Vaccine Molecule Design Based on Phage Display and Computational Modeling against Rhabdovirus.

Rhabdoviruses with rich species lead a variety of high lethality and rapid transmission diseases to plants and animals around the globe. Vaccination is one of the most effective approaches to prevent and control virus disease. However, the key antigenic epitopes of glycoprotein being used for vaccine development are unclear. In this study, fish-derived Abs are employed for a Micropterus salmoides rhabdovirus (MSRV) vaccine design by phage display and bioinformatics analysis. We constructed an anti-MSRV phage Ab library to screen Abs for glycoprotein segment 2 (G2) (G129-266). Four M13-phage-displayed Abs (Ab-5, Ab-7, Ab-8 and Ab-30) exhibited strong specificity to target Ag, and Ab-7 had the highest affinity with MSRV. Ab-7 (300 µg/ml) significantly increased grass carp ovary cell viability to 83.40% and significantly decreased the titer of MSRV. Molecular docking results showed that the key region of Ag-Ab interaction was located in 10ESQEFTTLTSH20 of G2. G2Ser11 and G2Gln12 were replaced with alanine, respectively, and molecular docking results showed that the Ag-Ab was nonbinding (ΔG > 0). Then, the peptide vaccine KLH-G210-20 was immunized to M. salmoides via i.p. injection. ELISA result showed that the serum Ab potency level increased significantly (p < 0.01). More importantly, the challenge test demonstrated that the peptide vaccine elicited robust protection against MSRV invasion, and the relative percentage survival reached 62.07%. Overall, this study proposed an approach for screening key epitope by combining phage display technology and bioinformatics tools to provide a reliable theoretical reference for the prevention and control of viral diseases.

Rationally Designed Self-Assembling Nanovaccines Elicit Robust Mucosal and Systemic Immunity against Rhabdovirus.

Viral diseases have constantly caused great threats to global public health, resulting in an urgent need for effective vaccines. However, the current viral vaccines often show low immunogenicity. To counter this, we report a smart strategy of a well-designed modular nanoparticle (LSG-TDH) that recapitulates the dominant antigen SG, low-molecular-weight protamine, and tetralysine-modified H-chain apoferritin (TDH). The constructed LSG-TDH nanovaccine could self-assemble into a nanocage structure, which confers excellent mucus-penetrating, cellular affinity, and uptake ability. Studies demonstrate that the LSG-TDH nanovaccine could strongly activate both mucosal and systemic immune responses. Importantly, by immunizing wild-type and TLR2 knockout (TLR2-KO) zebrafish, we found that TLR2 could mediate LSG-TDH-induced adaptive mucosal and systemic immune responses by activating antigen-presenting cells. Collectively, our findings offer new insights into rational viral vaccine design and provide additional evidence of the vital role of TLR2 in regulating adaptive immunity.

Antiviral, antioxidant, and anti-inflammatory activities of rhein against white spot syndrome virus infection in red swamp crayfish (Procambarus clarkii).

IMPORTANCE: Aquaculture is essential for ensuring global food security by providing a significant source of animal protein. However, the spread of the white spot syndrome virus (WSSV) has resulted in considerable economic losses in crustacean industries. In this study, we evaluated the antiviral activity of rhein, the primary bioactive component of Rheum palmatum L., against WSSV infection, and many pathological aspects of WSSV were also described for the first time. Our mechanistic studies indicated that rhein effectively arrested the replication of WSSV in crayfish by modulating innate immunity to inhibit viral gene transcription. Furthermore, we observed that rhein attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes while enhancing innate immunity by reducing total protein levels and increasing phosphatase activity. Our findings suggest that rhein holds great promise as a potent antiviral agent for the prevention and treatment of WSSV in aquaculture.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.

4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin: The first inhibitor of fish rhabdovirus glycoprotein.

