Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL^?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.     

Lumpfish (Cyclopterus lumpus,Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail‐biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV‐infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.     

The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo

Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well‐known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers.     

Antiviral abilities of Curcuma kwangsiensis ingredients against grouper iridoviral infection in vitro and in vivo

As one of the most serious pathogens in mariculture, the outbreaks of grouper Iridovirus (SGIV‐Gx) caused high mortality rates in cultured groupers in Guangxi, China. Hence, effective medicines for fighting against grouper Iridovirus are urgently needed. The possible application of Curcuma kwangsiensis ingredients against SGIV‐Gx infection was evaluated in vitro and in vivo in this study. The safe working concentration of each C. kwangsiensis ingredient was identified (C. kwangsiensis ethanol ingredient, CKEE ≤5 mg/ml; curcumin ≤20 μg/ml; curdione ≤500 μg/ml; curcumenol ≤500 μg/ml; curcumol displayed no cytotoxic effects even at 2 mg/ml) in vitro and in vivo. The inhibitory activities of each C. kwangsiensis ingredient against SGIV‐Gx infection were analysed using aptamer (Q2)‐based fluorescent molecular probe (Q2‐AFMP) and RT‐qPCR. The results showed that C. kwangsiensis ingredient (CKEE, curcumin, curcumol, curdione and curcumenol) displayed antiviral activities against SGIV‐Gx infection in a dose‐dependent manner. Furthermore, according to the inhibitory percentage analysed using RT‐qPCR results, CKEE and curdione had the best antiviral activity against SGIV‐Gx above 93%. Overall, the results suggest that some C. kwangsiensis ingredients have excellent antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling SGIV‐Gx outbreaks in mariculture.     

In vivo antiviral efficacy of moroxydine hydrochloride against grass carp reovirus and the drug safety assessment

Our previous studies have verified that moroxydine hydrochloride (Mor) could inhibit replication of grass carp reovirus (GCRV) and suppress apoptosis of Ctenopharyngodon idella kidney cells, but be lack of information whether exists on antiviral activity in vivo. The paper was undertaken to explore the antiviral response of Mor against GCRV in grass carp and investigate the safety of drug for aquatic organisms. The results showed that injection treatment of Mor could more effectively inhibit GCRV replication than immersion administration. All the RNA systheses of vp3 and vp6 on day 7 in head kidney, gill, hepatopancreas and dorsal muscle in the Mor injection group were lesser than 0.07‐fold than that of in control group. And the GCRV‐inducing grass carp mortality was effectively controlled within 7 days post Mor injection therapy. Additionally, the reduction of superoxide dismutase activity, total antioxidant capacity and catalase activity in serum was effectively controlled by Mor. Moreover, drug safety assessment results showed that 500 mg/L of Mor was safe to C. idella, Bacillus subtilis, Chlorella vulgaris and Tetrahymena thermophila, which was far higher than the therapeutic concentration. The present study proved Mor as harmless formulations or products had potential value in the control of GCRV in aquaculture, with the advantage of super in vivo antiviral activity and environmental safety.     

Molecular docking and simulation studies of 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 against VP26 and VP28 proteins of white spot syndrome virus

White spot syndrome virus (WSSV), an aquatic virus infecting shrimps and other crustaceans, is widely distributed in Asian subcontinents including India. The infection has led to a serious economic loss in shrimp farming. The WSSV genome is approximately 300 kb and codes for several proteins mediating the infection. The envelope proteins VP26 and VP28 play a major role in infection process and also in the interaction with the host cells. A comprehensive study on the viral proteins leading to the development of safe and potent antiviral therapeutic is of adverse need. The novel synthesized compound 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 is proved to have potent antiviral activity against WSSV. The compound antiviral activity is validated in freshwater crabs (Paratelphusa hydrodomous). An in silico molecular docking and simulation analysis of the envelope proteins VP26 and VP28 with the ligand 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 are carried out. The docking analysis reveals that the polar amino acids in the pore region of the envelope proteins were involved in the ligand binding. The influence of the ligand binding on the proteins is validated by the molecular dynamics and simulation study. These in silico approaches together demonstrate the ligand's efficiency in preventing the trimers from exhibiting their physiological function.     

Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

Effect of repeated exposure to AQUI‐S® on the viability and growth of Neoparamoeba perurans

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28‐day period to AQUI‐S^® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post‐smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non‐attached amoeba in the culture was assessed with a vital stain. AQUI‐S^® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.     

