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.

Epidermal growth factor receptor promotes infectious spleen and kidney necrosis virus invasion via PI3K-Akt signaling pathway.

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

Characterization and function of mandarin fish c-Myc during viral infection process.

c-Myc is a transcription factor and master regulator of cellular metabolism, and plays a critical role in virus replication by regulating glutamine metabolism. In this study, the open-reading frame (ORF) of c-Myc, designated as Sc-c-Myc, was cloned and sequenced. Multiple alignment of the amino acid sequence showed that the conserved domain of Sc-c-Myc, including the helix-loop-helix-zipper (bHLHzip) domain and Myc N-terminal region, shared high identities with other homologues from different species. Sc-c-Myc mRNA was widely expressed in the examined tissues of mandarin fish, and the higher mRNA levels was expressed in hind kidney. Moreover, mRNA and protein level of Sc-c-Myc was significantly increased in the Chinese perch brain (CPB) cells and spleen of mandarin fish post infection with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Sc-c-Myc overexpression promoted ISKNV and SCRV replication, on the contrary, knocking down Sc-c-Myc restrained ISKNV and SCRV replication. These results indicated that Sc-c-Myc involved in ISKNV and SCRV replication and proliferation, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

The composition and antiviral activity of scTRIM59 in Mandarin fish.

The tripartite motif (TRIM) proteins play critical roles in viral infection by modulating innate immunity. However, the molecular and antiviral activity of TRIM59 in mandrain fish is not fully understood. In present study, we cloned and sequenced the TRIM59 core sequence and explored its characteristics in Mandarin fish. The Siniperca chuatsi TRIM59 (scTRIM59) showed relatively high expression in immune-related organs. scTRIM59 expression was significantly down-regulated post ISKNV infection in vivo and vitro, but up-regulated at the early stages of SCRV infection in CPB cells. The overexpression of scTRIM59 inhibited ISKNV and SCRV infection, but decreased the expression of IRF3/IRF7-mediated signal genes. However, knockdown of scTRIM59 promoted the ISKNV and SCRV infection, but increased the expression of IRF3/IRF7-mediated signal genes. Those results indicated that scTRIM59 negatively regulated ISKNV, SCRV infection and IRF3/IRF7-mediated signal genes. This study provided new ideas about the function of scTRIM59.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

Identification of intron in ORF003 gene and its application for inactivation test of ISKNV.

The virus inactivation test is a critical skill in inactivated vaccine production. Active viruses produced viral mRNA in susceptible cells or the host can be used to infer whether a DNA virus is replicating by RT-PCR. But it is generally difficult to avoid genomic DNA contamination in the samples. However, the use of primers spanning an intron is an effective alternative for virus inactivation test. Therein, a nested RT-PCR was developed to detect active ISKNV in the inactivated vaccine. At first, the transcriptome analysis of CPB cell infected with ISKNV revealed several gaps in some viral transcripts compared to ISKNV genome. One intron in ORF003L with 80 bp (designated IN-3) was confirmed by PCR and sequencing analysis. Then, two primer sets (primer A and primer B) spanning the IN-3 intron were designed to detect ISKNV transcription. The nested RT-PCR conditions were optimized with 0.4 µM primer A and 0.2 µM primer B, and 68 °C and 55 °C for annealing temperature, respectively. The sensitivity results indicated that the nested RT-PCR could detect one copy of live ISKNV propagating in CPB cells for seven days. The nested RT-PCR method was more sensitive and accurate than the method of blind passages in cells and fish challenge experiments. Together, above results indicate that this assay is a time-saving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.

