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.

NK-lysin peptides ameliorate viral encephalopathy and retinopathy disease signs and provide partial protection against nodavirus infection in European sea bass.

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry.

Peptide inhibitors of Macrobrachium rosenbergii nodavirus.

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant (KDrel) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a KDrel value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a KDrel value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.

Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus.

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.

In vitro screening of selected antiviral drugs against betanodavirus.

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

Inhibition of nervous necrosis virus by ribavirin in a zebrafish larvae model.

The guanosine analog ribavirin is a broad-spectrum antiviral drug, mostly used in human clinical practice. It has in vitro and in vivo activity against a broad range of RNA and DNA viruses. Here, we report that treatment of zebrafish larvae with ribavirin prior to infection with nervous necrosis virus (NNV) significantly reduces the mortality caused by the virus during the first 10 days post-infection. The RNA genome of NNV harvested from ribavirin-treated infected larvae contains three synonymous and one single non-synonymous mutation, resulting in the replacement of a serine codon with a glycine codon in the RNA-dependent RNA polymerase gene. Adding increasing amounts of guanosine to ribavirin prior to larvae infection did not impede the antiviral activity. Ribavirin treatment of uninfected larvae reduces the basal level of IFNγ, but increases the level of IL-1ß mRNA expression. Furthermore, infecting larvae with NNV following ribavirin treatment reduces the expression levels of IFNγ, IFN-I, Mx, and TNF-α genes, while the expression of IL-1ß is increased. These results suggest that cytokine modulation plays an important role in the activity of ribavirin against NNV. Mortality of more than 40 species of teleost fish, mostly larvae and juveniles, from NNV is a major obstacle in hatcheries, and impedes the supply of young fish to farms. Hence, cost-effective ribavirin treatment should be considered as an efficient means to reduce the peril of NNV.

Glutamine is required for red-spotted grouper nervous necrosis virus replication via replenishing the tricarboxylic acid cycle.

Glutamine, one of the most important nutrients, plays a vital role in carbon metabolic pathway and has been reported to be required for the replication of several human DNA viruses. However, whether glutamine is required for RNA virus replication and the related mechanism remains elusive. Nervous necrosis virus (NNV), a positive-stranded RNA virus, can infect a number of important aquatic species and has caused great economic losses in aquaculture industry worldwide. In this study, the effects of glutamine on red-spotted grouper nervous necrosis virus (RGNNV) replication were investigated. The results showed that lack of glutamine did not affect the cell viability, but dramatically inhibited RGNNV replication, indicating that glutamine was required for RGNNV replication. Glutamine can be converted to α-ketoglutarate (α-KG) by glutaminase (GLS) to join in the tricarboxylic acid (TCA) cycle. Inhibiting the activity of GLS by a GLS inhibitor: bis-2-5-phenylacetamido-1,3,4-thiadiazol-2-ethyl sulfide (BPTES) significantly inhibited RGNNV replication, while adding the TCA cycle intermediates: α-KG, oxaloacetic acid (OAA), or pyruvate significantly restored RGNNV replication in glutamine-free medium, indicating that the requirement of glutamine for RGNNV replication was due to replenishing the TCA cycle. Taken together, these data revealed that glutamine could regulate RGNNV replication via TCA cycle, which will pave a new way for the prevention of the RGNNV infection in the future.

Dialysis buffer with different ionic strength affects the antigenicity of cultured nervous necrosis virus (NNV) suspensions.

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus (Nodaviridae). It is highly pathogenic to various marine fishes. Here, we investigated the antigenicity changes of cultured NNV suspensions during 14days of dialyses using a dialysis tube at 1.4×10(4) molecular weight cut off (MWCO) in three different buffers (Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), and deionized water (DIW)). Total NNV antigen titers of cultured NNV suspension varied depending on different dialysis buffers. For example, total NNV antigen titer during D-PBS dialysis was increased once but then decreased. During Tris-HCl dialysis, it was relatively stable. During dialysis in DIW, total NNV antigen titer was increased gradually. These antigenicity changes in NNV suspension might be due to changes in the aggregation state of NNV particles and/or coat proteins (CPs). ELISA values of NNV suspension changed due to changing aggregates state of NNV antigens. NNV particles in suspension were aggregated at a certain level. These aggregates were progressive after D-PBS dialysis, but regressive after Tris-HCl dialysis. The purified NNV particles self-aggregated after dialysis in D-PBS or in Tris-HCl containing 600mM NaCl, but not after dialysis in Tris-HCl or DIW. Quantitative analysis is merited to determine NNV antigens in the highly purified NNV particles suspended in buffer at low salt condition.

Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells.

Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.

Transcriptome analysis of medaka following epinecidin-1 and TH1-5 treatment of NNV infection.

