GHSC70 is involved in the cellular entry of nervous necrosis virus.

UNLABELLED: Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein. IMPORTANCE: Fish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

Decreasing salinity of seawater moderates immune response and increases survival rate of giant groupers post betanodavirus infection.

Giant groupers (Epinephelus lanceolatus), an important aquaculture fish in Asia, are attacked by nervous necrosis virus (NNV), belonging to betanodavirus. Environmental salinity can affect fish immunity and physiology. We examined whether decreasing salinity from 30 to 15 ppt during acclimation of groupers could affect survival with NNV infection and the associated factors. Although NNV infection decreased muscle moisture, up-regulated the gene expression of Na(+)-K(+)-2Cl(-) cotransporter isoform 2, and elevated plasma cortisol level in groupers, these factors were not related to the higher mortality of groupers reared at 30-ppt salinity (S30-groupers), compared to 15-ppt reared groupers (S15-groupers). Infected S30-groupers exhibited high leukocyte count and innate immune gene expression level. Moreover, NNV-infected dead S30-groupers showed high IL-1ß gene expression level but low NNV load in the brain. The high or excess IL-1ß gene expression levels in the brain of NNV-infected S30-groupers may be the factor in high mortality.

Grouper voltage-dependent anion selective channel protein 2 is required for nervous necrosis virus infection.

Nervous necrosis virus (NNV) is a non-enveloped virus with 2 segmented positive-sense single-stranded RNAs. NNV-induced mass mortality has occurred worldwide in many species of cultured marine fish and resulted in substantial economic losses. In our previous study, we cloned the gene of voltage-dependent anion selective channel protein 2 (GVDAC2), and the NNV RNA2 expression level decreased in GVDAC2-knockdown GF-1 cells 24 h after infection. Here, we investigated the role of GVDAC2 in the NNV infection in GF-1 cells. NNV infection did not considerably affect GVDAC2 gene expression. After performing immunostaining, we detected GVDAC2 at the mitochondria and GVDAC2 was colocalized with NNV-RNA-dependent RNA polymerase. However, these 2 proteins did not interact with each other in immunoprecipitation assay. The cellular ATP level in GVDAC2-downregulated cells was lower than that in control cells, and NNV-induced apoptosis was delayed in GVDAC2-siRNA-transfected cells. Therefore, we suggest that GVDAC2 is required for NNV infection for maintaining the cellular ATP level and has positive impact on virus-induced apoptosis.

Dietary supplementation of Pediococcus pentosaceus enhances innate immunity, physiological health and resistance to Vibrio anguillarum in orange-spotted grouper (Epinephelus coioides).

Groupers (Epinephelus spp.) are economically important fish species in Southeast Asian aquaculture. Vibriosis caused by Vibro spp. is one of the severe bacterial diseases that devastate the grouper aquaculture industry. Probiotics have been reported to show the potential to enhance fish immunity and to antagonize pathogens. In our previous study, a lactic acid bacterium Pediococcus pentosaceus strain 4012 (LAB4012), isolated from cobia intestine, protects cobia from photobacteriosis after a 2-week feeding. In this study, we examined the potential of LAB4012 to be a probiotic for the orange-spotted grouper through feeding, thus to guard against vibriosis. In vitro, LAB4012 culture supernatant with low pH suppressed the growth of Vibrio anguillarum, and lactic acid in the metabolite of LAB4012 appeared to be the major factor to the growth inhibition of V. anguillarum. In vivo, the challenge test showed that the cumulative mortality of the LAB4012-fed groupers was significantly lower than that of the control fish after V. anguillarum infection. Supplementation of LAB4012 in commercial diet not only enhanced the growth rate and erythrocyte numbers of the groupers, but also regulated the gene expression of the pro-/anti-inflammatory cytokines. One day post-infection of V. anguillarum, the leukocyte numbers in the peripheral blood and the phagocytic activity of the head-kidney phagocytes in the LAB4012-fed groupers were found significantly increased, when compared with those without LAB4012-feeding. These results suggested that LAB4012 can be a dietary probiotic for groupers in modulating the immunity and protecting the groupers from V. anguillarum infection.

Immune gene expressions in grouper larvae (Epinephelus coioides) induced by bath and oral vaccinations with inactivated betanodavirus.

