The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.

Self-assembling ferritin nanoplatform for the development of infectious hematopoietic necrosis virus vaccine.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus.

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Mucosal immunity and microbiota change in the rainbow trout (Oncorhynchus mykiss) gills after being challenged with infectious hematopoietic necrosis virus.

Respiratory structures are crucial for vertebrate survival, as they serve not only to perform gas-exchange processes but also as entry points for opportunistic pathogens. Previous studies have demonstrated that fish contain gill mucosal-associated lymphoid tissue, and harbor a large number of commensal bacteria on their surface and contribute to maintaining fish health. However, by far, very limited information is known regarding the effects of viral infection on gill mucosal immunity and microbiota homeostasis. In this study, we conducted an infection model by bath with infectious hematopoietic necrosis virus (IHNV) and revealed a 27 % mortality rate among rainbow trout in the first two weeks after infection. Moreover, we found that diseased fish with the highest IHNV loads in gills exhibiting severe damage, as well as increased goblet cell counts in both primary lamellae (PL) and secondary lamellae (SL). Additionally, RT-qPCR and RNA-seq analyses revealed that IHNV infection induced a strong innate and adaptive antiviral immune responses. Interestingly, an antibacterial immune response was also observed, suggesting that a secondary bacterial infection occurred in trout gills after viral infection. Furthermore, 16S rRNA analysis of trout gills revealed a profound dysbiosis marked by a loss of beneficial taxa and expansion of pathobionts following IHNV infection. Overall, our finding demonstrates that IHNV infection induces significant changes of the microbial community in the fish respiratory surface, thus triggering local antiviral and bacterial mucosal immunity.

Integrative mRNA-miRNA interaction analysis associated with the immune response in the head kidney of rainbow trout (Oncorhynchus mykiss) after infectious hematopoietic necrosis virus infection.

Rainbow trout (Oncorhynchus mykiss) is an important cold-water fish widely cultivated in China. The frequent occurrence of viral diseases caused by infectious hematopoietic necrosis virus (IHNV) seriously restricted the healthy development of the rainbow trout farming industry. However, the immune defense mechanism induced by IHNV in rainbow trout has not been fully elucidated. In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection using RNA-seq and identified key immune-related genes and miRNAs. The results showed that a total of 7486 genes and 277 miRNAs were differentially expressed, and numerous differentially expressed genes (DEGs) enriched in the immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway were significantly up-regulated, including LGP2, MDA5, TRIM25, IRF3, IRF7, TLR3, TLR7, TLR8, MYD88, and IFN1. Integration analysis identified six miRNAs (miR-141-y, miR-200-y, miR-144-y, miR-2188-y, miR-725-y, and miR-203-y) that target at least six key immune-related genes (TRIM25, LGP2, TLR3, TLR7, IRF3, and IRF7). Further, we verified selected immune-related mRNAs and miRNAs through qRT-PCR and confirmed the reliability of the RNA-seq results. These findings improve our understanding of the immune mechanism of rainbow trout infected with IHNV and provide basic data for future breeding for disease resistance in rainbow trout.

In-vitro immunomodulatory responses and antiviral activities of antimicrobial peptide octominin against fish pathogenic viruses.

Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 µg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 µg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 µg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 µg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.

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.

In vitro propagation of infectious hypodermal hematopoietic necrosis virus (Penaeus stylirostris penstyldensovirus) in PmLyO-Sf9 cells.

Infectious hypodermal hematopoietic necrosis virus (IHHNV/PstDVI) was isolated and propagated in the hybrid shrimp-insect cell line PmLyO-Sf9. A few hours after inoculation with an infected tissue extract or virus suspension, cytopathic changes could be observed in the cell line, including clustering, enlargement, syncytium formation, granulation, vacuole formation, tapering, irregularities in the plasma membrane with extended tails, detachment, cell death, and accumulation of cellular debris. Expression of viral genes, the presence of virions, and cytological changes observed using transmission electron microscopy suggested replication of the virus in these cells. The virus was purified by ultracentrifugation, negatively stained, and examined using an electron microscope, and the purified virus was found to be infectious both in vitro and in vivo. This development opens avenues for the study of the basic molecular mechanism of IHHNV infection, pathogenesis, and replication, which is much needed for developing an antiviral strategy in aquaculture.

Pathogenicity of two lineages of infectious hematopoietic necrosis virus (IHNV) to farmed rainbow trout (Oncorhynchus mykiss) in South Korea.

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Characterization of maternal immunity following vaccination of broodstock against IHNV or Flavobacterium psychrophilum in rainbow trout (Oncorhynchusmykiss).

