Peroxiredoxins are a family of antioxidant proteins that protect cells from oxidative damage caused by reactive oxygen species (ROS). Herein, the peroxiredoxin 3 gene from grass carp (Ctenopharyngodon idellus), named CiPrx3, was cloned and analyzed. The full-length cDNA of CiPrx3 is 1068 bp long, with a 753 bp open reading frame (ORF) that contains a thioredoxin-2 domain, two peroxiredoxin signature motifs, and two highly conserved cysteine residues. CiPrx3 was ubiquitously expressed in all the tested tissues, while its expression level was altered significantly after exposure to grass carp reovirus (GCRV) and pathogen-associated molecular patterns (PAMPs). CiPrx3 was localized in the mitochondria of transfected cells and concentrated in the nucleus after poly (I:C) treatment. Transformation of CiPrx3 into Escherichia coli enhanced host resistance to H₂O₂ and heavy metals. Purified recombinant CiPrx3 proteins could protect DNA against oxidative damage. Overexpression of CiPrx3 in fish cells reduced intracellular ROS, increased cell viability, and decreased cell apoptosis caused by H₂O₂ stimulation and GCRV infection. Further study indicated that CiPrx3 induced autophagy to inhibit GCRV replication in fish cells. Collectively, these results imply that grass carp Prx3 elevates host antioxidant activity and induces autophagy to inhibit GCRV replication.

Grass carp reovirus (GCRV) is the most virulent aquareovirus, inducing hemorrhagic disease that results in significant economic losses for the grass carp aquaculture industry in China. It has been reported that the cytokine storm, resulting from the viral-induced acute immune-inflammatory response, is the primary cause of death associated with viral infections. Therefore, exploring the negative regulatory factors of the immune response may provide important targets for virus prevention and control. In this study, we demonstrated that GCRV infection induces an acute immune-inflammatory response in yearling grass carp, while a milder immune response is observed in 3-year-old grass carp by assessing key gene expression at both the mRNA and protein levels. Furthermore, we found that the upregulation of autophagy core gene, autophagy-related gene 5 (ATG5), inhibits GCRV replication and reduces the excessive immune-inflammatory response induced by the virus. Notably, co-immunoprecipitation and bimolecular fluorescence complementation reveal that ATG5 interacts with RIG-I and MDA5, inhibiting the downstream immune response. Mechanistically, ATG5 promotes autophagy and K48-linked polyubiquitination to degrade RIG-I and MDA5. Importantly, ATG5 reduces IRF7 phosphorylation through the degradation of RIG-I and MDA5, which, in turn, leads to a decreased immune response during GCRV infection. Collectively, our findings suggest that ATG5 serves as an important negative regulatory factor of the viral-induced acute immune-inflammatory response, providing a promising target for the prevention and control of GCRV. IMPORTANCE: Grass carp reovirus (GCRV) is a highly virulent pathogen responsible for hemorrhagic disease in grass carp, leading to devastating losses in aquaculture. The study reveals that GCRV triggers an acute immune-inflammatory response in young grass carp, which is linked to high mortality, while a milder immune response occurs in older, resistant fish. Importantly, the autophagy-related gene ATG5 was identified as a critical regulator that suppresses excessive immune-inflammatory responses by promoting the degradation of RIG-I and MDA5, two key viral sensors. ATG5 not only reduces viral replication but also limits IRF7 phosphorylation, a key step in interferon signaling. This research uncovers a novel mechanism by which autophagy moderates viral-induced immune response, suggesting that ATG5 could be a valuable target for therapeutic interventions aimed at controlling GCRV infection and preventing cytokine storms in aquaculture. These findings could have broader implications for managing viral infections in fish farming.

As a member of the pattern recognition receptor (PRR) class, scavenger receptor class B type 1 (SRB1) plays a key role in innate immunity. Grass carp (Ctenopharyngodon idellus) ranks among the most extensively cultivated freshwater aquaculture species in China. However, little is known about the function of SRB1 in C. idellus. In this research study, a SRB1 gene was identified in C. idellus, named CiSRB1. The full-length cDNA of CiSRB1 is 2486 bp long, with an open reading frame (ORF) of 2486 bp encoding a 497 amino acid (aa) protein containing a conserved CD36 domain. The identified genomic DNA length of CiSRB1 is 20,042 bp, including 12 exons and 11 introns. The predictive analysis of protein interactions revealed that CiSRB1 could interact with the outer capsid proteins of typical GCRV strains. The tissue distribution of CiSRB1 exhibited age-dependent characteristics. CiSRB1 displayed the highest expression in the intestines and moderate levels in muscle, spleen, liver, and brain of one-year-old grass carp while maintaining relatively low levels in three-year-old grass carp. Following grass carp reovirus (GCRV) infection, notable upregulation of CiSRB1 transcripts was observed in major immune tissues (gills, intestines, spleen, and liver). Furthermore, significant differences were found between one-year-old and three-year-old grass carp, with lower CiSRB1 expression levels being detected in the older group. Additionally, a distinct response to GCRV infection was observed in one-year-old and three-year-old grass carp. It was found that one-year-old individuals had a mortality rate of up to 84% 6 days post-infection (dpi), whereas all three-year-old counterparts survived after GCRV infection. The analysis of GCRV copy numbers across tissues revealed substantially higher levels in one-year-old grass carp compared with their older counterparts, confirming the existence of age-dependent susceptibility to GCRV infection in grass carp. Combined with these results, it was speculated that the decline in cell-surface CiSRB1 expression with age may impede reovirus binding to host cells, potentially explaining why older grass carp demonstrated enhanced resistance to GCRV infection. This observation accentuates the importance of CiSRB1 in the context of GCRV infection and provides insights into age-dependent susceptibility to reovirus.

Spring viremia of carp virus (SVCV) severely threatens cyprinid aquaculture, yet the role of autophagy in SVCV infection remains unclear. Here, SVCV infection was associated with enhanced autophagic activity and autophagic flux in common carp and EPC cells. Pharmacological perturbation of autophagy revealed that rapamycin treatment increased SVCV gene expression and aggravated SVCV-induced cytopathic effects, whereas chloroquine blocked autophagic flux, suppressed viral gene expression, and mitigated virus-induced cellular damage. Transcriptomic analysis identified 1,868 differentially expressed genes after chloroquine treatment, with major changes in lysosome/autophagy-related pathways, glycan degradation, ion transport, cell cycle, DNA replication, and DNA repair. Metabolomic and integrated multi-omics analyses revealed changes in amino sugar and nucleotide sugar metabolism, steroid biosynthesis, riboflavin metabolism, and amino acid metabolism. Functional validation showed that diflorasone and all-trans-retinal reduced SVCV-G expression, alleviated cytopathic effects, and improved survival after SVCV infection. These findings suggest that SVCV infection is associated with enhanced autophagic flux, whereas chloroquine treatment blocks autophagic flux and is accompanied by reduced SVCV gene expression.