peripheral blood lymphocytes (PBLs) of vaccinated and challenged fish were significantly upregulated within 1-3 dpv and post-SDDV challenge. Fish were challenged with SDDV at 42 dpv (challenge 1) and 70 dpv (challenge 2). In the first challenge, the group that received booster vaccinations demonstrated notably higher survival rates than the control group (60% versus 20%, P < 0.05). However, in the second challenge, while there was an observable trend towards improved survival rates for the booster group compared to controls (42% versus 25%), these differences did not reach statistical significance (P > 0.05). These findings suggest that the SDDV-FIV vaccine effectively stimulates both humoral and cellular immune responses against SDDV. Booster vaccination enhances this response and improves survival rates up to 42 dpv. CONCLUSIONS: This research provides valuable insights into the development of efficient SDDV vaccines and aids in advancing strategies for immune modulation to enhance disease management in the aquaculture of Asian seabass.

Refolded recombinant major capsid protein (MCP) from Infectious Spleen and Kidney Necrosis Virus (ISKNV) effectively stimulates serum specific antibody and immune related genes response in Nile tilapia (Oreochromis niloticus).

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1ß and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.

Epitope screening of the major capsid protein within grouper iridovirus of Taiwan and the immunoprotective effect with SWCNTs as the vaccine carrier.

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L-1) and SWCNTs-P2 (5, 10, 20 mg L-1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L-1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Optimization of the efficacy of a SWCNTs-based subunit vaccine against infectious spleen and kidney necrosis virus in mandarin fish.

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which brings substantial economic losses to the mandarin fish culture industry in China. This study was aimed at optimizing the efficacy of a SWCNTs-based immersion subunit vaccine (SWCNTs-M-MCP) which as a promising vaccine against ISKNV. Mandarin fish were vaccinated by immersion, then we designed an orthogonal experiment to optimize different parameters affecting vaccination such as immune duration of bath immunization, immune dose, and fish density when immunized. Our results showed that the highest relative percent survival (86.7%) was found in the group 6 with 8 h of immune duration, 20 mg/L of immune dose, and 8 fish per liter of fish density. And other immune responses (serum antibody production, enzyme activities, and immune-related genes expression) also demonstrated similar results. In addition, the expression of IRF-I in group 6 (8 h, 20 mg/L, 8 fish per liter) was significant extents, and about 16-folds increases were obtained than the control group at 21 d post-vaccination. And the highest specific antibody response was significantly increased (more than 4-folds) than control group which was found in group 6. The optimum immune duration, immune dose, and fish density of SWCNTs-M-MCP were 8 h, 20 mg/L, 8 fish per liter, respectively. Importantly, our results also showed that immune duration had the greatest effect on the immune response of our vaccine, followed by immune dose. The study reported herein provides a helpful reference for the effective use of vaccine in fish farming industry.

Single-walled carbon nanotubes enhance the immune protective effect of a bath subunit vaccine for pearl gentian grouper against Iridovirus of Taiwan.

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-ß, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Protective immunity of largemouth bass immunized with immersed DNA vaccine against largemouth bass ulcerative syndrome virus.

To reduce the largemouth bass ulcer syndrome (LBUSV) aquatic economic losses, it must take effective preventive measures and coping strategies should be urgently investigated. In this research, the effects of a functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in largemouth bass (Micropterus Salmoides) against LBUSV were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to largemouth bass against LBUSV. We found more than 10 times increase in serum antibody levels, enzyme activities and immune-related genes (IL-6, IL-8, IFN-γ, IgM and TNF-α) expression, in the SWCNTs-pcDNA-MCP immunized groups compared with PBS group and the pure SWCNTs group. The survival rates for control group (PBS), pure SWCNTs groups (40 mg L-1), four pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) and four SWCNTs-pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) were 0%, 0%, 0%, 2.77%, 11.11%, 19.44%, 27.78%, 38.89%, 52.78% and 61.11%, respectively. Our results demonstrate that the SWCNTs-DNA vaccine can be used as a new method against LBUSV showing protection following challenge with LBUSV.

A voting mechanism-based linear epitope prediction system for the host-specific Iridoviridae family.

