Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by the procine epidemic diarrhea virus (PEDV), primarily characterized by acute vomiting, watery diarrhea, dehydration, and a high mortality rate of 80 %–100 % in piglets [[1], [2], [3]]. Due to rapid variations in the viral genome and immune evasion mechanisms, the protective effects of existing vaccines are limited [4]. Consequently, a comprehensive understanding of the pathogenic mechanisms of PEDV, particularly regarding its destruction of host cells and induction of cell death, as well as the identification of key targets for viral infection, has become a critical scientific issue in PED prevention. Evidence has confirmed that PEDV infection can cause damage to intestinal epithelial cells in piglets, a process closely linked to the interactions between viral proteins and host factors [5,6]. Zong et al. demonstrated that overexpression of interferon regulatory factor 8 (IRF8) reduces reactive oxygen species (ROS) levels in cells, thereby mitigating PEDV-induced oxidative damage and inhibiting the expression of apoptotic genes [7]. Overexpression of spike 1 protein in PEDV strains BJ2011, SM98, and CV777 S1 has been shown to induce cell apoptosis [8]. Furthermore, studies have reported that Portulaca oleracea water extract can inhibit PEDV-induced pyroptosis by downregulating the expression of Caspase 1 and GSDMD [9]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been shown to cause necroptosis in lung tissue through Z-nucleic acid (NA)-binding protein 1 (ZBP1) [10].

PANoptosis, a recently proposed mode of cell death, is characterized by the synergistic activation of pyroptosis, necroptosis, and apoptosis, which is tightly regulated by the PANoptosome complex. ZBP1 plays a crucial role in mediating interactions between inflammatory cell death pathways and was initially identified as a sensor for necroptosis [[11], [12], [13], [14]]. Subsequent studies have revealed that ZBP1 also regulates various inflammatory cell death pathways [15,16]. Viral infections are a significant trigger for PANoptosis, exhibiting a dual mechanism and biological significance in antiviral immunity. These reactions can restrict viral replication by eliminating infected cells while potentially causing pathological damage due to overactivation of PANoptosis [17]. For instance, ZBP1-dependent PANoptosis has been shown to limit influenza A virus (IAV) replication while also contributing to virus-induced lung lesions [18]. SARS-CoV-2 infection can induce PANoptosis in bystander cells, thereby maintaining the inflammatory response [19]. ZBP1 is a key gene in virus-induced PANoptosis of host cells. During IAV infection, ZBP1 not only stimulates NLRP3 inflammasome activation but also triggers apoptosis and necroptosis, indicating its critical regulatory role across these three cell death pathways [20]. Additionally, research has identified Z-RNA as an activator of ZBP1 in cells infected with influenza virus and poxvirus [21,22]. Oxidative stress, a pathological state characterized by excessive ROS production beyond the antioxidant defense capacity, can induce various types of cell death. NFS1 deficiency, in conjunction with oxaliplatin, has been shown to trigger PANoptosis by increasing intracellular ROS levels [23]. Reprogramming regulatory T cell-derived exosomes loaded with antioxidants can effectively prevent intestinal inflammation by blocking mitochondrial oxidative stress [24]. Furthermore, oxidative stress plays a significant role in viral infection processes. For instance, the aqueous leaf extract of M. oleifera has been shown to inhibit PEDV replication by suppressing oxidative stress-mediated apoptosis [25]. Moreover, it has been described that the influenza virus infects macrophage cell lines in mice through the ROS/NLRP3/pyroptosis pathway [26].

In recent years, the impact of oxidative stress on viral infection has received considerable attention from researchers. However, the roles of ROS and PANoptosis in PEDV-induced intestinal damage remain poorly understood. In the present study, we established a PEDV infection model in piglets and developed a ZBP1 knockout model, as well as an oxidative stress inhibition model utilizing in vitro cultured Zbp1 KO Vero E6 cells. Our results showed significantly elevated intracellular ROS levels and upregulated expression of PANoptosis genes in the PEDV infection model. Moreover, antioxidants such as N-acetylcysteine (NAC) partially alleviated cell death, highlighting the critical role of oxidative stress in PEDV pathogenesis. Additionally, we discovered that PEDV-N can colocalize with Z-nucleic acid (Z-NA) and ZBP1. These findings further elucidate the biological mechanisms underlying PEDV-induced intestinal damage in piglets and enrich our understanding of the role of oxidative stress in viral infection. This study focuses on the interaction between PEDV-N and ZBP1 and the molecular mechanism through which they induce PANoptosis in intestinal cells via oxidative stress, aiming to unveil new targets for PEDV pathogenesis and provide novel insights into preventing and treating diseases caused by coronaviruses.