Meisheng Yi

Frequent outbreaks of viral diseases and the increasing prevalence of polyviral co-infections have severely constrained the sustainable development of largemouth bass (Micropterus salmoides) aquaculture. Identifying conserved host factors exploited by diverse viruses is critical for developing broad-spectrum antiviral strategies, yet the key host molecules underlying cross-viral infection susceptibility in teleosts remain poorly characterized. Here, this research identifies Micropterus salmoides 14-3-3ε protein (Ms14-3-3ε) as a critical pro-viral host factor that facilitates the replication of multiple phylogenetically distinct viruses by negatively modulating NF-κB-mediated inflammatory responses. Ms14-3-3ε was ubiquitously expressed in healthy largemouth bass tissues and was significantly upregulated both in vivo and in vitro upon infection with viral hemorrhagic septicemia virus (VHSV), largemouth bass virus (LMBV), and red-spotted grouper nervous necrosis virus (RGNNV). Functional gain- and loss-of-function assays demonstrated that Ms14-3-3ε serves as a broad-spectrum pro-viral factor: its overexpression markedly enhanced the transcription and protein expression of core viral genes, whereas Ms14-3-3ε knockdown effectively suppressed the transcription expression of all three viruses and protein levels of VHSV G and RGNNV CP. Mechanistically, Ms14-3-3ε negatively regulates the NF-κB signaling pathway in a dose-dependent manner by specifically inhibiting the phosphorylation of p65 at the Ser536 residue. This suppression further downregulates the expression of downstream pro-inflammatory effector genes NLRP3 and IL-1β, thereby blunting host innate immune defense responses to multi-viral proliferation. Collectively, the results demonstrate that Ms14-3-3ε facilitates the replication of taxonomically distinct fish RNA and DNA viruses associated with reduced NF-κB pathway activity in largemouth bass. This study provides a novel, conserved host target for the development of broad-spectrum antiviral strategies in aquaculture.

The CD40–CD154 receptor-ligand axis is a core costimulatory regulator of antiviral immunity in mammals, but its functional role in teleosts remains largely unknown. Here, we identified the CD40 and CD154 homologs (MsCD40 and MsCD154) from largemouth bass (Micropterus salmoides), a globally farmed perciform teleost. Bioinformatic analysis confirmed that MsCD40 and MsCD154 harbor the conserved domain architectures of tumor necrosis factor receptor superfamily and TNF superfamily, respectively, with a teleost-specific phylogenetic clustering pattern. Both genes were ubiquitously expressed in immune-relevant tissues, and their transcription was dynamically regulated in response to viral hemorrhagic septicemia virus (VHSV) and largemouth bass virus (LMBV) challenge in vivo. Co-immunoprecipitation and immunofluorescence co-localization assays verified that MsCD40 and MsCD154 physically interact at the plasma membrane, forming a functional receptor-ligand complex. Functional assays showed that overexpression of either MsCD40 or MsCD154 significantly suppressed VHSV and LMBV infection in vitro. Furthermore, MsCD40 and MsCD154 overexpression dose-dependently activated nuclear factor-κB (NF-κB) reporter activity, and markedly upregulated the transcription of NF-κB downstream effector genes, including IL-8, NLRP3 and P105, under both VHSV and LMBV infection. Collectively, our findings demonstrate that the teleost CD40–CD154 costimulatory axis restricts both RNA and DNA viral infection in largemouth bass through NF-κB-mediated immune activation, which provides promising molecular targets for the development of broad-spectrum antiviral strategies in largemouth bass aquaculture.

We report the coding-complete genome sequence of a largemouth bass virus isolate obtained from diseased largemouth bass ( Micropterus salmoides ) in China. The genome is 99,378 bp in length with a GC content of 52.1% and contains 102 predicted coding DNA sequences.

Protein kinase C alpha (PKCα) is a central signaling molecule implicated in various cellular processes, including viral infections. However, its role in fish viruses, particularly nervous necrosis virus (NNV), remains elusive. Here, we report that PKCα from marine medaka (MmPKCα) facilitates red-spotted grouper NNV (RGNNV) entry by bridging viral receptor marine medaka myosin light chain 3 (MmMYL3, a known RGNNV receptor mediating macropinocytosis) binding to downstream actin dynamics. RGNNV infection upregulates MmPKCα expression and activates its phosphorylation. Gain- and loss-of-function studies demonstrated that MmPKCα enhances NNV entry, dependent on its kinase activity. We found that MmPKCα interacts with the RGNNV capsid protein via its C-terminal (CT) domain. Although MmPKCα localized to the cell surface, it did not function as an RGNNV receptor, evidenced by unaltered viral binding, inability to render non-susceptible cells permissive, and lack of inhibition by anti-PKCα antibodies/recombinant MmPKCα protein. Instead, MmPKCα directly interacts with MmMYL3 and is essential for MmMYL3-mediated macropinocytosis. Furthermore, MmPKCα binds to marine medaka cofilin (MmCFL1/2) through its CT domain and suppresses cofilin phosphorylation at Ser3, thereby activating cofilin and promoting actin rearrangement critical for macropinocytosis. Our study uncovers a novel MmMYL3-MmPKCα-cofilin signaling axis that RGNNV exploits to enter host cells, highlighting PKCα as a potential therapeutic target for controlling NNV infection. IMPORTANCE: Nervous necrosis virus (NNV) causes devastating losses in global aquaculture. Here, we identify a novel MmMYL3-MmPKCα-cofilin signaling axis that NNV exploits to enter host cells via macropinocytosis. This finding advances our understanding of fish virus invasion mechanisms, uncovering how NNV hijacks host signaling for entry. Beyond basic virology insights, our work highlights protein kinase C alpha (PKCα) as a promising therapeutic target, laying the groundwork for developing targeted antivirals to control NNV.