BACKGROUND: The Iridoviridae family is categorized into five genera and clustered into two subfamilies: Alphairidovirinae includes Lymphocystivirus, Ranavirus (GIV), and Megalocystivirus (TGIV), which infect vertebrate hosts and Betairidovirinae includes Iridovirus and Chloriridovirus, which infect invertebrate hosts. Clustered Iridoviridae subfamilies possess host-specific characteristics, which can be considered as exclusive features for in-silico prediction of effective epitopes for vaccine development. A voting mechanism-based linear epitope (LE) prediction system was applied to identify and endorse LE candidates with a minimum length requirement for each clustered subfamily RESULTS: The experimental results showed that four conserved epitopes among the Iridovirideae family, one exclusive epitope for invertebrate subfamily and two exclusive epitopes for vertebrate family were predicted. These predicted LE candidates were further validated by ELISA assays for evaluating the strength of antigenicity and cross antigenicity. The conserved LEs for Iridoviridae family reflected high antigenicity responses for the two subfamilies, while exclusive LEs reflected high antigenicity responses only for the host-specific subfamily CONCLUSIONS: Host-specific characteristics are important features and constraints for effective epitope prediction. Our proposed voting mechanism based system provides a novel approach for in silico LE prediction prior to vaccine development, and it is especially powerful for analyzing antigen sequences with exclusive features between two clustered groups.

Immersion vaccination of Mandarin fish Siniperca chuatsi against infectious spleen and kidney necrosis virus with a SWCNTs-based subunit vaccine.

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.

Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination.

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Chitosan and anisodamine improve the immune efficacy of inactivated infectious spleen and kidney necrosis virus vaccine in Siniperca chuatsi.

Siniperca chuatsi is an economically important fish in China, but infectious spleen and kidney necrosis virus (ISKNV) causes high mortality and significant economic losses. Currently, vaccination is the most promising strategy to prevent infectious diseases, while adjuvant can effectively enhance immune responses. In this study, inactivated ISKNV vaccine was prepared, then poly (I:C), chitosan, anisodamine and ims1312 were used as adjuvants to evaluate the effect on the immune responses and ISKNV replication. Chitosan could strongly boost the protection of liver and spleen tissues by pathological sections. In serum, poly (I:C) and chitosan group had protective effect on catalase, acid phosphatase, blood urea nitrogen. mRNA expressions showed these adjuvants induced the cytokines of early immune responses (TNF-α, Viperin) in both spleen and mesonephron by real time quantitative RT-PCR assays. Meanwhile, poly (I:C), chitosan and anisodamine were significantly improved the antiviral function and inhibited ISKNV replication. Chitosan and anisodamine played a significantly protective role in the immune protective rate test. The results indicated that all the four adjuvants are valid in the inactivated ISKNV vaccine, and chitosan is recommended preferentially. The present study provides reference for other animal vaccine adjuvants.

Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi).

Hepcidin is a liver-derived peptide hormone that is related to iron balance and immunity in humans. However, its function in Siniperca chuatsi has not been well elucidated. In this study, we analyzed the expression and function of the S. chuatsi hepcidin (Sc-hep) gene. Sc-hep was specifically expressed in the liver and appeared to be one of the most highly expressed genes in the liver. After spleen and kidney necrosis virus (ISKNV) infection and lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C) stimulation, the expression of Sc-hep in the liver increased by approximately 110, 6500, and 225 times, respectively. After ferrous sulfate (FS) injection, the expression of Sc-hep in the liver increased approximately 520-fold. We found that miR-19c-5p could inhibit Sc-hep expression. Five CpG dinucleotides distributed in the promoter region showed no differential methylation between the liver and the stomach, both presenting high methylation rates. After FS or LPS injection, the expression of three iron balance-related genes (FPN1, TFR1, and FTN) and five immune-related cytokine genes (IL-1ß, IL8, TNF-α, TLR22, and SOCS3) significantly changed. These results indicate that Sc-hep participates in the regulation of iron balance and plays an important role in the immune system. Sc-hep increased approximately 52-fold when mandarin fish were domesticated with artificial diets. Sc-hep might be used as a real-time biomarker of mandarin fish liver because its expression markedly varies under different physiological conditions.

Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro.

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV⁻host interaction and might be promising inhibitors of LCDV infection in fish.

LAMP-1-chimeric DNA vaccines enhance the antibody response in Japanese flounder, Paralichthys olivaceus.

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination.

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection.

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL-8 and COX-2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL-1ß, IL-8 and COX-2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)-induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation-related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.

Display of ISKNV orf086 protein on the surface of Aeromonas hydrophila and its immunogenicity in Chinese perch (Siniperca chuatsi).

