Effects of H9N2 avian influenza virus infection on metabolite content and gene expression in chick DF1 cells.

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.