Xenobiotics at environmental levels modulate influenza virus replication in a lineage- and subtype-dependent manner in vitro

The influence of environmentally relevant concentrations of aryl hydrocarbon receptor (AhR)-active pollutants on low-pathogenic avian influenza virus (LPAIV) replication remains poorly resolved, particularly under realistic co-exposure scenarios. Here, we quantified how single and mixed AhR agonists modulate replication of multiple LPAIV subtypes in avian fibroblasts at field-relevant concentrations. Chicken (DF-1) and duck (CCL-141) fibroblasts were exposed to 9-chlorophenanthrene (0.1-50 nM), PCB126 (0.01-10 nM), and TCDD (0.001-1 nM), with or without the endogenous AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ), and subsequently infected with four LPAIV subtypes (H16N3, H9N2, H7N3, H5N3). Viral replication was quantified by qPCR (ΔCt). Factorial models emphasized host lineage × virus subtype interactions and mixture-versus-single comparisons across a shared concentration window. Host lineage × virus subtype interactions explained the largest share of variance. Single-agent effects were highly context-dependent; statistically supported antiviral activity was observed only for PCB126 in DF-1 cells infected with H7N3 (q = 0.006). In contrast, a three-compound pollutant mixture significantly enhanced replication in CCL-141 cells infected with H7N3 (q = 3.8 × 10^-4), whereas this supported trend was not observed with FICZ co-exposure. Within the tested environmentally plausible concentration window, concentration-matched Highest Single Agent comparisons more often yielded mixture responses below the strongest matched single-agent response among the evaluated comparisons. Within the tested concentration window, responses were graded and lacked clear thresholds. Overall, AhR-active xenobiotics can tune LPAIV replication in a host- and subtype-specific manner, highlighting the need for mixture-aware, context-dependent ecological infection-risk assessment.