Co-infection with infectious spleen and kidney necrosis virus (ISKNV) and Aeromonas hydrophila is becoming ever more widespread in Chinese perch (Siniperca chuatsi) aquaculture industry, so that it's necessary to develop the combined vaccine against ISKNV and A. hydrophila disease. The surface display of heterologous on bacteria using anchoring motifs from outer membranes proteins has already been explored as an effective delivery system of viral antigens. In present study, the ISKNV orf086 gene, which is verified as a protective antigen, was inserted into ompA gene cassette of A. hydrophila GYK1 strain by homologous recombination. And an ompA-orf086 fusion A. hydrophila mutant strain K28 was constructed. Then the ISKNV orf086 was verified to express on the surface of A. hydrophila K28 by RT-PCR, western blot and indirect immunofluorescence assay. Next, Chinese perch were intraperitoneally inoculated with formalin inactivated A. hydrophila k28 emulsified with ISA763 adjuvant with a dose of 9 × 10(8) CFU per fish. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, Lysozyme and IgM were strongly up-regulated in Chinese perch post-inoculation. In addition, specific antibodies were detected by ELISA, and the results showed that antibody titer against ISKNV or A. hydrophila reached the highest with 1:800 or 1:1200 on 14dpv, respectively. Lymphocyte proliferation were detected by MTT methods, and the results showed that the SI values of AH-K28 vaccinated group to three different stimulators were significantly higher than those of control group. At last, protective efficacy were determined by challenge trials. The cumulative mortality rates of vaccinated groups were significantly lower than the control one (P < 0.05) after ISKNV or A. hydrophila challenge, and the relative percentage survival (RPS) value was 73.3% and 60%, respectively. This system provides a novel approach to the surface display of heterologous antigenic proteins on A. hydrophila and suggests the possibility to use the recombinant K28 strain as a combined vaccine against ISKNV and A. hydrophila infection.

Characterization of the gilthead seabream (Sparus aurata L.) immune response under a natural lymphocystis disease virus outbreak.

Lymphocystis or lymphocystis disease virus (LCDV) is distributed worldwide and affects many fresh and marine water fish species. LCDV is commonly found in aquaria fish species but also in farmed fish species, among them the gilthead seabream (Sparus aurata L.). The immune status of gilthead seabream (S. aurata) specimens under a natural outbreak of LCDV was studied. The replication of the virus was demonstrated in infected fish, but not in control fish. The results showed decreased total serum IgM levels and increased innate cellular immune response (peroxidase and respiratory burst activities) of head kidney leucocytes in LCDV-infected fish, compared to the values obtained in uninfected specimens. In addition, transcription of antiviral genes (ifn and irf3) was down-regulated in the skin of LCDV-positive fish as well as genes involved in cellular immunity (csf1r, mhc2a, tcra and ighm) that were down-regulated in skin and head kidney of infected fish. By contrast, the transcription of nccrp1 was up-regulated in head kidney after LCDV infection. These present results show that head kidney leucocytes are activated to encounter the virus at the sites of replication.

Protein encoded by ORF093 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in Chinese perch Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality and economic losses in Chinese perch, Siniperca chuatsi in China. Little information about the antigenicity of ISKNV proteins is available. In this study the ORF093 gene of ISKNV was cloned into the prokaryotic expression vector pET32a(+) and eukaryotic expression vector pcDNA3.1(+), and designated as pET-093 and pcDNA-093, respectively. A recombinant 51-kDa protein was detected in Escherichia coli BL21 (harboring pET-093) after IPTG inducement. Polyclonal antibodies were raised in rabbits against the purified ORF093 protein and the reaction of the antibodies was confirmed by western blotting using the purified recombinant protein. Expression of the pcDNA-093 in muscle tissue of vaccinated fish was confirmed by western blotting. The protection efficacy of ORF093 was investigated and results showed that cumulative mortality of Chinese perch was significant differences between immune groups and control (P<0.05) after ISKNV challenge, and the RPS value of 093 recombinant protein, pcDNA093 and pcDNA093+MCP was 47%, 50% and 57%. The results suggested that ORF093 is an effective vaccine candidate for ISKNV and it can be used in the control of ISKNV disease in Chinese perch.

Early protein ORF086 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in mandarin fish Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant loss in the Mandarin fish (Siniperca chuatsi) aquaculture industry. Vaccination is an important measure to prevent fatal ISKNV infection. In this study, the ORF086 gene encoding an early protein helicase of ISKNV was cloned into the prokaryotic pET32a (+) and eukaryotic pcDNA3.1 (+) expression vectors and designated as pET086 and pcDNA086, respectively. A recombinant 36 kDa protein was detected in Escherichia coli BL21 (harboring pET086) after isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction. Polyclonal antibodies against the purified ORF086 protein were raised in rabbits. The antibody reaction and the pcDNA086 expression in muscle tissues of vaccinated fish were confirmed using Western blot analysis. The protective efficacy of ORF086 was also investigated. The cumulative mortality rates of Mandarin fish were significantly different between immune and control groups (P < 0.05) after ISKNV challenge. The relative percentage survival (RPS) values of the recombinant ORF086 protein emulsified with ISA763A adjuvant and pcDNA086 added with QCDC adjuvant were 73% and 63%, respectively. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, and IgM were strongly up-regulated in the vaccinated groups post-immunization. In particular, the expression levels in the QCDC + pcDNA086 group was higher than those in the control groups (P < 0.05). These results indicated that the early protein ORF086 could be an effective antigen candidate for controlling ISKNV disease in Mandarin fish.