Infectious hematopoietic necrosis (IHN) is a significant viral disease affecting salmonids, whereas Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD), remains one of the most significant bacterial pathogens of salmonids. We explored maternal immunity in the context of IHN and BCWD management in rainbow trout (Oncorhynchus mykiss) aquaculture. Two experimental trials were conducted where different groups of female broodstock were immunized prior to spawning with an IHNV DNA vaccine or a live attenuated F. psychrophilum (Fp B.17-ILM) vaccine alone, or in combination. Progeny were challenged with either a low or high dose of IHNV at 13 days post hatch (dph) and 32 dph or challenged with F. psychrophilum at 13 dph. Mortality following a low-dose IHNV challenge at 13 dph was significantly lower in progeny from vaccinated broodstock vs. unvaccinated broodstock, but no significant differences were observed at 32 dph. Mortality due to BCWD was also significantly reduced in 13 dph fry that originated from broodstock immunized with the Fp B.17-ILM vaccine. After vaccination broodstock developed specific or neutralizing antibodies respectively to F. psychrophilum and IHNV; however, antibody titers in eggs and fry were undetectable. In the eggs and fry mRNA transcripts of the complement components C3 and C5 were detected at much higher levels in progeny from vaccinated broodstock and showed a significantly increased and rapid response post-challenge compared with unvaccinated broodstock. After challenges pro-inflammatory cytokine expression was immediately and considerably elevated in the fry from vaccinated broodstock vs. unvaccinated broodstock, whereas adaptive immune genes were elevated to a lesser degree. Results suggest that maternal transfer of innate and adaptive factors at the transcript level occurred because development of lymphomyeloid organs is not complete in such young fry. In addition to documenting maternally derived immunity in teleosts, this study demonstrates that broodstock vaccination can confer some degree of protection to progeny against viral and bacterial pathogens.

Coproduction and modeling spatial contact networks prevent bias about infectious hematopoietic necrosis virus transmission for Snake River Basin salmonids.

Much remains unknown about variation in pathogen transmission across the geographic range of a free-ranging fish or animal species and about the influence of movement (associated with husbandry practices or animal behavior) on pathogen transmission. Salmonid hatcheries are an ideal system in which to study these processes. Salmonid hatcheries are managed for endangered species recovery, supplementation of threatened or at-risk fish stocks, support of fisheries, and ecosystem stability. Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus of significant concern to salmon aquaculture. Landscape IHNV transmission dynamics previously had been estimated only for salmonid hatcheries in the Lower Columbia River Basin (LCRB). The objectives of this study were to estimate IHNV transmission dynamics in a unique geographic region, the Snake River Basin (SRB), and to quantitatively estimate the effect of model coproduction on inference because previous assessments of coproduction have been qualitative. In contrast to the LCRB, the SRB has hatchery complexes consisting of a main hatchery and ≥1 satellite facility. Knowledge about hatchery complexes was held by a subset of project researchers but would not have been available to project modelers without coproduction. Project modelers generated and tested multiple versions of Bayesian susceptible-exposedinfected models to realistically represent the SRB and estimate the effect of coproduction. Models estimated the frequency of transmission routes, route-specific infection probabilities, and infection probabilities for combinations of salmonid hosts and IHNV lineages. Model results indicated that in the SRB, avoiding exposure to IHNV-positive adult salmonids is the most important action to prevent juvenile infections. Migrating adult salmonids exposed juvenile cohort-sites most frequently, and the infection probability was greatest following exposure to migrating adults. Without coproduction, the frequency of exposure by migrating adults would have been overestimated by 70 cohort-sites, and the infection probability following exposure to migrating adults would have been underestimated by∼0.09. The coproduced model had less uncertainty in the infection probability if no transmission route could be identified (Bayesian credible interval (BCI) width = 0.12) compared to the model without coproduction (BCI width = 0.34). Evidence for virus lineage MD specialization on steelhead and rainbow trout (both Oncorhynchus mykiss) was apparent without model coproduction. In the SRB, we found a greater probability of virus lineage UC infection in Chinook salmon (Oncorhynchus tshawytscha) compared to in O. mykiss, whereas in the LCRB, UC more clearly exhibited a generalist approach. Coproduction influenced estimates that depended on transmission routes, which operated differently at main hatcheries and satellite sites within hatchery complexes. Hatchery complexes are found outside of the SRB and are not specific to salmonid hatcheries alone. There is great potential for coproduction and modeling spatial contact networks to advance understanding about infectious disease transmission in complex production systems and surrounding free-ranging animal populations.

Comprehensive analysis of immune parameters, mRNA and miRNA profiles, and immune genes expression in the gill of rainbow trout infected with infectious hematopoietic necrosis virus (IHNV).

