Yu-Chi Wu

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.

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.

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-1β, 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-1β 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.

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.

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

ABSTRACT 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, CD8&agr; and IgM gene expression increased in the brains, indicating CD8&agr;+ 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)‐1&bgr; gene, a neurotoxic factor in the brain. Therefore, IL‐1&bgr; overexpression is likely to be associated with the death of NNV‐infected groupers. HighlightsNeutralizing antibody titer corresponded to survival rate of immunized fish.Extravascular IgM+ cells were detected in the brains of groupers.The NNV load was not the highest in dead grouper brains among all challenged fish.Dead fish brains showed the highest gene expression of IL‐1&bgr;, a neurotoxic factor.

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

ABSTRACT High interleukin (IL)‐1&bgr; 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‐1&bgr; and tumor necrosis factor‐&agr;, 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‐1&bgr; in the CS by using anti‐IL‐1&bgr; polyclonal antibodies. Moreover, pGB cells treated with recombinant IL‐1&bgr; showed microglial proliferation and neuronal death. Thus, NNV infection may activate microglial proliferation and stimulate microglial secretion of IL‐1&bgr;, which is a critical cytokine responsible for neuronal death in NNV‐infected grouper brains. HIGHLIGHTSNeurons and microglia in pGB cells were susceptible to NNV infection.NNV infection led to microglial proliferation and neuronal death in pGB cells.NNV infection induced microglial secretion of IL‐1&bgr; and TNF‐&agr; in pGB cells.The IL‐1&bgr; secreted from microglia caused neuronal death in pGB cells.Recombinant IL‐1&bgr; induced microglial proliferation and neuronal death in pGB cells.