Porcine epidemic diarrhea (PED), induced by porcine epidemic diarrhea virus (PEDV) infection, represents a highly contagious swine disease that causes substantial economic losses in the global pig industry. As the most prevalent RNA modification, N6-methyladenosine (mA) has emerged as a crucial epitranscriptomic regulator of host-pathogen interactions, although its functional significance in PEDV-infected porcine intestinal epithelial cells (IPEC-J2) remains to be fully elucidated. This study drew m6A maps before and after ALKBH5 interfering with IPEC-J2 cells, revealing the mechanism by which ALKBH5 mediated mA regulates PEDV infection in IPEC-J2 cells. This study employs an in vitro model of PEDV-induced IPEC-J2 cell damage to investigate the regulatory mechanisms of m6A methylation in host antiviral responses. Utilizing a comprehensive multi-omics approach, including MeRIP-seq, RNA-seq, qPCR, Western blot, MeRIP-qPCR, and RIP-qPCR, we systematically uncovered the critical role of m6A methylation in antiviral defense. Our findings reveal that ALKBH5, a key demethylase during PEDV infection, plays a pivotal role in modulating m6A modification levels. Specifically, gene silencing of ALKBH5 significantly upregulates m6A modification but downregulates expression of the antiviral effector genes IFIT3 and HERC5. Mechanistically, we demonstrate that the YTHDC2 protein selectively recognizes mA modification sites within IFIT3 and HERC5 transcripts, thereby mediating ALKBH5-dependent regulation of mRNA stability and protein expression. Functional analyses further reveal that IFIT3 activates the IRF3/TBK1 signaling axis, while HERC5 enhances antiviral responses by modulating the key effector molecule ISG15. Together, these findings establish a synergistic antiviral mechanism in IPEC-J2 cells. This study is the first to elucidate the novel ALKBH5-YTHDC2-m6A molecular axis, which orchestrates host antiviral immunity through a dual-pathway regulatory mode. These findings provide a theoretical foundation for understanding the role of RNA epigenetic modifications in enterovirus infections and offer a molecular basis for developing m6A-targeted strategies to prevent and control porcine infectious gastroenteritis. 6 6 6