Molecular characterization and function of EGFR during viral infectionprocess in Mandarin fishSiniperca chuatsi.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase protein and plays a critical role in virus infection by modulating innate immunity. In this study, we cloned and sequenced the EGFR coding sequence of mandarin fish, designed as scEGFR, and explored its characteristics. scEGFR mRNA was widely expressed in the tested tissues of mandarin fish, and the higher mRNA levels were expressed in kidney and spleen. scEGFR expression was up-regulated in spleen and CPB cells at early stage of ISKNV and SCRV infection. Gefitinib (EGFR inhibitor) inhibited ISKNV and SCRV replication, and increased the expression of the interferon-stimulated genes (ISG). However the EGF (EGFR activator) promoted ISKNV and SCRV replication, and decreased the interferon-stimulated genes. Those results indicated that scEGFR and its signaling involved in ISKNV and SCRV infection, and EGFR activation negatively regulated the interferon response, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

Transcriptomic profiles reveal that inactivated iridovirus and rhabdovirus bivalent vaccine elicits robust adaptive immune responses against lethal challenge in marbled sleepy goby.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Siniperca chuatsi rhabdovirus (SCRV) induces autophagy via PI3K/Akt-mTOR pathway in CPB cells.

Autophagy is an important mechanism for organisms to eliminate viruses and other intracellular pathogens. Siniperca chuatsi rhabdovirus (SCRV) is an agent that has caused devastating losses in Chinese perch (Siniperca chuatsi) industry. But the role of autophagy in Siniperca chuatsi rhabdovirus (SCRV) infection is not clearly understood. In this study, we identified that SCRV infection triggered autophagy in CPB cells, which was demonstrated by the appearance of the membrane vesicles, GFP-LC3 punctuate pattern, conversion of LC3-I to LC3-II, and the co-localization of autophagosomes and lysosomes. The changes of autophagy flux in SCRV infection indicated that autophagy was inhibited at the early stage of SCRV infection, but was promoted at the late stage. UV-inactivated SCRV can induce autophagy, suggesting that SCRV replication is not essential for the induction of autophagy. Furthermore, we found inducing autophagy with Rapa inhibited SCRV proliferation, but inhibiting autophagy with 3-MA or CQ increased SCRV production in CPB cells. Then we assessed the effects of PI3K/Akt-mTOR signaling pathway on SCRV induced autophagy. We found that SCRV infection activated PI3K/AKT signaling pathway at 4 hpi, but inhibited it at 8 hpi. SCRV-N mRNA and protein level were decreased by inhibiting PI3K with LY294002, but increased by activating PI3K with 740Y-P. Those results indicated that SCRV infection induced autophagy via the PI3K/Akt-mTOR signal pathway, which will provide new insights into SCRV pathogenesis and antiviral treatment strategies.

First report of megalocytivirus (iridoviridae) in cultured bluegill sunfish, Lepomis macrochirus, in China.

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.

Development of strand-specific real-time RT-PCR for the analysis of SCRV transcription and replication dynamics.

To distinguish between three types of Siniperca chuatsi rhabdovirus (SCRV) viral RNA (vRNA, cRNA, and mRNA) and investigate SCRV transcription and replication dynamics in Chinese perch brain CPB cells, a novel, strand-specific, reverse transcriptase quantitative real-time PCR (RT-qPCR) assay was established. The method is based on strand-specific reverse transcription, using tagged primers to add a 'tag' sequence at the 5' end. We used the 'tag' sequence as the forward primer and a strand-specific reverse primer to quantify the three types of RNA. Three types of synthetic viral RNA were used as reference standards for validation and quantification. These assays were optimized to produce a standard curve from 102 to 107 copies/µL, with an efficiency of 91-101% and an R2 value of 0.9949-0.9999. The coefficients of variation for repeatability and reproducibility were less than 2.85% and 5.52%, respectively. Using this method, specific target RNA was detected at a 3500-70,000 fold higher level than other types of RNA. This method was also used to evaluate the dynamics of vRNA, cRNA and mRNA synthesis in CPB cells infected with SCRV. The results indicate that the intracellular dynamics of vRNA, cRNA and mRNA are different. In the earliest phase of SCRV infection, all three types of viral RNA increased very slowly. The copy number of vRNA and mRNA increased exponentially from 4 h post infection, while cRNA increased from 6 h post infection. The amount of cRNA was lower than vRNA and mRNA throughout the infection. The novel, strand-specific RT-qPCR method developed in this study provides critical data to aid the understanding of transcription and replication during SCRV infection.

MicroRNAs profiles of Chinese Perch Brain (CPB) cells infected with Siniperca chuatsi rhabdovirus (SCRV).

MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection.

iTRAQ-based proteomic profile analysis of ISKNV-infected CPB cells with emphasizing on glucose metabolism, apoptosis and autophagy pathways.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant losses in the cultured mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie interaction between ISKNV and hosts are not fully understood. In this study, the proteomic profile of CPB cells at progressive time points after ISKNV infection was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2731 proteins corresponding to 6363 novel peptides (false discovery rate <0.01) were identified. In the samples harvested 24 h (early-stage) and 72 h (late-stage) post-infection, 232 and 199 differentially expressed proteins were identified comparing with mock-infected cells, respectively. Western-blotting analysis of several proteins as G6PDH, ß-tubulin and RPL11 were done to validate iTRAQ data. Among those differentially expressed proteins, several glucose metabolism-related enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase phosphatase (PDP) and fumarate hydratase (FH), were up-regulated, while pyruvate dehydrogenase kinase (PDK) and enolase (ENO) were down-regulated at 24 h poi, suggesting that ISKNV enhanced glucose metabolism in CPB cells in early-stage infection. Simultaneously, expression of apoptosis-related proteins including Caspase 8, phosphoinositide 3-kinases (PI3Ks), and regulatory-associated protein of mTOR-like isoform X3 changed upon ISKNV infection, indicating that ISKNV induced apoptosis of CPB cells. Autophagy-related proteins including LC3 and PI3Ks were up-regulated at 24 h poi, indicating that ISKNV induced autophagy of CPB cells in early-stage infection. These findings may improve the understanding of ISKNV and host interaction and help clarify its pathogenesis mechanisms.

The pathogenicity and biological features of Santee-Cooper Ranaviruses isolated from Chinese perch and snakehead fish.

Ranavirus has become a noticeable threat to both farmed and natural populations of fish and amphibians. Herein, we reported that 3 strains of novel viruses, designated as ScRIV-GM-20150902, CmRIV-XT-20150917 and ScRIV-ZS-20151201, were isolated from diseased Chinese perch and snakehead fish in China. Efficient propagation of these isolates were determined in Chinese perch brain (CPB) cell line by the means of cytopathic effect observation, PCR amplification and electron microscopy observation. And their viral titers in CPB cells reached 108.13 TCID50 ml-1, 107.71 TCID50 ml-1 and 107.94 TCID50 ml-1, respectively. While the challenge experiment results showed that 3 isolates resulted in 100% mortality of Chinese perch after virus infection. Electron microscopy analysis showed that two kinds of viral inclusion bodies (intracytoplasmic and intranuclear inclusion body) were observed in infected CPB cells. Sequence alignment and phylogenetic analysis of major capsid protein gene sequences of isolates revealed that these isolates belonged to the species Santee-Cooper Ranavirus.

Application and development of a TaqMan real-time PCR for detecting infectious spleen and kidney necrosis virus in Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) is one of the major epidemiological agents that had caused great economic loss in Chinese perch (Siniperca chuatsi). In this study, a specific TaqMan real-time PCR was developed using a pair of primers and a TaqMan probe specific to the ORF007 gene of ISKNV to rapidly detect ISKNV copies in Chinese perch samples. This assay was optimized to produce linearity from 8.75 × 108 to 8.75 × 101 copies in standard curve with an efficiency of 98% and a R2 value of 0.9999. Moreover, the minimum detection limit of this assay was 10,000 times more sensitive than that of conventional PCR method. The coefficients of variation of intra- and inter-assay repeatability were less than 2.4% and 3.3%, respectively. The viral distribution in different tissues of diseased Chinese perch was evaluated by TaqMan real-time PCR method and the highest level of viral copies was detected in spleen. Among the 76 diseased Chinese perch clinical samples, 35 and 29 were positive samples based on the TaqMan real-time PCR and conventional PCR methods, respectively, indicating that the TaqMan real-time PCR was more sensitive than conventional PCR. Therefore, the TaqMan real-time PCR should be a useful tool for the early surveillance and quantitation of ISKNV.

Autophagy promoted infectious kidney and spleen necrosis virus replication and decreased infectious virus yields in CPB cell line.