Antiviral activity of Illicium verum Hook. f. extracts against grouper iridovirus infection

Grouper iridovirus causes high mortality rates in cultured groupers, and effective treatment for grouper iridovirus infection is urgently required. Illicium verum Hook. f. is a well-known medicinal plant with a variety of biological activities. The aim of this study was to analyse the use of I. verum extracts to treat grouper iridovirus infection. The safe working concentration of each I. verum extract was identified both in vitro and in vivo as follows: I. verum aqueous extract (IVAE) ≤ 500 μg/ml; I. verum ethanol extract (IVEE) ≤ 250 μg/ml; shikimic acid (SKA) ≤ 250 μg/ml; trans-anethole (TAT) ≤ 800 μg/ml; 3,4-dihydroxybenzoic acid (DDBA) ≤ 400 μg/ml; and quercetin (QCE) ≤ 50 μg/ml. The inhibitory activity of each I. verum extract against grouper iridovirus infection was analysed using aptamer (Q2)-based fluorescent molecular probe (Q2-AFMP) and RT-qPCR. All of the I. verum extracts displayed dose-dependent antiviral activities against grouper iridovirus. Based on the achieved per cent inhibition, IVAE, IVEE, DDBA and QCE were associated with the greatest antiviral activity (all > 90%). Together, our results indicate that I. verum extracts have effective antiviral properties, making it an excellent potential source material for the development of effective treatment for grouper iridovirus infection.     

Chemical composition and in vitro antifungal activity of Sambucus ebulus and Actinidia deliciosa on the fish pathogenic fungus,Saprolegnia parasitica

The aim of the present study was to determine the chemical composition of Sambucus ebulus and Actinidia deliciosa ethanolic extracts as well as their in vitro antifungal activity on the mycelial growth of the water mold, Saprolegnia parasitica. The preliminary minimum inhibition concentration (MIC) and minimum lethal concentration (MLC) were determined by the HeMP method and finally, concentrations of each extract ranging from 1 to 10% were prepared by an agar dilution method to assess the in vitro antifungal activity, quantitatively. Both herbal extracts inhibited growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥5% for S. ebulus, whereas it was not observed even at 10% concentration of A. deliciosa. Based on gas chromatography coupled with mass spectrometry (GC/MS) analysis, the main constituents of S. ebulus include monophthalate (66.17%), fatty acids (26.47%), phytol (4.25%), and acetic acid (2.11%). Using colorimetric assays, A. deliciosa contained phenolic contents at 162 mg gallic acid (GAE)/g DW and flavonoid contents at 2.31 mg quercetin (QE)/g DW. In conclusion, the results of this study indicated that S. ebulus and A. deliciosa showed some antifungal activities against S. parasitica with formerly exhibiting stronger activity (p < 0.05).

     

Administration of hot‐water extract of Padina boergesenii via immersion route to enhance haemolymph‐immune responses of Fenneropenaeus indicus (Edwards)

Various polysaccharides extract from marine algae to increase the non‐specific immune system in crustacean. In this study, effects of hot‐water extract on total haemocyte count, total plasma protein, Phagocytic activity, bacterial clearance efficiency and bactericidal activity while the shrimp Fenneropenaeus indicus (10.12±2.18 g) were immersed in seawater (40 g L^?1 and 25±0.8 °C) containing hot‐water extract of brown algae Padina boergesenii at 100, 300 and 500 mg L^?1 for 1–4 h, were investigated. These parameters increased significantly (P<0.05) when the shrimp were immersed in seawater containing hot‐water extract at 100 mg L^?1 after 3 h and 300 and 500 mg L^?1 after 2 h. Fenneropenaeus indicus that were immersed in hot‐water extract at 300 and 500 mg L^?1 had significantly increased phagocytic activity and increased clearance efficiency to Vibrio harveyi after 2 h. But bactericidal activity increased after 1 h immersed in 500 mg L^?1 concentration.     

