Viperin as a putative antiviral effector in rainbow trout (Oncorhynchus mykiss) cells identified by CRISPR-Cas9 functional analysis following exposure to inactivated viral hemorrhagic septicemia virus
It is demonstrated that viperin plays a central role in coordinating antiviral responses in rainbow trout macrophages and is essential for proper activation of classical IFN–ISG pathways.
Abstract
Viperin is recognized for its potent broad-spectrum antiviral activity; however, its role in the antiviral immune response of rainbow trout (
Oncorhynchus mykiss
) remains poorly characterized. In this study, the contribution of viperin to the innate immune response against viral haemorrhagic septicaemia virus (VHSV) was investigated using CRISPR–Cas9 gene editing of the RTS11 cell line, a monocyte/macrophage-like cell line derived from rainbow trout spleen. Cells were edited via electroporation using ribonucleoprotein (RNP) complexes targeting viperin, achieving up to 50% editing efficiency. Edited cells and corresponding controls were subsequently stimulated with heat-inactivated VHSV, and RNA sequencing was performed to assess transcriptomic changes associated with partial viperin silencing. Transcriptomic analysis revealed disruption of canonical antiviral signalling pathways, particularly those associated with the MAVS–TBK1 axis, even in the absence of viral replication. While a robust inflammatory response mediated by TRAFs and NF-κB was maintained, genes involved in apoptosis, autophagy, and phagocytosis were predominantly downregulated, suggesting a shift toward cellular survival and adaptive responses. Alternative interferon-stimulated genes were also induced, including TRIM45, indicating activation of compensatory antiviral signalling pathways. Additionally, significant upregulation of S-adenosylmethionine synthetase (MAT) and cytidine deaminase (CDA) was observed, suggesting a compensatory metabolic response that may regulate nucleotide pools available to viral machinery while supporting viperin transcription and epigenetic antiviral regulation. Collectively, these findings demonstrate that viperin plays a central role in coordinating antiviral responses in rainbow trout macrophages and is essential for proper activation of classical IFN–ISG pathways. Even partial silencing of viperin, in the presence of a non-replicative viral stimulus, shifts macrophage responses toward compensatory inflammatory and metabolic mechanisms, indicating that loss of viperin may compromise direct antiviral defence.
Mosquitoes transmit arboviruses that represent major global public health challenges. Increasing insecticide resistance and absence of effective antiviral therapies underscore the need for novel vector control strategies. Insect-specific viruses have emerged as candidates for biological control, however, the cellular mechanisms underlying their interactions with mosquito hosts remain poorly understood. Here, we examined the immune response of Aedes albopictus U4.4 cells to Kamiti River virus (KRV) infection, an insect-specific flavivirus. Cells were infected with KRV, and transcriptomic and small RNA profiles were analyzed at 24, 48 and 72 h post-infection. KRV infection induced production of virus-derived small interfering RNAs (vsiRNAs) and virus-derived PIWI-interacting RNA (vpiRNAs) from 24 to 72 h. The vsiRNAs predominantly mapped to the 3′ untranslated region of the KRV genome, whereas vpiRNAs formed distinct hotspots in regions encoding the NS1, NS3, NS4A/B and NS5 proteins. Transcriptomic analysis revealed upregulation of genes associated with the humoral immune response, including defensin, cecropin, and glutathione S-transferase, and downregulation of Toll-like receptors and ecdysone-induced transcripts at later stages of infection. These gene expression patterns suggest an early activation followed by suppression of key immune signaling pathways. Collectively, the findings indicate that KRV leads to coordinated modulation of antiviral RNAi and host transcriptional responses, consistent with a balanced, commensal-like interaction in mosquito cells.