Spring viraemia of carp virus (SVCV), a highly pathogenic rhabdovirus, could cause spring viraemia of carp (SVC) with up to 90% lethality. Like other rhabdoviruses, the entry of SVCV into susceptible cells was mediated by a single envelope glycoprotein G. Specific inhibitors targeting the glycoprotein were the most effective means to alleviate the epidemic. The programs including SWISS-MODEL, I-TASSER, Phyre2 and AlphaFold2 were used to build a three-dimensional structural model of glycoprotein. The structural comparison between SVCV-G and homology protein VSV-G revealed that the SVCV glycoprotein ectodomain (residues 19 to 466) folded into four distinct domains. Based on the potential small molecule binding sites on glycoprotein surfaces, virtual screening of the anti-SVCV drug libraries was performed using Autodock software and 4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin (MOA) with a high binding affinity was identified. The solubility enhancer tags including trigger factor and maltose binding protein were fused with the ectodomain of glycoprotein, and the target protein with a purity of about 90% was successfully obtained. The interaction confirmation tests revealed that the fluorescence intensity of a characteristic peak induced by the endogenous chromophores in glycoprotein was decreased with the addition of MOA, indicating changes in the microenvironment of glycoprotein. Moreover, the interaction could cause a slight conformational change in glycoprotein, as shown by the content of ß-turn, ß-folding, and random coil of protein all increased with the decrease of α-helix content after the addition of MOA compound. These results demonstrated that MOA could act as a novel drug against fish rhabdovirus via direct targeting of glycoprotein.

Enterobacter asburiae E7, a Novel Potential Probiotic, Enhances Resistance to Aeromonas veronii Infection via Stimulating the Immune Response in Common Carp (Cyprinus carpio).

Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1ß, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-ß at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls (54%; P < 0.01). Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic. IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety.

Biomimetic nanovaccine based on erythrocyte membrane enhances immune response and protection against tilapia lake virus.

An infectious disease emerged in recent years, Tilapia Lake Virus Disease (TiLVD), has severely restricted the development of global tilapia industry. Vaccination has proved potential strategy to prevent its causative agent Tilapia Lake Virus (TiLV) infectious. However, the response intensity of subunit vaccine is limited by its low immunogenicity, thus inclusion of adjuvants is required. Thus, we prepared a biomimetic nano-system (Cs-S2@M-M) with a particle size of ∼100 nm and an encapsulation efficiency of about 79.15% based on erythrocyte membrane. The immune response was detected after intramuscular injection to assess the effectiveness of the vaccine. The biomimetic system significantly up-regulates the expression of immune genes, enhances the activity of non-specific immune-related enzymes (P < 0.05) and improved relative percentage survival by 17.4%-26.1% in TiLV challenge. The biomimetic nano-system based on erythrocyte membrane induced significant immune response in tilapia and enhanced protection against TiLV, promising as a model for fish vaccines.

Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis.

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

A therapeutic agent of ursolic acid demonstrates potential application in aquaculture.

Micropterus salmoides rhabdovirus (MSRV) has a high mortality rate and causes huge economic losses to the aquaculture industry. In this study, we identified that ursolic acid (UA) had antiviral efficacy against MSRV in vitro and in vivo. The results showed that UA inhibited MSRV replication in grass carp ovary (GCO) cells with a half-maximal inhibitory concentration (IC50) of 5.55 µM, reduced viral titers and decreased cytopathic effects (CPE). Mechanistically, UA does not directly damage viral particles. On the other hand, UA inhibits MSRV replication by altering viral binding and release. Furthermore, pre- and post-treatment assays revealed that UA had preventive and therapeutic effects. For in vivo studies, UA could enhance the survival rate of MSRV-infected largemouth bass. Similarly, UA reduced the viral load of MSRV in the heart, spleen and brain at 3, 5 and 7 d post-infection. In conclusion, UA is an effective inhibitor of rhabdovirus in aquaculture.

Dual-Targeting Polymer Nanoparticles Efficiently Deliver DNA Vaccine and Induce Robust Prophylactic Immunity against Spring Viremia of Carp Virus Infection.

Spring viremia of carp virus (SVCV) is highly contagious and lethal to most cyprinid fish, causing serious economic losses to the carp aquaculture industry. Although DNA vaccines can generate long-term humoral and cellular immune responses, which provide protective immunity against SVCV, the major drawback of DNA vaccines is their low immunogenicity in clinical tests. Here, we construct a dual-targeted polymer DNA vaccine delivery platform (MCS-PCHG) by using mannosylated chitosan to encapsulate the poly(d,l-lactide-co-glycolide)-loaded DNA vaccine containing the heavy-chain CH3 region (CH3) of common carp IgM and the antigenic domain (G131c). The developed nanovaccine delivery platform showed good biocompatibility in vivo and in vitro. With the modification of the mannose moiety and the modification of CH3, the constructed MCS-PCHG could efficiently activate the maturation of antigen-presenting cells. Moreover, we observe significantly high level of immune-related genes expression, serum antigen-specific IgM, SVCV-neutralizing antibody titers in fish vaccinated with MCS-PCHG. Next, the protective efficacy of MCS-PCHG was further evaluated by challenge test. The highest survival rate (ca. 84%) was observed in fish vaccinated with MCS-PCHG after challenging with SVCV. This study presents a novel design for smart, dual-targeted polymer nanoparticles, which are inherently biocompatible, promising for targeted vaccine delivery. IMPORTANCE Spring viremia of carp virus (SVCV) affects global cyprinid fish farming industry, with no available commercial vaccine. Herein, we developed a dual-targeting polymer nanovaccine (MCS-PCHG) by using mannose and common carp IgM heavy chain CH3 region (CH3) as antigen presenting cell (APCs) recognition moiety, attaining the effective delivery of antigen. This dual-targeting polymer vaccine can efficiently activate the APCs, and further induce robust and durable adaptive immune response with good protection against SVCV infection. Our study provides valuable theoretical basis for developing efficient vaccine against infectious diseases in aquaculture.