Rhabdoviruses exhibit a broad host range, yet the cellular receptors underlying their cross-species tropism remain poorly defined. Here, we identified tetraspanin CD9 as a conserved functional entry receptor for diverse rhabdoviruses across genera, including viral hemorrhagic septicemia virus (VHSV) ( Novirhabdovirus ), Siniperca chuatsi rhabdovirus (SCRV) ( Siniperhavirus ), and vesicular stomatitis virus (VSV) ( Vesiculovirus ). We demonstrated that the domain IV of VHSV glycoprotein G directly interacted with the large extracellular loop domain of Lateolabrax japonicus CD9 (LjCD9). CD9 knockout, CD9 protein, or CD9 antibody significantly reduced VHSV infection in vitro and CD9 knockout zebrafish, while HEK293T cells, which are nonsusceptible but permissive to VHSV, become susceptible when expressing LjCD9, suggesting that LjCD9 is an entry receptor for VHSV. We further confirmed that LjCD9 functions as a functional receptor of SCRV. Importantly, the human CD9 orthologue can serve as a receptor of VSV. Our findings also revealed that LjCD9 mediated VHSV entry via clathrin- and caveolae-mediated endocytosis. Notably, nitazoxanide (NTZ) was identified as a broad-spectrum inhibitor of VHSV, SCRV, and VSV likely by interfering with the G protein–CD9 interaction. This study establishes CD9 as a cross-species receptor for rhabdoviruses and highlights NTZ as a promising broad-spectrum antiviral agent.

Nervous necrosis virus (NNV) is a lethal aquatic pathogen that activates NF-κB signaling to manipulate host immune responses, yet the underlying molecular mechanisms remain poorly defined. Here, we identify the NNV capsid protein (CP) as a novel viral SUMO E3 ligase that promotes SUMOylation of Lateolabrax japonicus mitochondrial antiviral signaling protein (MAVS) to drive NF-κB activation. We show that CP induces p65 nuclear translocation and upregulates proinflammatory cytokine expression in a MAVS-dependent manner. Mechanistically, CP interacts with SUMO2 and the E2 conjugating enzyme L. japonicus UBC9 to promote MAVS SUMOylation at lysine 325 (K325), which stabilizes MAVS, facilitates its aggregation, and amplifies downstream NF-κB signaling. Notably, disruption of this modification via the K325R mutation or SUMOylation inhibition abrogates MAVS aggregation and inflammatory responses. Our findings reveal a previously unrecognized strategy by which NNV hijacks the host SUMOylation machinery to fine-tune MAVS function, promoting immune evasion and viral persistence.

Viral hemorrhagic septicemia virus (VHSV) is a major pathogen affecting freshwater and marine fish species, posing a significant threat to global aquaculture. Reverse genetics systems are essential for studying viral replication, and host interactions, as well as developing vaccines and therapeutics. In this study, we developed a reverse genetics platform for VHSVLB2018 strain, a genetically distinct VHSV genotype IVa strain which exhibits low genomic identity with other Asian isolates, using a dual RNA polymerase I/II transcription vector. We successfully rescued recombinant VHSV in mammalian (B7GG) and fish (FHM and EPC) cell lines, and engineered recombinant VHSV strains expressing EGFP (rVHSV-EGFP) and cherry (rVHSV-Cherry) fluorescent proteins. Phenotypic analysis revealed that unmodified recombinant VHSV (rVHSV) exhibited growth kinetics and virulence similar to the wild-type VHSV, while fluorescent protein-expressing variants showed attenuated replication and virulence, with the rVHSV-EGFP strain displaying the greatest attenuation. Utilizing the rVHSV-EGFP strain, we conducted antiviral compound screening and identified three promising inhibitors-xanthohumol, octyl gallate, and rottlerin that effectively inhibit VHSV replication. Time-of-addition assays further revealed that xanthohumol and rottlerin targeted the viral replication stage, while octyl gallate interfered with viral internalization. This reverse genetics system provides a versatile platform for studying VHSV pathogenesis, developing live-attenuated vaccines, and screening antiviral compounds, enhancing our understanding of this pathogen and offering new tools for aquaculture disease management.