Thermal stress modulates interferon regulatory factor 9 in giant grouper (Epinephelus lanceolatus) in the course of antiviral immunity.

Interferon regulatory factor 9 (IRF9) is a key antiviral gene regulation during innate immune activation in groupers. In this study, we unpack how thermal stress reshapes the antiviral machinery driven by giant grouper IRF9 (ggIRF9) under nervous necrosis virus (NNV) challenge. In vivo, ggIRF9 mRNA levels surged 2- to 12-fold above baseline in a tissue-specific manner following LPS and poly (I:C) stimulation, and NNV infection drove a roughly 2-fold expression spike as early as 3 h post-infection. The overexpression of ggIRF9 dramatically amplified the promoter activity of two frontline antiviral genes, e.g. Mx1 and PKR, reaching peak inductions of ∼25- and ∼30-fold over mock-treated controls, respectively. Additionally, the DNA-binding domain of ggIRF9 appears to physically engage the interferon-sensitive response element within the Mx1 promoter, providing a direct mechanistic link between IRF9 occupancy and Mx1 transcriptional output. Moreover, ggIRF9 expression proved highly vulnerable to thermal perturbation. Hypothermic conditions induced a consistent ∼0.6-fold suppression of ggIRF9, while hyperthermic stress triggered a more complex, context-dependent modulatory pattern. In vitro, both cold and heat stress significantly inhibited ggIRF9 promoter activity. Cold likely acting through heat shock factor 1a (HSF1a) and heat through HSF1b, converging on the same outcome that a weakened antiviral transcriptional program. In short, these findings position ggIRF9 as an essential factor of innate antiviral immunity in giant groupers, and reveal that thermally compromised IRF9 function translates directly into diminished host defense, a finding with pressing relevance given the climate-driven escalating thermal volatility of aquaculture environments.