Nervous necrosis virus (NNV) has caused mass mortality in many mariculture fish species. Bath vaccination of inactivated NNV and oral immunization of recombinant NNV coat protein are reported to protect grouper larvae against NNV infection. However, the information of immune gene expression in grouper larvae (Epinephelus coioides) after bath and oral immunizations is still limited. In this study, grouper larvae were respectively bath- and orally immunized with binary ethylenimine (BEI)-inactivated NNV, and the expression levels of immune genes were analyzed. Significant gene expressions of IL-1ß, Mx, MHC-I, MHC-II, CD8α, IgM and IgT were observed in bath- and orally immunized fish 1-4 weeks post immunization (wpi). Particularly, the up-regulation of IL-1ß and Mx gene expression lasted for 4 weeks. The IgT gene expression in gill was only induced by bath immunization, while that in gut was only stimulated by oral immunization. Both immunizations elicited MHC-I and CD8α gene expression relative to cellular immunity. Furthermore, NNV RNA genome, which was detected in inactivated NNV, could induce Mx gene expression in grouper brain (GB) cells, indicating that NNV RNA genome could be recognized by pathogen-recognition receptors (PRRs). In summary, bath and oral vaccinations with BEI-inactivated NNV triggered the gene expression of not only humoral immunity but also cellular immunity.

Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis.

Cobia (Rachycentron canadum) is an economically important fish species for aquaculture in tropical and sub-tropical areas. Cobia aquaculture industry has severely damaged due to photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp), especially in Taiwan. Antibiotics and vaccines have been applied to control Pdp infection, but the efficacy has been inconsistent. One species of lactic acid bacteria, Pediococcus pentosaceus strain 4012 (LAB 4012), was isolated from the intestine of adult cobia, and its culture supernatant can effectively inhibit Pdp growth in vitro. The acidic pH derived from metabolic acids in LAB culture supernatant was demonstrated to be an important factor for the suppression. After a 2-week feeding of LAB 4012, the growth rate of the fed cobia was 12% higher than that of the non-fed group, and the relative percentage of survival (RPS) of the fed cobia was found to be 74.4 in Pdp immersion challenge. In addition, the respiratory burst (RB) of peripheral blood leukocytes (PBL) in the LAB 4012-fed group was significantly higher than that of the non-fed group. Although feeding LAB 4012 did not improve specific antibody response in cobia after immunization with Pdp vaccine, it still significantly raised the survival rate by 22% over that of the non-fed group after Pdp immersion challenge. Judging by the quick induction of high protection against Pdp infection and promotion of growth in larvae, LAB 4012 was considered to be a viable probiotic for cobia aquaculture.

Cytotoxic CD8α+ leucocytes have heterogeneous features in antigen recognition and class I MHC restriction in grouper.

CD8 is a membrane glycoprotein found primarily on the surface of T lymphocytes such as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and γδ T lymphocytes. It helps T lymphocytes to kill the infected cells that presents microbial antigen on the cell surface. However, analysis of fish cellular immunity has been limited because of the lack of CD8 antibodies in grouper. In this present study, we cloned full-length CD8α cDNAs from orange-spotted grouper (Epinephelus coioides), an important fish species economically. The deduced protein of CD8α contained 227 amino acid residues in length and included one signal peptide, Ig superfamily V domain, hinge region, transmembrane domain, cytoplasmic tail and conserved binding motif associated with tyrosine kinase p56(lck). The molecular weight of the mature protein was estimated at 22.5 kDa and pI at 9.55. Phylogenetically, the predicted grouper CD8α protein was similar to CD8α from other marine fish species in which the identity was 50-60%. Real-time PCR revealed that CD8α transcript was constitutively expressed in thymus, head kidney, gill, spleen, gut and peripheral blood leucocyte (PBL); and the highest expression in thymus. CD8α transcript in the spleen of fish injected with nervous necrosis virus (NNV) was significantly up-regulated at 4 days post-injection compared to the untreated fish. Rabbit antiserum prepared against recombinant CD8α protein was able to recognize specifically the subset lymphocytes which have a diameter of 7 µm, a high nucleus/cytoplasm ratio and a ring-shaped cytoplasm. The cytotoxicity of CD8α(+) lymphocytes at one-week post-NNV infection was enhanced significantly against NNV-infected autologous fin cells in comparison with NNV-infected allogeneic or RSIV-infected autologous fin cells. Flow cytometry analysis revealed that both the number and mean fluorescence intensity (MFI) of CD8α(+) PBL were significantly increased at 7 days post-NNV infection. The specific cytotoxicity and MHC class I restriction of the lymphocytes sorted by rCD8α antibody are properties that can be attributed to CTL. In addition, low level of cytotoxicity was found in PBL against allogeneic targets as well as CD8α(+) effectors killed autologous targets nonspecifically, implicated presence of cytotoxic T subsets, possibly nonspecific cytotoxic cells (NCC) and γδ T lymphocytes, without MHC class I restriction. In conclusion, grouper cytotoxic CD8α(+) PBL have heterogeneous features in specific antigen recognition and class I MHC restriction.

