The initial sensing of infection by RNA viruses is mediated by Toll-like receptors 3,7,8 and RIG-like receptor signalling pathways. All these pathways lead to essential induction of type I interferon (IFN) via activation of interferon regulatory factor 3 and 7 (IRF3 and IRF7). These IRFs are activated by phosphorylation through kinases known as TANK binding kinase 1 (TBK1), and I-kappa-B kinase epsilon (IKK?). Viral hemorrhagic septicemia virus (VHSV) is an enveloped negative-sense single-stranded RNA virus that affects more than 50 marine and freshwater species. In this study, we identified AcIRF3, AcIRF7, AcTBK1, and AcIKK? identified from Amphirion clarkii and investigated the antiviral responses against (VHSV).
The genes were identified from a previously stipulated transcriptomic database, and in silico analysis was carried out for predicted genes and protein sequences. Spatial expressions of the gene transcripts were examined in twelve tissues in healthy yellowtail clown fishes. Four genes were cloned into pcDNA3.1(+) and EGFP-N1 vectors for the cell-based antiviral and sub-cellular localization assays.
According to the spatial expression analysis, all four genes are universally expressed in tested tissues and predominantly expressed in the cytoplasm of the cells. AcTBK1, AcIRF3 and AcIRF7 significantly induced IFNα expression upon VHSV infection. AcIKK? and AcTBK1 overexpression did not significantly change cell apoptosis, but AcIRF3 and AcIRF7 overexpression showed reduced apoptosis at 48 h of VHSV infection. However, prolonged infection (seven days of post-infection) of VHSV, AcTBK1, and AcIRF3 showed severe cytopathic effects same as the pcDNA control. AcIKK? and AcIRF7 showed less cytopathic effect than the pcDNA control. Therefore, AcIRF3, AcIRF7, AcTBK1, and AcIKK? might exert differential immune modulatory responses upon fish RNA virus infections.