Effect of replacement of dietary fish meal with silkworm pupae meal on growth performance,body composition,intestinal protease activity and health status in juvenile Jian carp (Cyprinus carpio var. Jian)

This study investigated the effect of replacement of graded dietary fish meal (FM) protein (0, 50%, 60%, 70% and 80%) with silkworm pupae meal (SP) in juvenile Jian carp. Triplicate groups comprising 18 fish (15.96 ± 0.66 g) were fed one of five isonitrogenous and isocaloric diets (designated SP0, SP50, SP60, SP70 or SP80) for 8 weeks. The final body weight and specific growth rate of fish in the SP60, SP70 and SP80 groups were significantly lower than those for fish in the SP0 group (P < 0.05). The muscle protein content in the SP50 group was significantly higher than in the SP80 group (P < 0.05). With increasing FM replacement levels, the hepatic superoxide dismutase (SOD) activity decreased and the malondialdehyde (MDA) content increased among the groups, with the significant difference appeared in SP80 group. The gene expression level of heat shock protein 70 in the SP70 group was significantly higher than in the SP0 group (P < 0.05), while that in the SP80 group was significantly less than in the SP70 group (P < 0.05). Significantly decreased intestinal protease activity, increased serum ALT and AST activities, irregular‐shaped hepatocytes and intestinal microvilli were found in the SP80 group. The study demonstrates that it is practical to replace 50% of the Jian carp dietary FM protein with SP, higher SP levels are not recommended and that oxidation status of the SP should be carefully assessed.     

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass,Dicentrarchus labrax

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues—eye, oesophagus, gills and skin—as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.     

Molecular characterization and expression analysis of two RING-between-RING (RBR) ubiquitin ligase orthologues from the Asian seabass (Lates calcarifer)

Post-translational modifications like ubiquitination determine the fate of many cellular proteins. There are evidences about the role of these ubiquitinated proteins in the antiviral immune response. E3 ubiquitin ligases like RBR (RING between RING) proteins play a crucial role in the regulation of immune signalling. In the present study, two RBR genes (RNF114 and RNF144A) from Asian seabass (Lates calcarifer) were cloned and characterized. Phylogenetic analysis reveals that these genes were closely clustered with their counterparts from teleost origin. Structurally they feature multiple domains with RING1 and RING2 domains and an IBR (in between RING) domain. The challenge studies with poly(I:C) and RGNNV virus gave evidence that both Lates RNF114 and RNF144A genes play a crucial role in antiviral immune response. Our studies suggest that RBR is an important gene in innate immune response and shed light on antiviral immune research in Lates calcarifer and its aquaculture.

     

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.     

In vivo and in vitro protein digestibility and growth performance of animal and no‐conventional plant ingredients for river prawn Macrobrachium americanum (Caridea: Palaemonidae)

The objectives of this study were to evaluate the effects of non‐traditional plant and animal protein for river prawn M. americanum on in vivo versus in vitro digestibility in addition to growth performance. In this study, ten ingredients were used: coconut meal, jackfruit meal, soybean meal, poultry by‐product meal, squid meal and fish meal. In vivo studies were conducted using the zeolite marker method, whereas in vitro digestibility was determined by the pH‐stat method using river prawn hepatopancreas enzymes. According to the findings, river prawns digest animals more efficiently than plant ingredients, with squid and fish meal showing a tendency of higher values. Fish meal (53.66%), squid meal (48.52%) and jackfruit meal (42.21%) were found to have a digestibility greater than 40% in in vivo and in vitro methods, thus suggesting that they are the best ingredients used in this study. This also validates their inclusion in practical diets. The correlation of digestibility of in vivo and in vitro was low (R² = .6749). The highest daily weight gain was recorded in the diets containing jackfruit, coconut and squid. Given that the potential of no‐conventional plant ingredients available regionally was significantly higher, this underscores the need to conduct further research to validate their inclusion in practical diets.     

Utilization of Shrimp By-Products by Bioconversion with Medical Fungi for Angiotensin I-Converting Enzyme Inhibitor and Antioxidant

ABSTRACT

A liquid fermentation process to bioconvert shrimp by-products was developed with two species of fungi, Boletus edulis and Suillus bovinus. Angiotensin I-converting enzyme (ACE) inhibitory, antioxidant activities of bioconverted products and the deodorization effect of fungal bioconversion were determined. Two species of fungi could grow quickly in shrimp by-products medium and deodorize shrimp by-products. Bioconverted products had higher ACE inhibitory and antioxidant activities than control medium. Water extracts without crude polysaccharides from mycelia of B. edulis had the highest ACE inhibitory activity (IC₅₀ = 0.084 ± 0.011 mg/mL). Crude exopolysaccharides of B. edulis had higher antioxidant activity. Bioconversion with the fungus was proven to be a novel, practicable way to recycle shrimp by-products.