Anti-viral mechanism of barramundi Mx against betanodavirus involves the inhibition of viral RNA synthesis through the interference of RdRp.

Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation.

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.

Humoral and cytokine responses in giant groupers after vaccination and challenge with betanodavirus.

Giant groupers were immunized with two dosages (Vhigh and Vlow) of inactivated nervous necrosis virus (NNV) and subsequently challenged with NNV at 4 weeks post vaccination (wpv). Several indicators were used to analyze the protective effects of the NNV vaccine. The neutralizing antibody titer of fish serum mostly corresponded to the survival rate of immunized fish in the NNV challenge test. Extravascular IgM+ cells were detected in the brains of both NNV-infected and noninfected groupers. After NNV infection, CD8α and IgM gene expression increased in the brains, indicating CD8α+ and IgM+ lymphocyte infiltration. Moreover, the NNV load was not the highest in dead grouper brains, suggesting that this load in the brain was not the key factor for the death of groupers. However, the brains of dead fish showed the highest expression of the interleukin (IL)-1ß gene, a neurotoxic factor in the brain. Therefore, IL-1ß overexpression is likely to be associated with the death of NNV-infected groupers.

Interleukin-1ß secreted from betanodavirus-infected microglia caused the death of neurons in giant grouper brains.

High interleukin (IL)-1ß gene expression was observed in dead giant grouper brains after nervous necrosis virus (NNV) infection. To investigate the neuronal death caused by NNV infection, primary tissue culture of giant grouper brains (pGB) was performed. In NNV-infected pGB cells, the viral capsid protein was detected in both neurons and microglia; furthermore, microglial proliferation and neuronal death were observed. The culture supernatant (CS) of NNV-infected pGB cells contained IL-1ß and tumor necrosis factor-α, which were mainly released from the microglia. A new batch of pGB cells was treated with CS, resulting in neuronal death, which could be prevented by blocking the IL-1ß in the CS by using anti-IL-1ß polyclonal antibodies. Moreover, pGB cells treated with recombinant IL-1ß showed microglial proliferation and neuronal death. Thus, NNV infection may activate microglial proliferation and stimulate microglial secretion of IL-1ß, which is a critical cytokine responsible for neuronal death in NNV-infected grouper brains.

Persistently betanodavirus-infected barramundi (Lates calcarifer) exhibit resistances to red sea bream iridovirus infection.

Nervous necrosis virus (NNV) and red sea bream iridovirus (RSIV) are two important pathogens that have caused acute, highly contagious, and widespread diseases among wild and cultured fish, especially at larval and juvenile stages. We discovered that the pathogenicity of NNV to the 80 days post-hatch (dph) barramundi is lower than that to the 14 dph barramundi. Following NNV challenge, no mortality occurred in the 80 dph barramundi, but NNV RNA2 and barramundi Mx (BMx) gene expression was detected in the brain and liver. The 80 dph barramundi pre-challenged with NNV became more resistant to the following RSIV challenge (mortality: 62%) compared to the NNV-free barramundi challenged with RSIV (mortality: 100%). A similar phenomenon was revealed in the cell culture system that RSIV proliferated less progeny in the barramundi brain (BB) cell line which exhibit persistent NNV infection than in NNV-free cured BB (cBB) cell line. The potential factors involved in the resistance of the persistently NNV-infected barramundi and BB cells to the secondary RSIV infection were examined in this study. We prove that barramundi anti-NNV polyclonal antibodies do not cross-neutralize RSIV, and NNV infection does not interfere with RSIV replication. However, the interferon (IFN) response and BMx gene expression in cBB cells suppresses the RSIV proliferation. Our study suggests that the NNV-induced IFN response and BMx expression are responsible for the resistance of barramundi to RSIV infection.