Protective immunity by DNA vaccine against Micropterus salmoides rhabdovirus.

Micropterus salmoides rhabdovirus (MSRV) is one of the common pathogens in the largemouth bass industry, which can cause lethal diseases in juvenile fish and enormous economic losses. To establish effective means to prevent MSRV infection, the pcDNA3.1-G plasmid containing the MSRV glycoprotein gene was successfully constructed and intramuscularly injected into the largemouth bass to evaluate the immune responses and protective effects in our study. As the results showed, the serum antibody levels of the fish vaccinated with different doses of pcDNA3.1-G were significantly higher compared with the control groups (PBS and pcDNA3.1). Meanwhile, the immune parameters (acid phosphatase and alkaline phosphatase) were also significantly up-regulated. Several immune-related genes (IgM, IL-8, IL-12p40 and CD40) were expressed in the pcDNA3.1-G groups at higher levels than in the control groups, which indicated that strong immune responses were induced. Besides, the survival percentages of fish in the control groups (PBS and pcDNA3.1) and pcDNA3.1-G groups (2.5, 5, 10 and 20 µg/fish) at 14 days after challenge experiment with MSRV were 0%, 0%, 6.1%, 15.2%, 29.0% and 48.5% respectively. This study indicated that pcDNA3.1-G was a prospective DNA vaccine candidate against MSRV-induced mortality.

Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

In vitro and in vivo inhibition of a novel arctigenin derivative on aquatic rhabdovirus.

Spring viraemia of carp virus (SVCV) poses a serious threat to aquaculture industry due to the lack of approved antiviral treatments. Therefore, a novel arctigenin derivative, 4-(2-methylimidazole) octanoxy-arctigenin (MON), was synthesized to assess the antiviral activity against SVCV in vitro and in vivo. The results indicated MON decreased the SVCV glycoprotein (G) gene expression in vitro by a maximum inhibitory rate of > 99% at 3.5 µM. Furthermore, MON showed the protective effect on epithelioma papulosum cyprinid (EPC) cells and considerably decreased the cytopathic effect (CPE). More importantly, MON inhibited SVCV G gene expression levels in vitro at the half-maximal activity (IC50) of 0.18 µM at 48 h. For in vivo studies, MON demonstrated anti-SVCV activity by enhancing the survival rate of zebrafish (Danio rerio) after infection via pelvic fin base injection. These results tended to be consistent with MON decreasing the SVCV titer of infected zebrafish. During this time, viral loads of the spleen and kidney have declined in SVSV-infected zebrafish. Based on the histopathological assay, MON exhibited the high protective effect in the spleen and kidney of SVCV-infected fish. Combined, MON is on track to become a novel agent to address SVCV infection in aquaculture.

Quinoline, with the active site of 8-hydroxyl, efficiently inhibits Micropterus salmoides rhabdovirus (MSRV) infection in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC50 ) value of 4.66 µM at 48 h. Moreover, 8-hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti-MSRV activity of 22 quinoline derivatives, we found that 8-hydroxyquinoline possessed the efficient active site of 8-hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8-hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV-infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post-infection. Overall, 8-hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.

Amantadine, a promising therapeutic agent against viral encephalopathy and retinopathy.

