Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

Orange-spotted grouper IFNh response to NNV or MSRV and its potential antiviral activities.

Type I interferon (IFN) plays a crucial role in the antiviral immune response. Nervous necrosis virus (NNV) and Micropterus salmoides rhabdovirus (MSRV) are the most important viruses in cultured larvae and juveniles, causing great economic losses to fish farming. To better understand the antiviral activities and immunoregulatory role of IFN from orange-spotted grouper (Epinephelus coioides), EcIFNh was cloned from NNV infected sample. EcIFNh has an open reading frame (ORF) of 552 bp and encodes a polypeptide of 183 amino acids. Phylogenetic tree analysis showed that EcIFNh was clustered into the IFNh branch. The tissue distribution analysis revealed that EcIFNh was highly expressed in the liver and brain of healthy orange-spotted grouper. The mRNA levels of EcIFNh were significantly upregulated after poly (I:C) stimulation and NNV or MSRV infection. Furthermore, the promoter of EcIFNh was characterized and significantly activated by EcMDA5, EcMAVS, EcSTING, EcIRF3, and EcIRF7 in the luciferase activity assays. We found that EcIFNh overexpression resisted the replication of NNV and MSRV, while EcIFNh silencing facilitated NNV replication in GB cells. In addition, EcIFNh recombinant protein (rEcIFNh) enhanced the immune response by inducing the expression of ISGs in vivo and in vitro, suggesting the potential application of rEcIFNh for anti-NNV and anti-MSRV. Taken together, our research may offer the foundation for virus-IFN system interaction in orange-spotted grouper.

Two cytokine receptor family B (CRFB) members in orange-spotted grouper Epinephelus coioides, EcCRFB3 and EcCRFB4, negatively regulate interferon immune responses to assist nervous necrosis virus replication.

Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 990 bp encoding 329 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I-induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFNRs to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV.

An affinity peptide exerts antiviral activity by strongly binding nervous necrosis virus to block viral entry.

Nervous necrosis virus (NNV) causes viral nervous necrosis (VNN), a disease that leads to almost 100% mortality among larvae and juvenile fish, severely affecting the aquaculture industry. VNN vaccines based on inactivated viruses or virus-like particles (VLPs) are unsuitable for fish fry with immature adaptive immune systems. Here, we applied an anti-NNV strategy based on affinity peptides (AFPs). Three phage display peptide libraries were screened against RBS, the VLP of orange-spotted grouper nervous necrosis virus (OGNNV). From the positive clones, a dodecapeptide with the highest binding capacity (BC) to RBS was selected. This AFP agglutinated or disrupted virion particles, inhibiting RBS entry into sea bass (SB) cells. To enhance BC and solubility, we amended the AFP sequence as "LHWDFQSWVPLL" and named as 12C. One to three copies of 12C in tandem were prokaryotically expressed with a maltose binding protein (MBP) linked by a flexible peptide. Of the recombinant proteins expressed, MBP-triple-12C (MBP-T12C) exhibited the highest BC, efficiently blocked RBS entry, and strongly inhibited OGNNV infection at viral entry. Moreover, MBP-T12C bound the VLPs of all NNV serotypes, displaying broad-spectrum anti-NNV ability, and recognized only OGNNV and mud crab virus, demonstrating binding specificity. Therefore, these anti-NNV AFPs specifically bound NNV, aggregating or disrupting the viral particles, to reduce the contact probability between the virus and cell surface, subsequently inhibiting viral infection. Our results not only provided a candidate of anti-NNV AFP, but a framework for the development of antiviral AFP.

Orange-spotted grouper nervous necrosis virus-encoded protein A induces interferon expression via RIG-I/MDA5-MAVS-TBK1-IRF3 signaling in fish cells.

IMPORTANCE: As a major pathogen, nervous necrosis virus (NNV) infects more than 120 fish species worldwide and is virulent to larvae and juvenile fish, hampering the development of the fish fry industry. Understanding virus-host interaction and underlying mechanisms is an important but largely unknown issue in fish virus studies. Here, using channel catfish ovary and fathead minnow cells as models for the study of innate immunity signaling, we found that NNV-encoded ProA activated interferon signaling via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway which was still suppressed by the infection of wild-type NNV. This finding has important implications for the comprehension of NNV protein function and the immune response from different cells. First, RIG-I is the key node for anti-NNV innate immunity. Second, the response intensity of RLR signaling determines the degree of NNV proliferation. This study expands our knowledge regarding the overview of signal pathways affected by NNV-encoded protein and also highlights potential directions for the control of aquatic viruses.

Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.