Nervous necrosis virus (NNV) infects a wide range of larval and juvenile fish species, thereby causing enormous economic losses in the aquaculture industry. Possible solutions to this problem include the use of antimicrobial peptides (AMPs), which directly inhibit bacterial growth, and also modulate host signaling mechanisms. The AMPs epinecidin (Epi)-1 and Tilapia hepcidin (TH) 1-5 have been demonstrated to be effective against Nervous necrosis virus infection in medaka (Oryzias latipes). However, the underlying molecular mechanisms are yet to be explored. Here, microarray analyses were performed to examine how NNV infection and/or epinecidin-1 or TH1-5 treatment affects gene expression in medaka; such analyses enabled the prediction of host signaling pathways affected by virus infection and/or regulated by epinecidin-1 and TH1-5. Transcriptome analysis revealed altered expression of genes involved in B cell activation, T cell activation, adipocytokine signaling, and mast cell activation. We subsequently used real-time PCR to analyze expression of key genes involved in these signaling mechanisms. Medaka infected with NNV exhibited up-regulation of PVALB, CEBPA, IFIM, IFN, IL-6ST, NF-kB2, SOC3, SP1, and TGFB1, and such increases were prevented by pre-treatment with epinecidin-1 or TH1-5. Immunohistochemistry using the anti-NNV antibody to stain brain and eye sections revealed that epinecidin-1 treatment during or after infection clears viral load, while TH1-5 treatment only reduces viral numbers if applied during infection. These observations demonstrate that epinecidin-1 and TH1-5 modulate NNV-induced host signaling mechanisms, thereby preventing viral multiplication in host organisms.

Determining anti-betanodavirus compounds through a GF-1 cell-based screening platform.

Betanodavirus is a highly contagious pathogen, responsible for severe losses incurred in the aquaculture industry. Currently, there are no commercially available antivirals against the virulence observed during very early stages of fish larvae development. Therefore, we developed a novel GF-1 (grouper fin cell) cell viability-based screening assay to facilitate the discovery of an anti-betanodavirus agent. The assay conditions were optimized and the robustness of the assay was confirmed by a Z' factor value ranging from 0.7 to 0.94. After screening a library of 2000 small molecule compounds, 43 compounds with a virus inhibition capacity of ⩾55% were identified. A cytochrome P450 inhibitor, proadifen hydrochloride, was validated with an EC50 value of 6.48µM and a CC50 value of 20.63µM. This compound inhibited the amplification of viral RNA by 99.68% 5days post-infection. Surprisingly, we found that 18 of 43 compounds act as neurotransmitter agents. These findings indicate a novel way of investigating the infection mechanism of betanodavirus, and suggest potential candidates for an anti-betanodavirus drug.

Evaluation of antiviral activity of Oligonol, an extract of Litchi chinensis, against betanodavirus.

Betanodaviruses, members of the family Nodaviridae, are the causal agents of viral nervous necrosis (VNN) in many species of marine farmed fish. In the aquaculture industry, outbreaks of betanodavirus infection result in devastating damage and heavy economic losses. Although an urgent need exists to develop drugs against betanodavirus infection, there have been few reports about antibetanodavirus drugs. In this study, we examined the inhibitory effect of Oligonol, a purified phenolic extract from lychee fruit, on betanodavirus infection in fish cells. Oligonol significantly inhibited replication of betanodavirus (EC(50) = 0.9-1.8 µg/mL) as shown by the reduction of the virus-induced cytopathogenic effect (CPE) and the protection of cells in the crystal violet staining assay. The inhibition was dose dependent. A time-of-addition assay indicated that Oligonol's action takes place at an early stage of the viral infection. According to an attachment inhibition assay, it is possible that Oligonol partially inhibits attachment of the virion to the cell. Our data show that Oligonol could serve as an antiviral agent against betanodavirus.

Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12-24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24-48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.

ATP-sensitive potassium channel (K(ATP))-dependent regulation of cardiotropic viral infections.

The effects of the cellular environment on innate immunity remain poorly characterized. Here, we show that in Drosophila ATP-sensitive potassium channels (K(ATP)) mediate resistance to a cardiotropic RNA virus, Flock House virus (FHV). FHV viral load in the heart rapidly increases in K(ATP) mutant flies, leading to increased viremia and accelerated death. The effect of K(ATP) channels is dependent on the RNA interference genes Dcr-2, AGO2, and r2d2, indicating that an activity associated with this potassium channel participates in this antiviral pathway in Drosophila. Flies treated with the K(ATP) agonist drug pinacidil are protected against FHV infection, thus demonstrating the importance of this regulation of innate immunity by the cellular environment in the heart. In mice, the Coxsackievirus B3 replicates to higher titers in the hearts of mayday mutant animals, which are deficient in the Kir6.1 subunit of K(ATP) channels, than in controls. Together, our data suggest that K(ATP) channel deregulation can have a critical impact on innate antiviral immunity in the heart.

Anti-fish nodaviral activity of furan-2-yl acetate extracted from marine Streptomyces spp.