Outbreaks of viral encephalopathy and retinopathy (VER) in marine and freshwater species severely devastate the aquaculture worldwide. The causative agent of VER is nervous necrosis virus (NNV), which mainly infects the early developmental stages of fish, limiting the effectiveness of vaccines. To counter this case, the anti-NNV potentials of nine drugs with broad-spectrum antiviral activity were explored using ribavirin as a positive drug. Toxicity of the selected drugs to SSN-1 cells and grouper was firstly evaluated to determine the safety concentrations. For screening in vitro, amantadine and oseltamivir phosphate can relieve the cytopathic effects and inhibit NNV replication with the 90% inhibitory concentrations (IC90 ) of 38.74 and 106.75 mg/L, respectively. Amantadine has a stronger anti-NNV activity than ribavirin at the with- and post-NNV infection stages, indicating that it is a potential therapeutic agent against VER by acting directly on NNV. Similarly, amantadine also has a strong anti-NNV activity in vivo with the IC90 of 27.91 mg/L at the 7 days post-infection, while that was 73.25 mg/L for ribavirin. Following exposure to amantadine (40 mg/L) and ribavirin (100 mg/L) for 7 days, the survival rates of NNV-infected grouper were increased to 44% and 39%, respectively. The maximum amantadine content (11.88 mg/Kg) in grouper brain was reached following exposure for 24 hr, and amantadine can be quickly excreted from fish, reducing the risk of drug residue. Results so far indicated that amantadine is a promising therapeutic agent against NNV in aquaculture, providing an effective strategy for VER control at the early developmental stages of fish.

A Nanobody-Mediated Virus-Targeting Drug Delivery Platform for the Central Nervous System Viral Disease Therapy.

Viral diseases of the central nervous system (CNS) represent a major global health concern. Difficulties in treating these diseases are caused mainly by the biological tissues and barriers, which hinder the transport of drugs into the CNS. To counter this, a nanobody-mediated virus-targeting drug delivery platform (SWCNTs-P-A-Nb) is constructed for CNS viral disease therapy. Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is employed as a disease model. SWCNTs-P-A-Nb is successfully constructed by employing single-walled carbon nanotubes, amantadine, and NNV-specific nanobody (NNV-Nb) as the nanocarrier, anti-NNV drug, and targeting ligand, respectively. Results showed that SWCNTs-P-A-Nb has a good NNV-targeting ability in vitro and in vivo, improving the specific distribution of amantadine in NNV-infected sites under the guidance of NNV-Nb. SWCNTs-P-F-A-Nb can pass through the muscle and gill and be excreted by the kidney. SWCNTs-P-A-Nb can transport amantadine in a fast manner and prolong the action time, improving the anti-NNV activity of amantadine. Results so far have indicated that the nanobody-mediated NNV-targeting drug delivery platform is an effective method for VER therapy, providing new ideas and technologies for control of the CNS viral diseases. IMPORTANCE CNS viral diseases have resulted in many deadly epidemics throughout history and continue to pose one of the greatest threats to public health. Drug therapy remains challenging due to the complex structure and relative impermeability of the biological tissues and barriers. Therefore, development in the intelligent drug delivery platform is highly desired for CNS viral disease therapy. In the study, a nanobody-mediated virus-targeting drug delivery platform is constructed to explore the potential application of targeted therapy in CNS viral diseases. Our findings hold great promise for the application of targeted drug delivery in CNS viral disease therapy.

Ophiopogon japonicus inhibits white spot syndrome virus proliferation in vivo and enhances immune response in Chinese mitten crab Eriocheir sinensis.

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control. Herbal medicines have been widely used to treat a number of viral infections, which could offer a rich reserve for antiviral drug discovery. Here, we evaluated the inhibition activities of 30 herbal medicines against WSSV in Chinese mitten crab Eriocheir sinensis. A WSSV infection model in E. sinensis was firstly established in order to determine the antiviral effects of the plant extracts and to explore the potential action mechanisms. Results showed that the highest anti-WSSV activity was obtained by the treatment of Ophiopogon japonicus extract (93.03%, 100 mg/kg). O. japonicus treatment decreased viral loads in a dose-dependent manner and significantly improved the survival of WSSV-challenged crabs. O. japonicus reduced the expression of vital genes in viral life cycle in vivo, particularly for the immediate-early stage gene ie1. Further results indicated that O. japonicus could repress the JAK-STAT signaling pathway to block ie1 transcription. Moreover, O. japonicus could modulate certain immune genes such as the myosin, toll-like receptor, crustin, and prophenoloxidase in the interactions between WSSV and crabs. The up-regulated expression of pro-autophagic factors (Gabarap and Atg7) and elevated levels of antioxidant enzymes (SOD, CAT and GSH) suggested that O. japonicus may induce autophagy and attenuate WSSV-induced oxidative stress. Taken together, O. japonicus could inhibit WSSV proliferation and improve the survival of WSSV-challenged crabs. Thus, O. japonicus may have the potential to be developed as a preventive or therapeutic agent against WSSV, and its effective compounds merit further isolation and identification.