Autophagy plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of infectious kidney and spleen necrosis virus (ISKNV), an agent that has caused devastating losses in Chinese perch (Siniperca chuatsi) industry. We found that ISKNV infection triggered the complete autophagic process, as demonstrated by microtubule-associated protein 1 light chain 3B II (LC3B-II) conversion, an increased accumulation of punctate GFP-LC3-expressing cells, a higher number of autophagosome-double-membrane vesicles in the cytoplasm, and increased levels of autophagic flux in CPB cells. Then, we investigated the role of autophagy in the process of ISKNV replication. Results showed that inducing autophagy by rapamycin promoted ISKNV replication and proteins synthesis but decreased extracellular virus yields. While, blocking autophagosome-lysosome fusion by chloroquine (CQ) promoted infectious virus yields in culture supernatant. These results offer insight into the complex interactions between ISKNV and host cell, providing new insights into viral pathogenesis and antiviral treatment strategies.

Mandarin fish p53: Genomic structure, alternatively spliced variant and its mRNA expression after virus challenge.

A number of size variants of the p53 protein have been described in mammal, but little is known about alternative splicing of p53 expression and function in the fish. In our previous study, the immune defense and antiviral responses of p53 had been determined in mandarin fish (Siniperca chuatsi). However, the role of its splicing variants remains unknown. In the present study, the organization of mandarin fish p53 (Sc-p53) genome sequence was determined and a novel splice variant was characterized. The Sc-p53 genomic sequence was composed of 5543 bp, containing 11 exons and 10 introns, which was similar to other species. Then, a 1106 bp full-length cDNA of a novel splice variant p53 from mandarin fish (designed as Sc-p53I6) was cloned and characterized. Quantitative real-time PCR assays revealed that Sc-p53I6 was expressed in all tissues examined, and it was most abundant in the gill, hemocyte and hind kidney. Western blotting analysis revealed that Sc-p53I6 protein was abundant in liver, trunk kidney, hind kidney, stomach and heart. In addition, the regulation of Sc-p53I6 gene expression after virus infection was determined and characterized. The results showed twice rise expression pattern of Sc-p53I6 in CPB cells and spleen of mandarin fish in response to infectious kidney and spleen necrosis virus (ISKNV). However, a different expression pattern, once rise, of Sc-p53I6 in response to Siniperca chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc-p53I6 was significantly up-regulated in CPB at 4 h and spleen of mandarin fish at 12 h post-infection. These results will shed a new light on antiviral response mechanisms of p53 in mandarin fish.

The biological features and genetic diversity of novel fish rhabdovirus isolates in China.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses which infects mammals, birds, reptiles, fish, insects and plants. Herein, we reported the isolation and characterization of 6 novel viruses from diseased fish collected from China including SCRV-QY, SCRV-SS, SCRV-GM, CmRV-FS, MsRV-SS, OmbRV-JM. The typical clinical symptom of diseased fish was hemorrhaging. Efficient propagation of these isolates in a Chinese perch brain cell line was determined by means of observation of cytopathic effect, RT-PCR and electron microscopy. Sequence alignment and phylogenetic analysis of the complete G protein sequences revealed that these isolates were clustered into one monophyletic lineage belonging to the species Siniperca chuatsi rhabdovirus.

Inhibition of Grass Carp Reovirus Infection by DNA Aptamers against S10 Protein.

Grass Carp reovirus (GCRV) is one of the most pathogenic agents among aquareovirus isolates and has the ability to cause a severe epidemic outbreak of hemorrhagic disease, thus resulting in both a high mortality rate during the culture of Grass Carp Ctenopharyngodon idella and an enormous economic loss. Aptamers have been demonstrated to have strong promising applications in antiviral drug development. In the present study, a complementary DNA fragment encoding the S10 gene of GCRV was cloned. The S10 protein was expressed and purified. Aptamers for S10 protein were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and their characteristics and antiviral actions were examined. All targeting-selected aptamers formed a similar structure, forming a 5-7 base loop at the terminus. The results show that the aptamers could inhibit the GCRV infection. The most significant inhibitory effect was obsereved when the aptamers were added to the cell culture for 1 h before the cells were infected by GCRV. Our data showed that these novel molecular agents could be considered suitable candidates for anti-GCRV therapy. Received August 23, 2016; accepted February 5, 2017.

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.