Binding kinetics of grouper nervous necrosis viruses with functionalized antimicrobial peptides by nanomechanical detection.

We report the binding kinetics of fish-infected grouper nervous necrosis viruses (NNV) and selected antimicrobial peptides (AMPs) by nanomechanical detection. AMPs, the vital member in an innate immunity, are promising candidates in the fight against pathogens due to their broad range of antimicrobial activity and low toxicity. Grouper NNV primarily cause mass mortality of many marine cultured fish species, and two selected AMPs in this study were found to inhibit viruses by agglutinating its virions to form aggregates. The binding activity of NNVs with functionalized AMPs onto a sensing microcantilever yielded induced surface stresses, indicating high binding strength of molecular interaction. The binding affinity and kinetic rate constants of molecular recognition events calculated for NNV-AMP(TH1-5) compared to NNV-AMP(cSALF) were found to be 2.1-fold and 4.43-fold, respectively, indicating TH1-5 effectively bind with NNV more than cSALF. Moreover, a microscopic X-ray photoelectron spectroscopy technique was employed for further validation of pre- and post-NNV binding onto peptides-functionalized sensing surface. An increase in the spectrum and intensity of the P 2p and N 1s elements for the post-NNV binding was clearly shown to ensure the existence of phosphate groups and nitrogen-containing ring structures of specific NNV-TH1-5 interaction. Therefore, the microcantilever biosensing technique provides a potential and useful screening of AMPs for affinity to NNVs.

Vaccination of grouper broodfish (Epinephelus tukula) reduces the risk of vertical transmission by nervous necrosis virus.

Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured fish at larval stage. Strong evidence of vertical transmission of NNV has been reported in the carrier broodstock of striped jack and sea bass. An effective immunization program was developed and monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at 10(9)TCID(50)kg(-1), and the enhanced 2-fold neutralization activity could sustain up to 17 months post-vaccination (mpv). An immunization program was applied in the broodstocks of grouper (Epinephelus tukula) with body weight of 35-60kg. The levels of NNV-specific antibodies detected, from 1 to 5 mpi, in the homogenates of the eggs from the vaccinated broodfish were elevated than that from non-vaccinated fish. By nested RT-PCR, NNV was detectable in the eggs from the non-vaccinated fish at Month 5, but not in the eggs from vaccinated fish. It is therefore suggested that vaccination will be a potentially practical measure to reduce the risk of vertical transmission of NNV of grouper broodfish under stress of repeated spawning.

Efficacies of inactivated vaccines against betanodavirus in grouper larvae (Epinephelus coioides) by bath immunization.

Betanodavirus is the pathogen of viral nervous necrosis (VNN) disease that has caused mass mortality among many species of marine fish at larval stage. In this study, the efficacy of inactivated betanodavirus was evaluated by bath-immunization and bath-challenge of orange-spotted grouper (Epinephelus coioides) at early larval stage. Two kinds of chemicals were used for inactivation of the virus, and the relative percent survival (RPS) values of 0.4mM binary ethylenimine (BEI)-inactivated vaccine was revealed to be 79-95, higher than that of 0.1-0.2% formalin-inactivated vaccines (39-43). Three lengths of bath immunization time were tested, and 20 min immersion of BEI-inactivated betanodavirus at a concentration of 10(6)TICD(50)/ml was sufficient to induce high protection (RPS > 75). Protection of the BEI-inactivated vaccine was evaluated at different time post immunization, and the peak of protection was observed 30 days post vaccination, and retained for at least 3 months. The efficacies of formalin-inactivated vaccines with or without encapsulation were compared, and the result revealed that the efficacy of formalin-inactivated vaccine could be significantly improved by nano-encapsulation (RPS = 85). All these data strongly suggested that bath immunization with nano-encapsulated formalin-inactivated or BEI-inactivated betanodavirus vaccines is an effective strategy to protect grouper larvae against VNN.