Nervous necrosis virus (NNV) is a major viral pathogen that causes viral nervous necrosis in a wide range of fish species and poses a serious threat to global aquaculture. However, the antiviral responses of mandarin fish (Siniperca chuatsi) to NNV infection remain poorly understood. In this study, we investigated host antiviral responses using Chinese perch brain (CPB) cell line and in vivo infection models. Transcriptome sequencing of red-spotted grouper nervous necrosis virus (RGNNV)-infected CPB cells revealed extensive transcriptional reprogramming, with numerous interferon-stimulated genes (ISGs) significantly upregulated. Comparative transcriptomic analysis across multiple teleost species identified a conserved set of ISGs induced during RGNNV infection, including CMPK2, RSAD2, DHX58, IRF3, and EPSTI1. Among these genes, CMPK2 showed strong induction in both infected CPB cells and mandarin fish brain tissues. Functional assays further demonstrated that CMPK2 overexpression significantly inhibited RGNNV replication in CPB cells and was accompanied by increased expression of several interferon-related genes. These findings provide the first functional evidence that CMPK2 restricts RGNNV infection in S. chuatsi. Our study highlights a conserved interferon-responsive antiviral module in teleost fish and identifies CMPK2 as a potential antiviral effector involved in host defense against RGNNV infection.
Chanxia Qin, Jinlong Huang, Linmiao Li et al.· Developmental and Comparativ...· 0 citations
Aeromonas hydrophila is a major pathogen of Motile Aeromonad Septicemia (MAS) in Nile tilapia, with pathogenesis and immunity poorly understood. In early 2026, a > 70% mortality outbreak occurred at a Nanning tilapia farm. The dominant strain NN0116 was identified as A. hydrophila by 16S rRNA and whole-genome ANI analysis. It exhibited high virulence (LD50 = 5.2 × 103 CFU/fish). Its 5.37 Mb genome encodes 5065 proteins and contains T2SS, T3SS, T6SS, and pore-forming toxins. Virulence and antimicrobial resistance genes co-localized on genomic islands GI2 and GI7. The strain was resistant to 12 antibiotic classes but susceptible to third-generation cephalosporins and fluoroquinolones. Head kidney transcriptomics at 24 h identified 1066 differentially expressed genes, including 61 immunoglobulin (Ig) genes. KEGG enrichment revealed 43 significant pathways; the top ten were driven by 26 Ig genes. Network analysis of the top 20 pathways identified B cell receptor signalling (ko04662) and COVID-19 (ko05171) as central nodes, alongside 10 secondary core pathways, all Ig-driven. Integration of bacterial genomic and host transcriptomic data characterizes hypervirulent A. hydrophila NN0116 and demonstrates that Ig-mediated B cell responses are central to host defence, offering targets for vaccines and therapeutics.
Fuhao Geng, Jifeng Zhang, Zulin Fang et al.· Journal of Fish Diseases· 0 citations
Mactra veneriformis is an economically important bivalve mollusc in China, but its aquaculture is frequently threatened by Vibrio infections, particularly Vibrio alginolyticus. To investigate the molecular immune response of M. veneriformis to V. alginolyticus, we performed RNA-seq analysis of hepatopancreatic tissues collected at 48 h post-infection, the peak mortality time point, with PBS-injected individuals used as controls. Infection with V. alginolyticus caused severe histopathological damage in the hepatopancreas and resulted in a cumulative mortality of 53.3% over 14 d, compared with 3.3% in the control group. Transcriptomic analysis identified 2623 differentially expressed genes (DEGs), including 1585 significantly up-regulated genes and 1038 down-regulated genes. KEGG enrichment analysis demonstrated that DEGs were significantly enriched in immune related and metabolism pathways, including the JAK-STAT signaling pathway, RIG-I-like receptor (RLR) signaling pathway, and cytochrome P450 (CYP450) signaling pathway. Collectively, these findings revealed candidate immune related genes (tlr3, tlr5, myd88, nfkb1, il-17d, and ifi44l), a putative TLR-MyD88-NF-κB signaling axis, and KEGG signaling pathways, including JAK-STAT, RLR and CYP450, that may be involved in the innate immune response of M. veneriformis to V. alginolyticus infection. These results provide a transcriptomic basis for understanding host-pathogen interactions in this species and highlight candidate genes and pathways for future functional validation and potential application in disease-resistance breeding.
Hongda Li, Tao Liu, Qiang Li et al.· Comparative Biochemistry and...· 0 citations