The antiviral activity of furan-2-yl acetate (C6H6O3) extracted from Streptomyces VITSDK1 spp. was studied in cultured Sahul Indian Grouper Eye (SIGE) cells infected with fish nodavirus (FNV). The nodavirus infection in the SIGE cells was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and the antiviral activity of furan-2-yl acetate was assessed by cytopathic effect, as well as reduction in nodaviral titre (TCID50 mL⁻¹, where TCID50) is the 50% tissue culture infective dose) in the cultured SIGE cells under in vitro conditions. Furan-2-yl acetate (20 µg mL⁻¹) effectively inhibited the replication of the FNV-infected SIGE cell lines and the viral titre was reduced from 4.3 to 2.45 log TCID50 mL⁻¹ on treatments. Furan-2-yl acetate (20 µg mL⁻¹)- treated SIGE cell survival was found to be 90%, as determined by methyl thiazol tetrazolium assay. The results of an immunofluorescent assay revealed a strong association between the viral capsid protein inhibition and a decline in viral replication. The results suggest that furan-2-yl acetate suppressed FNV replication in cultured fish cells, providing a potential approach for the control of nodaviral diseases in marine fishes.

Antiviral activity by fish antimicrobial peptides of epinecidin-1 and hepcidin 1-5 against nervous necrosis virus in medaka.

The nervous necrosis virus (NNV)-medaka infection model was used in this study for analysis of NNV infection and treatment of NNV with the antimicrobial peptides (AMPs) of epinecidin-1 and hepcidin 1-5 at the organismal level. Our results showed that co-treatment of AMPs with the virus was effective in promoting a significant increase in medaka survival. Re-challenge with the virus also showed high survival suggesting that these two AMPs enhanced fish survival. However, pretreatment or post-treatment with AMPs showed that both of these AMPs increased medaka survival and suggested that AMPs can be used as drugs to rescue infected medaka. The data presented here indicate that epinecidin-1 and hepcidin 1-5 have in vivo antivirus activity against the NNV, and hepcidin 1-5 functions like a lytic peptide after an in vitro assay. Infection after pretreatment, co-treatment, and post-treatment with epinecidin-1 or hepcidin 1-5 was verified by RT-PCR which showed both peptides can downregulate NNV and interferon gene expressions. In addition, our results suggest that epinecidin-1 or hepcidin 1-5 may prove to be an effective chemotherapeutic agent for aquaculture in the future.

Structure and function of a genetically engineered mimic of a nonenveloped virus entry intermediate.

Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membrane-disrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.

Antimicrobial peptides (AMP) with antiviral activity against fish nodavirus.

Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. Antimicrobial peptides (AMPs) play an important role of innate immunity either against bacterial pathogens or viruses. Up to date, little is known if any AMP could effectively inhibit fish nodaviruses and its mechanism. In this study, the antiviral activities of three antimicrobial peptides (AMPs) against grouper NNV (GNNV) were screened in the fish cell line. Two of the three AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able to agglutinate purified NNV particles into clump, and the clumps were further confirmed to be viral proteins by TEM and Western blot. The NNV solution, separately pre-mixed with AMP (TH 1-5 or cSALF) or deionized-distilled water for 1 h, was used to infect GF-1 cells, and the levels of capsid protein in the GNNV-AMP-infected cells at 1 h post infection were much lower than that in the GNNV-H(2)O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infected the cells. Treatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression; however, grouper epinecidin-1 (CP643-1) could induce the expression of Mx in the pre-treated cBB cells. This study revealed three AMPs with anti-NNV activity through two different mechanisms, and shed light on the future application in aquaculture.

Inactivation of nervous necrosis virus infecting grouper (Epinephelus coioides) by epinecidin-1 and hepcidin 1-5 antimicrobial peptides, and downregulation of Mx2 and Mx3 gene expressions.

Betanodaviruses are one of the serious pathogens in nervous necrosis viral (NNV) disease that brings about mortality in the larval stage of grouper (Epinephelus coioides). In this study, the efficacy of pretreatment, co-treatment, and posttreatment with the antimicrobial epinecidin-1 and hepcidin 1-5 peptides against a betanodavirus was evaluated by intraperitoneal inoculation in grouper. The results showed that co-treatment of epinecidin-1 or hepcidin 1-5 with the virus was effective in promoting a significant decrease in grouper mortality. Re-challenge with virus again after 30 day in co-treated grouper groups showed high survival suggesting that epinecidin-1 and hepcidin 1-5 enhanced fish survival. However, grouper inoculated with NNV and then inoculated with epinecidin-1 8 h later showed significantly different survival from the group inoculated with virus alone, suggesting that epinecidin-1 can be used as a drug to rescue infected grouper. Infection after pretreatment, co-treatment, and posttreatment with epinecidin-1 or hepcidin 1-5 was verified by RT-PCR which showed downregulation of Mx2 and Mx3 gene expressions. All these data strongly suggest that epinecidin-1 and hepcidin 1-5 are effective peptides for protecting grouper larvae by reducing NNV infection.