Rainbow trout (Oncorhynchus mykiss) is a species of cold-water fish with important economic values, widely cultivated worldwide. However, the outbreak of infectious hematopoietic necrosis virus (IHNV) caused the large-scale death of rainbow trout and seriously restricted the development of the trout farming industry. In this study, the changes of immune parameters in different periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 h post-infection (hpi)), transcriptome profiles of 48 hpi (T48G) compared to control (C48G), and key immune-related genes expression patterns were measured in rainbow trout gill following IHNV challenge through biochemical methods, RNA sequencing (RNA-seq), and quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that alkaline phosphatase (AKP), acid phosphatase (ACP), total superoxide dismutase (T-SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities, as well as lysozyme (LZM) and malonaldehyde (MDA) content decreased and then increased during infection, and remained at a high level after 48 hpi (P < 0.05), whereas catalase (CAT) activity showed a significant peak at 48 hpi (P < 0.05). The mRNA and miRNA analysis identified 4343 differentially expressed genes (DEGs) and 11 differentially expressed miRNAs (DEMs), and numerous immune-related DEGs involved in the Toll-like receptor signaling pathway, apoptosis, DNA replication, p53 signaling, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway and expression were significantly up-regulated in T48Gm group, including tlr3, tlr7, tlr8, traf3, ifih1, trim25, dhx58, ddh58, hsp90a.1, nlrc3, nlrc5, socs3, irf3, irf7, casp7, mx1, and vig2. The integrated analysis identified several important miRNAs (ola-miR-27d-3p_R+5, gmo-miR-124-3-5p, ssa-miR-301a-5p_L+2, and ssa-miR-146d-3p) that targeted key immune-related DEGs. Expression analysis showed that tlr3, tlr7, traf3, ifih1, dhx58, hap90a.1, irf3, irf7, and mx1 genes increased and then decreased during infection, and peaked at 72 hpi (P < 0.05). However, trim25 expression peaked at 96 hpi (P < 0.05). This study contributes to understanding immune response of rainbow trout against IHNV infection, and provides new insights into the immune regulation mechanisms and disease resistance breeding studies.

Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia and IHNV by immunization with VHSV genotype IVa backbone-based single-cycle viruses.

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

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.

Teleost Eye Is the Portal of IHNV Entry and Contributes to a Robust Mucosal Immune Response.

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout's OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish.

Andrographolide Alleviates Oxidative Damage and Inhibits Apoptosis Induced by IHNV Infection via CTSK/BCL2/Cytc Axis.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that causes significant economic losses to salmon trout farming. Although vaccines have been invented for the treatment of IHNV, findings from our previous survey show that breeding enterprises and farmers require effective oral drugs or immune enhancers. However, studies on the development of oral drugs are limited. In the present study, we used bioinformatics methods to predict the protein targets of andrographolide (Andro) in IHNV. Cells were infected with IHNV, and the effect of andrographolide was explored by evaluating the expression levels of genes implicated in oxidative stress, activities of antioxidant enzymes, and the expression of genes implicated in apoptosis and necrosis. In the present study, cells were divided into NC, IHNV, IHNV+10 µM andrographolide, and IHNV+20 µM andrographolide groups. qRT-PCR was performed to determine the expression level of genes, and an antioxidant enzyme detection kit was used to evaluate the activities of antioxidant enzymes. Fluorescent staining was performed using a reactive oxygen species detection kit (ROS) and Hoechst 33342/PI double staining kit, and the mechanism of alleviation of apoptosis and oxidative stress andrographolide after IHNV infection was determined. The results indicated that andrographolide inhibits viral growth by binding to the NV protein of IHNV and increasing the antioxidant capacity of the body through the CTSK/BCL2/Cytc axis, thereby inhibiting the occurrence of IHNV-induced apoptosis. This is the first study to explore the antagonistic mechanism of action of andrographolide in alleviating IHNV infection. The results provide valuable information on alternative strategies for the treatment of IHNV infection during salmon family and provide a reference for the use of andrographolide as an antioxidant agent in agricultural settings.

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.

Comparative Study on Immune Function of the Head and Trunk Kidney in Rainbow Trout Responding to IHNV Infection.

A teleost's kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost's head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost's trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts.

Alterations of the Mucosal Immune Response and Microbial Community of the Skin upon Viral Infection in Rainbow Trout (Oncorhynchus mykiss).

The skin is the largest organ on the surface of vertebrates, which not only acts as the first line of defense against pathogens but also harbors diverse symbiotic microorganisms. The complex interaction between skin immunity, pathogens, and commensal bacteria has been extensively studied in mammals. However, little is known regarding the effects of viral infection on the skin immune response and microbial composition in teleost fish. In this study, we exposed rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus (IHNV) by immersion infection. Through pathogen load detection and pathological evaluation, we confirmed that IHNV successfully invaded the rainbow trout, causing severe damage to the epidermis of the skin. qPCR analyses revealed that IHNV invasion significantly upregulated antiviral genes and elicited strong innate immune responses. Transcriptome analyses indicated that IHNV challenge induced strong antiviral responses mediated by pattern recognition receptor (PRR) signaling pathways in the early stage of the infection (4 days post-infection (dpi)), and an extremely strong antibacterial immune response occurred at 14 dpi. Our 16S rRNA sequencing results indicated that the skin microbial community of IHNV-infected fish was significantly richer and more diverse. Particularly, the infected fish exhibited a decrease in Proteobacteria accompanied by an increase in Actinobacteria. Furthermore, IHNV invasion favored the colonization of opportunistic pathogens such as Rhodococcus and Vibrio on the skin, especially in the later stage of infection, leading to dysbiosis. Our findings suggest that IHNV invasion is associated with skin microbiota dysbiosis and could thus lead to secondary bacterial infection.