Acyclovir inhibits white spot syndrome virus replication in crayfish Procambarus clarkii.

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control WSSV. To address the urgent need for finding effective antiviral agents against WSSV, we examined the anti-WSSV activities of 11 common antiviral agents in crayfish Procambarus clarkia. The results showed that acyclovir displayed the highest inhibition on WSSV replication in vivo (92.59%, 50 mg/kg). Acyclovir repressed WSSV proliferation followed a dose-dependent fashion and pre- or post-treatment of acyclovir exerted strong inhibition on the viral loads. Further, we observed a markedly reduced expression levels of WSSV genes (immediate-early IE gene ie1, DNA polymerase gene DNApol and envelope protein gene Vp28) that are crucial in viral life cycle with the acyclovir treatment during the early infection. Meantime, we also found a significantly increased expressions of anti-oxidative as well as apoptosis related genes, suggesting that acyclovir could effectively suppress WSSV replication in vivo. Finally, acyclovir treatment could significantly improve the survival rate of WSSV-challenged crayfish by 56%. Taken together, acyclovir has the potential to be developed as a promising preventive or therapeutic agent against WSSV infection, and this finding may provide a reference for rapid discovery anti-WSSV agent in crustacean aquaculture.

Optimization of immunization procedure for SWCNTs-based subunit vaccine with mannose modification against spring viraemia of carp virus in common carp.

Immersion vaccination of single-walled carbon nanotubes loaded with mannose-modified glycoprotein (SWCNTs-MG) vaccine has been proved to be effective in preventing spring viraemia of carp virus (SVCV). Immunization procedure has immense consequence on the immune effect of the immersion vaccine. However, immunization procedure optimization for SWCNTs-MG vaccine against SVCV has not been reported. In this study, accordingly, a full-factor experiment was designed to optimize the immunization procedure of SWCNTs-MG vaccine by three aspects of vaccine dose (30 mg/L, 40 mg/L and 50 mg/L), immunization density (8 fish L-1 , 24 fish L-1 and 48 fish L-1 ) and immunization time (6, 12 and 24 hr). Furthermore, we used the immunization group (A1B2C1, 30 mg/L, 24 fish L-1 and 6 hr) in the previous study as a positive control (PC) to evaluate the immunization effect optimized conditions from the expression of immune-related genes and relative percentage survival (RPS). At 28 days post-vaccination (DPV), common carps were intraperitoneal injected SVCV challenged test indicated that the A1B2C2 group (30 mg/L, 24 fish L-1 , 12 hr) displayed superiority of protective efficacy compare with other groups and the RPS with 77.9%, which was 15.6% higher than the PC group of RPS with 62.3%. Moreover, the expression of immune-related genes such as IL-10, CD4 and MHC-II was also significantly higher than PC group. The specific experimental flow chart is shown in Figure 1. Conclusively, these results demonstrated that vaccine dose, immunization density and immunization time are 30 mg/L, 24 fish L-1 and 12 hr, which is the more appropriate immunization programme with juvenile carp for SWCNTs-MG vaccine. This study provides a profitable reference for improving the immune efficiency of aquatic immersion vaccine. [Figure: see text].

Immersion immunization of common carp with bacterial ghost-based DNA vaccine inducing prophylactic protective immunity against spring viraemia of carp virus.

The interactive applications of immunization route, vaccine type and delivery vectors are emerging as a key area of research within the field of mass immunization in fishery production. In an effort to improve DNA vaccine's immune efficiency in large-scale immunization, a promising bacterial ghost-loaded DNA vaccine was constructed based on Escherichia coli DH5α. In common carp was investigated the immune response to immersion immunization via related indicator analysis, and the challenge test of spring viraemia of carp virus (SVCV) was carried out. The result indicated that BG-loaded DNA vaccine induced higher serum antibody level than naked pEG-G. Simultaneously, the immunophysiological indicators and genes change at the more advanced levels in the BG/pEG-G immune group. At the treatment concentration of 20 mg/L of the BG/pEG-G group, IgM and IgZ expressions in vivo were markedly increased by 21.62 times and 6.91 times, respectively, and the relative percentage survival reached the peak of 59.57%. This study paves the way for future aquatic animal vaccine research, which aimed to develop the highly effective immersion vaccine system by delivery vectors, with the ultimate aim to prevent and restrict SVCV in actual production.